i

Detailed Understanding of Dark State Dynamics of
Far-Red Fluorescent ProteinsbySamantha L. Allen
B.S., Agnes Scott College, 2014

A thesis submitted to the
Faculty of the Graduate School of the
University of Colorado in partial fulfillmentof the requirements for the degree ofDoctor of Philosophy
Department of Chemistry
2020

ii

This thesis entitled:
Detailed Understanding of Dark State Dynamics of Far-Red Fluorescent Proteins.
Written by Samantha L. Allen
has been approved for the College of Arts & Sciences Department of Chemistry________________________________________
Ralph Jimenez________________________________________
Amy E. Palmer

Date________________

The final copy of this thesis has been examined by the signatories, and we find that boththe content and the form meet acceptable presentation standards of scholarly work in theabove mentioned discipline.

iii

Allen, Samantha L. (Ph.D., Chemistry)
Detailed Understanding of Dark State Dynamics of Far-Red Fluorescent Proteins
Thesis directed by Prof. Ralph Jimenez

The development of fluorescent proteins in the far-red and near-infrared has
unlocked new potential for deep-tissue imaging, significantly reducing scatter, cellular
autofluorescence, and absorption by cellular constituents compared to their blue-shifted
counterparts. These proteins are characterized by low quantum yields, which must be
improved to fully utilize these proteins for imaging. The dark state photophysics of nearinfrared fluorescent proteins have yet to be characterized, which is necessary to renderthem useful for imaging and to inform generation of mutants with improved quantum
yields. This work discusses my study of the dark state conversion of two far-red
fluorescent proteins, smURFP and miRFP670, which both use biliverdin as achromophore. I designed and optimized the fabrication of microwell devices for theisolation of single proteins. Use of these devices speeds data collection and decreases
measurement variability compared to the preceding technique. The microwell devices
were used to perform time-resolved fluorescence measurements of smURFP andmiRFP670, the results of which indicate the proteins’ effectiveness and guide future
generations of far-red mutants.

iv

Dedication
To Rob, thank you for always pushing me to succeed

v

Acknowledgments
There are many people who have helped me throughout my graduate studies, andwithout whom I would not have been able to complete this work. I would like to thank Dr.
Leon Venable, my chemistry advisor at Agnes Scott College, who first encouraged me topursue research opportunities and apply to graduate school. I would also like to thank Dr.
Christopher Elles and Dr. Amanda Houk, who first mentored me in research andspectroscopy at the University of Kansas.
Special thanks to my advisor, Dr. Ralph Jimenez, for providing me with interesting
research directions to pursue. His guidance has made me into a better scientist. I would
also like to thank Pia Friis, who first taught me the basic biology skills that I needed. I
would also like to thank Dr. Nancy Douglas, who continued this education. I would like tothank Dr. Brett Fiedler, who recruited me to the group and taught me how to makemicrofluidics, and to Premashis Manna, for introducing me to dark state conversion. I
would like to think Dr. Sheng-Ting Hung and Srijit Mukherjee, for countless discussions
on the dark states of fluorescent proteins. Thank you to Dr. Alex Mikhaylov, Kristen
Parzuchowski, and Ryan Wilson, for always giving me the physics perspective. Thankyou to Emma Simmerman, Dr. Richard Erickson, and Dr. Josh Slocum for your helpful
feedback and scientific wisdom throughout this journey.
My friends and family have provided vital support throughout the way. Thank you
to Alina Mateo, Zenyth Sheppard, Kathryn Randall, Vaidehi Mehta, Curtis Beimborn, andRandall Chiu for providing me with laughs and support along the way. Thank you to myfamily for supporting me. Thank you to Amelia Allen, my first friend and sister, whoprovided me with endless emotional support.
This thesis would not have been possible without the help from my husband, Rob
Wells, without whom I could not have completed this degree. One PhD student in ahousehold is bad enough. In addition to coding advice he has given me emotional support
throughout thesis writing, providing encouragement along the way, all of this despite
working on his own graduate studies at the same time.

viContents

Chapter
1. Introduction…………………………………………………………………1
1.1 Discovery and Initial Development of Fluorescent Proteins..............................1
1.2 Evolution of Far-Red Fluorescent Proteins………………………..……………...5
1.3 Fluorescent Protein Photophysics……………………………………………….12
1.4 Advances in Super-Resolution Imaging…………………………………………22
1.5 Thesis Objectives…………………………………………………………...……..27

2. Design and Fabrication of Microwell Devices………………………….28
2.1 Introduction…………………………………………………………………………28
2.2 Droplets for Protein Confinement………………………………………………...30
2.3 Optimization of Microwell Device Design……………………………………….. 33
2.4 Troubleshooting Microwell Device Fabrication………………………………….38
2.5 Microwell Filling Investigation…………………………………………………….46
2.6 Pre-illumination Study……………………………………………………………..50
2.7 Conclusion and Future Outlook…………………………………………………..52

3. Data Collection and Analysis Methods…………………………………54
3.1 Introduction…………………………………………………………………………54
3.2 Sample Preparation……………………………………………………………….54

vii

3.3 Experimental Setup………………………………………………………………..56
3.4 Optical Measurements of Dark State Photophysics…………………………….59
3.5 Calculation of Irradiance……………………………………………………..……61
3.6 Analysis of Continuous Illumination Data……………………………………..…64

4. Results of Dark State Analysis…………………………………………..73
4.1 Introduction…………………………………………………………………………73
4.2 smURFP Continuous Illumination Study………………………………………...74
4.3 miRFP670 Continuous Illumination Study……………………………………….80
4.4 mCherry Continuous Illumination Study………………………………………....86
4.5 Comparison of smURFP and miRFP670 to RFPs ……………………...………91
4.6 Kinetic Modeling of Continuous Illumination Results …..…………………..…..93
4.7 Discussion………………………………………………………………………...105
4.8 Conclusion………………………………………………………………………..110
4.9 Future Directions…………………………………………………………………110

Bibliography…………………………………………………………………....112

Appendix………………………………………………………………………..121
A. Steady State Fluorescence Measurements………………………121
A.1 Publication Status……………………………………………………………121
A.2 Introduction…………………………………………………………………...121
A.3 Methods……………………………………………………………………….122
A.4 Results……………………………………………………………………..…122

viii

B. Detailed Protocols……………………………………………….…..125
B.1 Microfluidic Master Fabrication…………………………………………..…125
B.1.1 Selecting Materials and Design………………………………………125
B.1.2 Wafer Cleaning………………………………………………………...126
B.1.3 Spin-Coating the Wafer……………………………………………….126
B.1.4 Soft-Bake……………………………………………………………….128
B.1.5 UV Exposure…………………………………………………………...128
B.1.6 Post-Exposure…………………………………………………………130
B.1.7 Development…………………………………………………………...131
B.1.8 Hard Bake………………………………………………………………132
B.1.9 Silanization……………………………………………………………..132

B.2 Preparation, Cleaning, and Storage of Microwell Devices….133
B.3 Bacterial Expression……………………………………………135
B.4 Fluorescent Protein Expression, Purification, and Storage…136
B.4.1 Expression……………………………………………………………...136
B.4.2 Purification and Concentration……………………………………….137

ix

List of Tables

Table
1.1 Properties of BV-based fluorescent proteins…………………………………………….9
4.1 Time constants obtained from fitting smURFP continuous illumination data………..80
4.2 Time constants obtained from fitting miRFP670 continuous illumination data………85
4.3 Time constants obtained from fitting mCherry continuous illumination data……..….90

x

List of Figures

Figure1.1 Structure of GFP-like fluorescent protein………………………………………..……….2
1.2 Comparison of light absorption by tissue………………………………………...……….6
1.3 Biliverdin structure and binding……………………………………………………..…….7
1.4 Biliverdin photoconversion reaction…………………………………………….…..…….8
1.5 Crystal Structures of miRFP670 and smURFP Y56R………………………………….10
1.6 smURFP and miRFP670 excitation and emission………………………...…………...11
1.7 Three-level system diagram……………………………………………...……………...17
1.8 Fluorescence trajectory for a single fluorescent molecule…………………………….21
1.9 Illustration of image collection through SOFI…………………………….……..………26
2.1 Droplet and microwell Images…………………………………….………………………31
2.2 Depiction of a single microwell device………………………………….……………….33
2.3 Picture of a single microwell within a laser beam………………………………………36
2.4 Microwell master diagram…………………………………………………………………37
2.5 Fabrication process schematic…………………………………………………………...38
2.6 Flow diagram of microwell device fabrication process………………………………….40
2.7 Comparison of microwell depth distribution to fluorescence signal…………..……….47
2.8 Results of microwell time lapse experiment……………………………………………..49

xi

2.9 Results of pre-illumination study………………………………………………………….51
3.1 Optical setup……………………………………………………………………………….58
3.2 Flow chart summary of data analysis scheme………………………………………….65
3.3 Sample miRFP670 and smURFP fluorescence decays………………………………..67
3.4 Overview of data analysis scheme……………………………………………………….72
4.1 smURFP experimental data………………………………………………………………75
4.2 smURFP fits………………………………………………………………………………..76
4.3 Two-exponential fit to the first 10 ms of the smURFP fluorescence decays………….78
4.4 miRFP670 experimental data…………………………………………………………….81
4.5 miRFP670 fits………………………………………………………………………………83
4.6 Two-exponential fit to miRFP670 fluorescence decays………………………………..84
4.7 mCherry continuous illumination data……………………………………………………87
4.8 mCherry fits………………………………………………………………………………...88
4.9 Two-exponential fit of mCherry fluorescence decays..……………..……………..…...89
4.10 Comparison of rate constants………………………………………………………..….92
4.11 Two-level model diagram and equations……………………………………………….93
4.12 Comparison of miRFP670 two-level model to experimental results…………………94
4.13 Comparison of two-level simulation to smURFP experimental data…………………96
4.14 Three-level model diagram and equations…………………………………………….97
4.15 Comparison of smURFP simulated results using a three-level model………………98
4.16 Effect of kDSC on simulated data……………………………………………………..100
4.17 Three-level model with excited-state bleaching……………………………………...102
4.18 Effect of switching bleaching terms on simulated data………………………………103

xii

4.19 Protein environment around chromophore for miRFP proteins……………………106
4.20 Protein environment of smURFP chromophore…………………………...…………109
A.1 Excitation-dependent emission spectra………………………………………………..123

1

Chapter 1: Introduction

1.1 Discovery and Initial Development of Fluorescent Proteins
The generation of fluorescent proteins with emission wavelengths spanning thevisible spectrum enabled knowledge of the inner workings of cells to a degree notpreviously thought possible. Green fluorescent protein (GFP) was first isolated in 1962 by
Osamu Shimomura. He was attempting to isolate the green fluorescence from the jellyfish
Aequorea victoria, and instead extracted a blue emitting protein, named aequorin. GFP,
the protein responsible for the green fluorescence of Aequorea victoria, was isolated asa byproduct of the aequorin purification.1 Shimomura extracted and characterized thestructure of the GFP chromophore in 1979,2 and following this there were no major
developments in GFP until the 1990‘s when the gene encoding GFP was cloned andsequenced, and the chromophore formation was characterized, leading to rapid progressin the field.3-5 Martin Chalfie then used the isolated gene to express GFP in E. coli and C.
elegans. Surprisingly, he found that the E. coli and C. elegans expressing GFP was
fluorescent, indicating that GFP required no species-specific cofactors or enzymes forfluorescence.6 Prior to this success, it was not believed that the protein alone would
generate fluorescence in another organism.

2

The isolation of the GFP gene and expression in E. coli enabled Roger Y. Tsienand his lab to generate new fluorescent proteins (FPs) by making mutations to the aminoacid sequence of existing FPs and observing the spectral properties of the resulting
protein. Mutants were found with improved brightness, greater photostability, and weregenerated with a range of wavelengths including ones that were spectrally distinct for the
purpose of multicolor imaging.7-9 The crystal structure of GFP revealed the structure to
be an 11-stranded β-barrel with a coaxial helix, with the chromophore forming from thecentral helix, enabling directed mutagenesis of GFP.9 This structure is shown in Figure
1.1. Fluorescent proteins which share these structural features are referred to as GFPlike.

Figure 1.1 Structure of a GFP-like fluorescent protein. (a) Crystal Structure of EGFP (PDB # 2Y0G) (b) Top view of fluorescent proteinwith chromophore in gray.(c) GFP chromophore

Although mutagensis of GFP was able to generate GFP-like proteins with a rangeof wavelengths, it was unable to access red wavelengths. It was not until the isolation of
the tetrameric fluorescent protein DsRed (558/583 nm ex/em) from coral that red
fluorescence was achieved.10,11 The spectral properties have been found to be caused

3by the protein environment surrounding the chromophore in addition to the amino acidresidues forming the chromophore. The cyan FP amFP486 has a chromophore formed
from Lysine, Tyrosine, and Glycine residues, with Histidine and Alanine sidechains key
to the blue shift in fluorescence.12 Yellow fluorescent protein zFP538 also exhibited a
Lysine-Tyrosine-Glycine chromophore, with a red shift caused by extended chromophore
conjugation and π-stacking.13 A structural study of the YFP chromophore found that its
Serine-Tyrosine-Glycine chromophore, while identical to the GFP chromophore, is redshifted by nearly coplanar π-stacking interactions.14,15 Red-emitting FPs have beenshown to undergo an additional multistep reaction for chromophore formation, resulting
in delocalization of electron density over the polypeptide backbone and a red-shift in
excitation and emission.10,16 Multiple mutations of DsRed resulted in the generation of amonomeric red fluorescent protein, mRFP1 (584/607 nm ex/em), a protein more usefulas a genetically encoded fusion tag compared to the tetrameric DsRed.17 From thisprotein, the mFruit series of red fluorescent proteins (RFPs) was developed with improved
brightness and photostability, including mCherry (587/610 nm ex/em).18,19 Considerable
work has been done to improve RFPs for spectroscopic study, but no single RFP fulfills
all desired qualities (monomeric, bright, minimal blinking at a wide range of irradiances,
pH stability, photostability) for optimal use across single molecule and other spectroscopic
applications.20
In addition to achieving red fluorescence from proteins with the discovery of
DsRed, mutagenesis of this protein and others from coral and other sea creatures havealso led to the development of GFP-like proteins in the far-red portion of the spectrum.
Proteins developed in this region of the spectrum include E2-Crimson (611/646 nm

4ex/em),21 mNeptune (600/650 nm ex/em)22, TagRFP657 (611/657 nm ex/em),23 andeFP670 (605/670 nm ex/em).24 GFP-like far-red fluorescent proteins have been
demonstrated to be useful in imaging in vivo and in mammalian cells.21-24 Theoretical
study of mPlum (590/649 ex/em) found that the large Stokes shift is increased bydisplacements of water H-bonds to the imidazolinone ring oxygen in addition to aminoacid side chain rotation, with the largest red shift achieved with the highest negative
charge accumulation on the acylmine.25
Fluorescent proteins possess several advantages compared to small moleculedyes. Fluorescent proteins are genetically econded, meaning they can be incorporated
into cells by transforming those cells with DNA that encodes for the fluorescent protein ofinterest. Once incorporated into cells, they are reproduced by the cellular apparatus andcan be used to target subcellular compartments and to interact with an analyte. 7,26,27
Fluorescent proteins exhibit lower brightness and lower photostability compared to small
molecule dyes.
Dyes can leak out of cells and are difficult to load in thicker tissues, often requiring
invasive techniques to import them into cells. While dyes alone cannot target subcellularcompartments like fluorescent proteins, hybrid biosensors have been introduced to
combine the benefits of synthetic dyes with the benefits of the genetic encodability offluorescent proteins. Hybrid biosensors utilize bright, photostable synthetic dyes toimprove upon the imaging capabilities, which can then be bound to a fluorescent protein
tag to target specific cellular locations. These hybrid sensors are still in the early phasesof development and suffer from the same cellular delivery issues dyes face with many ofthem demonstrated in cell culture only.28

5Fluorescent proteins have been used as sensors for pH, temperature, and analyte
concentration.27,29,30 Using

fluorescentproteins

tomonitorcellularactivityisadvantageous because the protein genetic fusions often preserve biochemical functions
and cellular localization of the fused protein.29
The discovery of GFP and subsequent development into a range of wavelengths
and into a variety of uses transformed the field of fluorescence microscopy. Recognizing
the significance of this discovery and acknowledging the implications, the 2008 Nobel
Prize in Chemistry was awarded to Osamu Shimomura, Martin Chalfie, and Roger Y.
Tsien for their contributions to this field. These accomplishments made multicolor imaging
possible and generated new insights into biological targets and pathways, but a different
approach was needed to generate FPs with excitation and emission wavelengths in the
far-red and near-infrared portion of the spectrum, a challenge for deep-tissue imaging.

1.2 Evolution of Far-Red and Near-infrared Fluorescent ProteinsAs described in the previous section, fluorescent proteins revolutionized the fieldof cellular imaging and revealed the inner workings of cells to an extent not previously
thought possible. Despite the broad availability of fluorescent proteins at a wide range of
wavelengths, the generation of suitable fluorescent proteins in the far-red (FR) and nearinfrared (NIR) region of the spectrum has been difficult to achieve. The longest
wavelength excitation spectrum of a GFP-like fluorescent proteins is 611 nm.21-24 Thissection will discuss the evolution of FR and NIR FPs from bacteriophytochromes andcyanobacterial proteins to generate a series of biliverdin-binding fluorescent proteins.

6Light absorption by tissue

Figure 1.2 Comparison of light absorption by tissue. Figure adapted from Ref. 31. Light absorption was
Calculated assuming normally oxygenated tissue, 50 mM hemoglobin, and a composition of 50% water and
15% lipids.

Deep-tissue imaging is limited by a deficiency of fluorescent proteins exhibiting
excitation and emission in the far-red and near-infrared region of the spectrum. Lower
absorption by hemoglobin, water, and lipids (Figure 1.2), lower tissue autofluorescencein the 650-900 nm region, and low scattering at longer wavelengths necessitates the useof far-red fluorescent proteins for imaging deeper than a few millimeters in a tissue
sample.31,32 For improved deep-tissue imaging, proteins with excitation above 600 nm
and emission above 650 nm are needed.

7

(a)

(b)

Figure 1.3 Biliverdin structure and binding (a) Biliverdin molecular structure. (b) Biliverdin withinbacteriophytochrome binding pocket. Crystal structure showing BV within Deinococcus radiodurans (IFP1.4
parent bacteriophytochrome). Figure from Ref. 37.

Phytochromes are poorly fluorescent photoreceptors found in plants, fungi,
bacteria and cyanobacteria that use tetrapyrroles as chromophores. 33 Of this group,
bacteriophytochrome photoreceptors (BphPs) are the most promising precursor for farred imaging because they have the most red-shifted absorption among thephytochomes.34 Nearly all BphPs bind autocatalytically and covalently to biliverdin Ixα(BV, Figure 1.3 (a)) via a cysteine amino acid residue.35,36 A crystal structure illustratingbiliverdin binding within a bacteriophytochrome is shown in Figure 1.3 (b).37 BphPs absorb
light in the near-infrared, resulting in signal transductions, conformational changes, andprotonation reactions of the chromophore.33 Bacteriophytochromes are composed of thePAS, GAF and PHY domains. The absorption maximum of BphPs varies depending on
the photochemical state of the molecule, exhibiting photoconversion between a red
absorbing (~700 nm) and a far-red absorbing ground state (~750 nm).33,35 This
photoconversion occurs via isomerization around a C15=C16 double bond and asubsequent proton transfer process across several amino acid side chains. The

8photoconversion from the red-absorbing from to the formation of an intermediate
photoproduct is shown in Figure 1.438,39

Figure 1.4. Biliverdin photoconversion reaction. The reaction results in formation of the Lumi-R
photoproduct, which further reacts to form the far-red absorbing form (Pfr) in R. palustris P3
bacteriophytochrome. Adapted from Ref. 39.

Biliverdin is a flexible molecule and exhibits photoconversions in the excited stateand intramolecular proton transfer in the ground or excited state. BV in solution adopts
several different conformations and has a quantum yield of 10-4 in the far-red portion of
the spectrum, and must be conformationally restricted via binding to a protein in order toinduce fluorescence in this region.40,41 Biliverdin is a byproduct of heme degradation andis abundant in eukaryotic cells, indicating BV-binding fluorescent proteins can ideally be
used in mammalian cells without additional cofactors. In practice, many of these
fluorescent proteins require the co-expression with heme oxygenase to produce a higher
concentration of biliverdin or the addition of exogenous biliverdin to achieve brightfluorescence.41-43

9Bright fluorescent proteins that bind biliverdin were generated by first truncating
the wild-type protein to the PAS and GAF domains of a bacteriophytochrome andsubsequently performing several rounds of mutagenesis. Truncation of the protein to thePAS,GAF domain in addition to mutations made around the chromophore was found to
inhibit the photoconversion process and increase the quantum yield.34,41,42 The PAS and
GAF domains are necessary for effective BV incoroporation.36,41 Initial iterations of BphPbased fluorescent proteins required exogenous BV to be added to achieve brightfluorescence,41-43 but later versions efficiently incorporated cellular BV to produce brightfluorescence without added BV.34,44,45 The first monomeric BphP-derived FP, IFP1.4 was
dim without the addition of BV.41 A series of dimeric fluorescent proteins were developedthat demonstrated high brightness in mammalian cells without the requirement ofadditional BV that included iRFP670 (643/670 nm ex/em).34 The properties of these
proteins are summarized in Table 1.1.

Excitation

Emission

Quantum

Extinction Coefficient

(nm)

(nm)

Yield

(M-1 cm-1)

IFP1.4

684

708

0.07

92,000IFP2.0

690

711

0.08

86,000

mIFP

683

704

0.08

82,000iRFP670

643

670

0.11

114,000

miRFP670

642

670

0.14

87,400smURFP

642

670

0.18

180,000

Protein

Table 1.1. Properties of BV-based fluorescent proteins.

Through additional mutations to a bacteriophytochrome, the monomeric protein
miRFP670 is generated (Figure 1.5 a). miRFP670 was 5-times brighter than the10previously developed monomer mIFP in mammalian cells and has the highest molecularbrightness of any BphP-derived fluorescent protein. It has a quantum yield of 0.14 and an
extinction coefficient of 87,400 M-1cm-1, with an excitation maximum of 642 nm, and anemission maximum of 670 nm. miRFP670 is bright without additional biliverdin in
mammalian cells but becomes 7-fold brighter when co-expressed with heme
oxygenase.46

(a)

(b)

Figure 1.5. Crystal structures of miRFP670 and smURFP Y56R (a) Crystal Structure of miRFP670 with thebiliverdin chromophore in green (PDB #5VIV) (b) smURFP Y56R crystal structure with the biliverdin
chromophore in blue (PDB #6FZN)

smURFP (Figure 1.5 b) was evolved from an allophycocyanin (APCα) subunit fromcyanobacteria.47 APC is a light-harvesting biliprotein optimized for highly efficient energy
transfer via natural evolution, resulting in a higher natural fluorescence quantum yield.48,49
The wild-type protein covalently binds phycocyanobilin (PCB) via a cysteine residue withthe help of a lyase to incorporate the chromophore.49 The protein was evolved to attach
PCB without the help of a lyase and was further evolved to bind biliverdin. Biliverdin is11

preferable to PCB as a chromophore due to its abundance in mammalian cells. The
resulting protein, smURFP, has the highest molecular brightness of any biliverdin-binding
far-red fluorescent protein. It has a quantum yield of 0.18 and an extinction coefficient of
180,000 M-1cm-1. The excitation maximum is 642 nm, and the emission maximum is 670
nm.47 APC-based NIR FPs show low affinity for binding BV, leading to lower effective
brightness in cells. Enhancing the binding efficiency increases the fluorescence of these
proteins.50 Excitation and emission spectra for miRFP670 and smURFP (Figure 1.6)
illustrate the similarities in excitation and emission for these two proteins.

Normalized Signal

smURFP and miRFP670 Excitation and Emission
1.00

smURFP
Excitation

0.80

smURFP
Emission

0.60

miRFP670
Excitation

0.40

miRFP670
Emission

0.20
0.00
450

550

650

750

850

Wavelength (nm)Figure 1.6. smURFP and miRFP670 excitation and emission. Normalized spectra for excitation andemission.

12

The crystal structure of IFP1.4 showed that a dense, hydrophobic binding pocket
restricts movement of the BV D-ring and prevents C15=C16 isomerization as a relaxation
pathway.38 NIR FPs with even higher effective brightness were obtained via mutations
that enhance fluorescence that may be caused by a reduction in motion of the BV A ring
via van der Waals interactions and introducing a Hydrogen bond between thechromophore and protein.50
The utility of FR and NIR BV-based FPs has been demonstrated in vivo and incells.36,39,40 BphP-based FPs are often genetically encoded alongside a heme oxygenase
gene to convert heme into biliverdin in order to increase the amount of biliverdin in cells,
allowing use in cells such as E. coli, which possess heme but not BV.30,32

1.3 Fluorescent Protein Photophysics
Fluorescence occurs following excitation, when a fraction of molecules in the
excited electronic state returns to the ground electronic state. Additionally, somemolecules will become trapped in a long-lived dark state, and some will be permanently
photobleached. The emission lifetime is the average time between excitation and returnto the ground state, and is typically on the order of 10-9 s for fluorescent proteins, much
faster than light absorption, which is on the order of 10-15 s.51 This section will discuss thephotobleaching and dark state conversion processes observed in fluorescent proteins,and potential mechanisms.
Fluorescence emission occurs at longer wavelengths compared to excitation in aphenomenon known as the Stokes shift. At each electronic energy level, there are several

13vibrational energy levels. Molecules are typically excited from the ground electronic statefrom the lowest vibrational state to a higher vibrational state of S1, since at room
temperature the excited vibrational energy are not thermally populated to a significant
extent. Excited molecules in condensed phase, such as proteins in solution, rapidly relax
to the lowest vibrational state in ~10-12 s resulting in emission from a lower vibrational
state of the excited electronic state to a higher vibrational level of the ground state. Other
factors that further decrease emission energy relative to excitation include solvent
relaxation effects, conformational changes, and excited state proton transfer. 51
Brightness is a key factor determining the utility of fluorescent proteins, influencing
the lowest concentration that can be used in experiments as well as the sensitivity andcontrast from background fluorescence in live cells.52,53 Brightness can be expressed aseither molecular brightness or cellular brightness. Molecular brightness determines thefluorescent protein performance in solution and is defined as the product of extinction
coefficient and fluorescence quantum yield (Mb =ε∙ϕ). Equation 1.1 describes how
fluorescence quantum yield is calculated. Cellular brightness refers to the brightness offluorescent proteins within cells and is dependent on the molecular brightness in addition
to protein expression level, protein folding, and intracellular folding and stability of theprotein (sensitivity to pH, ionic strength, etc.). For the NIR FPs, chromophore affinity andspecificity is an additional concern for cellular brightness, with some NIR FPs exhibiting
higher molecular brightness, but lower cellular brightness.46,47,50,54,55 This effect occurs
due to competition between biliverdin and related molecules for the binding site for the
FP apoprotein.56,57

14

𝜙=

𝑘𝑟
𝑘𝑟 + 𝑘𝑛𝑟

Equation 1.1. Quantum yield. Ratio of emitted photons to total number absorbed. k r is the radiative rate
constant and knr is the rate constant for non-radiative decay.

Another key factor determining fluorescent protein utility is photostability, the abilityof fluorescent proteins to absorb and emit photons while resisting photoreactions that leadto degradation. A photobleached molecule no longer produces fluorescence.
Mechanisms for photobleaching have been characterized in various fluorescent dyes andin select fluorescent proteins but have yet to be characterized in NIR FPs.
One mechanism that has been proposed for photobleaching in certain dyes andfluorescent proteins is photoreaction via a dark triplet state. Both small molecule dye
Rhodamine 6G and fluorescent proteins EGFP, EYFP, DsRed, and mCherry have shown
evidence of photobleaching occurring via a triplet state and leading to photoproductformation.58-62 Rhodamine 6G photobleaching involves a multistep photoreaction from thetriplet state that results in radical anion.59-61 Single-molecule spectroscopy on EYFP,
EGFP, and DsRed similarly supported a mechanism of multistep reaction from the triplet
state and leading to photoproduct formation.62 These measurements on EGFP, EYFP,
and DsRed reported photobleaching time constants of 2.8 ± 0.2 ms, 3.5 ± 0.5 ms, and
0.4 ± 0.1 ms at saturating irradiances of 13 ± 3 kW/cm2, 6 ± 1 kW/cm2, and 50 ± 10
kW/cm2, respectively.62 The single-molecule results were reinforced by a fluorescencecorrelation study that also found EYFP to be more photostable than EGFP.63
Fluorescence measurements on mCherry reported photobleaching time constants of 3.5± 0.7 s and 57.0 ± 4.6 ms at 2.5 and 25 kW/cm2 respectively.58

15

Photobleaching via a singlet state has also been proposed as a mechanism insome fluorescent dyes. A fluorescence correlation spectroscopy study of photobleachingof fluorescein and borondipyrromethene (BODIPY) dyes determined that bleaching forthese dyes occurred primarily via an excited singlet state, by comparing rates ofphotobleaching and rates of intersystem crossing in multiple of these dyes. 64
In addition to irreversible photobleaching, fluorescent proteins also exhibit
reversible loss of fluorescence, known as dark state conversion (DSC). Dark state
conversion is a light-driven, reversible process. A fluorescent protein in the initially excitedelectronic state enters a dark (nonfluorescent) state. The process by which the excitedstate of a fluorescent protein returns to the ground state is called Ground State Recovery(GSR). Once a fluorescent protein has returned to the ground state, it can resume
fluorescing. The steady-state population of molecules in the dark state is determined by
the rate at which the molecule enters and exits the dark state.
A three-level system diagram depicts the cyclic process of excitation, emission, withthe possibility of entering and exiting the dark state as well as photobleaching (Figure1.7). Previous studies of fluorescent proteins photophysics have employed threelevel,65,66 four-level,58,67,68 and more complex models69 to describe the photophysics ofvarious fluorescent proteins. The four-state model includes two anionic and two neutral
states of the chromophore, with the neutral forms designated nonfluorescent. and was
initially proposed by Dickson et al to describe single-molecule observations of a GFPvariant.68 An analogous model was used to interpret fluorescent measurements on RFPs,
with two bright and two non-fluorescent states, with the dark states interpreted as a
combination of neutral and isomeric states of the chromophore. This model agreed with

16

experimental findings of a linear relationship between dark state fraction and DSC time
constant for the RFPs studied.58 A similar four-state model was used to describe TagRFPT fluorescence results with two fluorescent and two dark states.67 Another study found athree-state model with a bright ground and excited state and a dark state was sufficient
to describe fluorescent measurements on several RFPs.65 A similar three-level model with
2 bright and one dark state was used to describe the flickering dynamics observed fromFCS experiments on red fluorescent protein eqFP611.66 Sinnecker et al proposed amechanism with a protonated form, deprotonated form, and a dark form to describe thekinetics observed in fluorescence correlation spectroscopy (FCS) measurements of
ECFP and EYFP.69 A model with fewer states such as the three-level model may be morepractical to use, it also risks simplifying the complex photophysics in the dark state. On
the other hand, a more complicated model with several levels may have so many
parameters as to risk overinterpreting the data. In practice, we will use the three-level
model as a starting point to interpret our data since it can combine multiple dark stateprocesses into one state. This is done in an effort to use the simplest model that can
describe experimental observations.

17

kIC

Figure 1.7 Three-level system diagram. The ground state (S0), the first excited state (S1), and D indicating
a dark state. kex is the excitation rate constant, kem is the emission rate constant, kS1Bleach and kDBleach are thebleaching rate constants, and kDS1, kS1D, and kGSR are the rate constants for entering and exiting the darkstate.

Many of the photophysical parameters used to describe fluorescent proteins areinterrelated. A higher dwell time in the dark state conversion results in lower fluorescencebrightness and lower quantum yields, since a greater fraction of the molecules isnonfluorescent at any given observation time. The relatively low quantum yields of RFPs
mCherry, mRuby2, and mKate has been linked to the relatively large fraction of moleculesin the dark state compared to mScarlet, an RFP with a much higher quantum yield. 70 A
correlation between excitation rate and sensitivity to irradiance of reversible andirreversible photobleaching rate constants has been demonstrated in RFPs.58 As a result,when comparing photophysical properties of different fluorescent proteins, it is necessary
to do measurements at the same excitation rate to accurately compare relevant

18parameters. Excitation rate (Equation 1.2) is dependent on extinction coefficient andirradiance. Irradiance is reported as power divided by area (e.g. kW/cm 2) and is ameasure of excitation intensity.

𝑘𝑒𝑥 =ln⁡(10) ∙ 1000 ∙ 𝜀 ∙ 𝜆 ∙ 𝐼ℎ ∙ 𝑐 ∙ 𝑁𝐴

Equation 1.2. Excitation rate, kex. ε is the extinction coefficient. λ is the wavelength. I is the irradiance, h is
Planck’s constant, c is the speed of light, and NA is Avogadro’s number. This calculation is an average forkex over the volume illuminated based on the irradiance calculation

Among fluorescent proteins that exhibit conversion to the dark state, fluorescentproteins exhibit a variety of mechanisms for entry into the dark state, including electrontransfer reactions71,72, chromophore conformational changes,71-73 reactions via a triplet
state,71,72 and excited-state proton transfer.69,74-76 Dark state conversion and ground state
recovery processes have been reported on timescales ranging from hundreds ofmicroseconds to seconds.76-79
Several fluorescent proteins were found to exhibit dark state conversion that could
be attributed to proton transfer reactions. Proteins undergoing these reactions include
both variants of GFP and a protein derived from a sea anemone. Fluorescence studies
of several GFP variants found that ECFP, EGFP, EYFP, and Citrine exhibited light-driven
dark state conversion that increased at lower pH values in both cells and solution.69,76 For
these proteins, return from the ground state is spontaneous, but it can be accelerated bylight. This pH dependent effect was attributed to proton transfer between amino acidresidues exposed to the surrounding solution and the chromophore, whereas at high pHinternal proton transfer is thought to be predominant.69,76 Timescales for dark state19

conversion range from 2.4 s for ECFP, 130 s for EYFP, and 28 s for Citrine at irradiancesof 2.5 W/cm2 for ECFP and 2.35 W/cm2 for EYFP and Citrine.69 In contrast with thesevariants derived from GFP, DsRed exhibited pH-independent (4.4-9.0 range) light-driven
dark state conversion.74 Excitation-dependent, pH-independent flickering observed inboth DsRed and Citrine indicated the presence of an additional dark state, distinct fromthe protonated dark state observed in many GFP variants.74 Blinking of dsRed and Citrine
was reported on the timescale of hundreds of µs for the irradiance range 0.4-7.3
kW/cm2.74,80 The T203Y and T203F mutants of GFP exhibited both the pH-dependentand light-dependent dark state conversion effects discussed above. Flickering from these
proteins was observed over a µs-ms time scale for irradiances from 0.6-5000 kW/cm2.75
Dark state conversion occurring via conformational changes in the chromophore
has been observed in IrisFP, a protein derived from a coral protein. X-ray radiolysis wasused to generate a radical dark state. This indicates that intermolecular electron transfer
affects the blinking of IrisFP.71 Quantum Mechanics/Molecular mechanical calculations
on IrisFP indicated two mechanisms from which the distorted chromophore dark state canarise. The two mechanisms involve a proton transfer reaction that can occur via a triplet
state or a radical ground state and results in a distorted chromophore with disrupted πconjugation, resulting in loss of fluorescence.72 Both mEos2 and Dendra2 also show
evidence of dark state conversion via chromophore distortion and twisting motions. 73
Blinking timescales also give indications of the mechanism behind dark state
conversion.81 Fast blinking behavior (tens of microseconds to ms) has been attributed toconversion to a triplet state, while slower blinking (0.1-10s range) is associated withexcited-state conformational dynamics.73 mCherry and TagFPP-T have blinking reported

20

on the sub-ms to ms timescale.65 eqFP611 was found to have blinking on two timescales,a fast 300 µs timescale and a slower blinking on a 10 ms timescale.66
Blinking refers to the phenomenon of individual fluorescent protein molecules
entering the dark state and then returning to an emissive state. Blinking of fluorescentproteins was first observed in a study of single molecules green fluorescent protein.68
Single molecule measurements enabled visualization of this phenomenon that is
obscured in ensemble experiments. Single molecules of GFP were discovered to turn on
and off several times over a timescale of seconds, eventually entering a long-lived darkstate in which no emission is observed.68 A blinking trajectory for a single fluorescentmolecule shows it alternating between on and off states with lifetimes τON and τOFF,
respectively (Figure 1.8). Equations 1.3 and 1.4 show how the on- and off-times can becalculated based off the rate constants for dark state conversion (DSC) and ground state
recovery (GSR), respectively. Histograms of single molecule trajectories with on and offtimes can be constructed to determine average behavior. Fluorescent proteins with shortdark state lifetimes will blink on and off several times within an observation period. The
average number of blinks that can be observed is the ratio of the rate constant for
conversion to the dark state compared to the rate constant for irreversible photobleaching.
(kDSC/kBleach).73

21

Figure 1.8 Fluorescence trajectory for a single fluorescent molecule. τ ON is the time the molecule spends
fluorescing, and τOFF is the time the molecule spends in the dark state.

τon⁡=⁡

𝟏𝒌𝒆𝒙 ∗𝒌𝑫𝑺𝑪 ∗𝝉𝒆𝒎

Equation 1.3. Equation to calculate the on time (τon) for a fluorescence trajectory. kex is the excitation rate
constant, kDSC is the dark sate conversion rate constant, and τem is the fluorescence lifetime.

τoff⁡=⁡𝟏

𝒌𝑮𝑺𝑹

Equation 1.4. Equation to calculate the off time (τoff) for a fluorescence trajectory. kGSR is the ground state
recovery rate constant.

Photoswitchable fluorescent proteins exhibit highly efficient transfer to the darkstate, allowing the molecules to be switched on and off between nonfluorescent andfluorescent states. A fluorescent molecule in the “on” state can be switched off throughlight-driven conversion into a nonfluorescent or “off” state. Photoswitching is caused byreversible conversion between these on and off states.82 Studies have demonstrated that
pulsed excitation with a carefully selected frequency can be used to depopulate the dark

22

state

or ensure relaxation to the ground state between pulses and thus increase

fluorescence.83-85 Optical depopulation of dark state population has been utilized toincrease the measured fluorescence signal of FPs, resulting in improved sensitivity andselectivity for imaging, as demonstrated with AcGFP86 and protein rsFastLime.85
Depending on the application, the dark state can be a hindrance or a benefit. Whenhigh brightness over long, uninterrupted time intervals is needed such as cellular tracking
experiments, a fluorescent protein with little to no dark state conversion would be
preferable. The super-resolution techniques discussed in the next section utilize the darkstate to generate imaging resolution above the diffraction limit. Understanding dark state
photophysics can help guided evolution of fluorescent proteins by informing which
mutations can be linked to dark state conversion. Improved knowledge of dark statebehavior for a range of FPs allows evaluation of the utility of those FPs for imaging
techniques to determine whether the FP is better suited for cellular tracking experiments
or for super-resolution imaging techniques. The next section discusses super-resolution
imaging techniques and how they take advantage of dark state conversion.

1.4 Advances in Super-Resolution Imaging
While microscopy using fluorescent proteins is a powerful technique for imaging
living systems, standard light microscopy techniques could not distinguish emitters below
200 nm until the 1990s when techniques were introduced that used the dark state toovercome this limit. This diffraction limit was first described by Ernst Abbe in 1873 as theclosest distance that two objects can be and still be distinguishable (Equation 1.5).87 As

23

a result of this limit, objects smaller than approximately half a wavelength of light cannot
be resolved with traditional fluorescence microscopy techniques, preventing detailed
observation of subcellular objects below this size. This section discusses several of thesetechniques, referred to as super-resolution imaging, and how they utilize the dark state toovercome the diffraction limit.

∆𝑥 ≥𝜆2𝑛𝑠𝑖𝑛𝛼Equation 1.5. Diffraction limit. Δx is the distance between two objects that can be distinguished. n is therefractive index. α is the aperture angle under which the point source is observed. nsinα is also known as
the numerical aperture of the lens.

A key achievement in the development of super-resolution capabilities was the
successful detection of single molecules. This work was pioneered by W.E. Moerner in1989 when he successfully detected absorption of single molecules of pentacene in a pterphenyl crystal.88 Single-molecule fluorescence is observed when only one molecule ispresent within the irradiating volume, and when the signal from that molecule is larger
than the background signal.89 Single-molecule methods were used to reveal
photoswitchable behavior of GFP in 1997, which demonstrated the first use of a room
temperature photoswitchable molecule.68 Ensemble measurements observe behavior ofa large number of molecules and report the average value of parameters across the wholegroup, whereas single-molecule studies report on parameters of individual emitters.
Effective use of single-molecule fluorescence requires knowledge of total number ofphotons a fluorescent molecule can emit on average and rate of fluorescence, which isaffected by trapping in dark states. Additionally, single molecule signal is weak and easily

24lost in the background.89 Utilization of single molecule techniques and photoswitchable
molecules is essential for the localization-based super-resolution techniques.
Stefan W. Hell and Jan Wichmann introduced a method in 1994 to overcome thediffraction limit using pointillistic scanning technique with shaped beams and Hell
implemented the technique in 2000.90,91 This technique, Stimulated Emission Depletion(STED) fluorescence microscopy, requires the use of a saturable molecular transition to
inhibit fluorescence in a limited area, resulting in higher resolution. This is achieved byusing light of one wavelength to excite the sample, and a second beam to saturate thetransition, reducing the number of excited molecules. The second beam of light inhibits
fluorescence everywhere except the innermost region of the sample, so only thisinnermost region can contribute to the fluorescence signal.90 The initial application of thistechnique required irradiances in GW/cm2 to suppress fluorescence sufficiently to reach
sub-diffraction limit resolution.91 Ground-State Depletion (GSD) fluorescence microscopy
is a related technique that overcomes the diffraction limit by depleting the ground state ofmolecule in the outer region of the focus by using high intensity irradiation (MW/cm2) toquickly cycle molecules between excitation and emission, with a fraction caught in thedark state each time. The reduced number of excitable molecules leads to a decrease in
full-width half maximum (FWHM) and an increase in resolution.92 Reversible Saturable
Optical Fluorescence Transitions (RESOLFT) is an iteration of this technique which has
achieved resolution as low as 30-40 nm with irradiances on the scale of W/cm 2 byselecting saturable processes and fluorophores that require lower intensities, using
reversible photoswitches in this case.93

25

In 2006, two similar super-resolution fluorescence microscopy localization
techniques were developed independently of one another, Photoactivated Localization
Microscopy (PALM) proposed by Eric Betzig and Stochastic Optical Reconstruction
Microscopy (STORM) proposed by Xiaowei Zhuang. Both methods were demonstrated
to achieve ~20 nm resolution.94,95 Both techniques use the selective localization of sparse
subsets of fluorophores in which only one molecule in a diffraction limited volume islocalized at a time. This is done through switching fluorophores on for localization with
one wavelength of light, and then switching them off with another wavelength of light.
Through repetitive cycles of imaging, a total image with nanometer resolution can bereconstructed. The set of fluorophores that are turned on at any time is controlled so that
there is no overlap, and no two within a diffraction-limited volume are on at the sametime94,95 This can theoretically be done with any fluorophore that exhibits blinking, but
photo-switchable ones are the most straightforward to use for this application.
Super-resolution Optical Fluctuation Imaging (SOFI) was first published in 2009and obtained resolution beyond the diffraction limit in all three dimensions.96 SOFI
requires less sophisticated equipment compared to STED, PALM, and STORM, using
only a widefield microscope and CCD camera, compared to the requirement of totalinternal reflection fluorescence (TIRF) microscopy and an EMCCD camera for single
molecule for PALM and STORM.94-96 SOFI uses statistical analysis of temporalfluctuations caused by fluorophore blinking to enhance contrast over a series of images.
This process is illustrated in Figure 1.9. The resulting super-resolution image is virtually
background-free and is collected from seconds of acquisition time, improving upon
temporal resolution compared to PALM and STORM.96 Successful application of SOFI

26

requires a label with two spectrally distinct emission states, the label must switch between
these two states independently and stochastically, and the pixels used should be smaller
than the diffraction limit.96

Figure 1.9 Illustration of image collection through SOFI. At time t1, three fluorophores are shown in the ON
state, too close to exactly localize all three of them. At time t2, the middle fluorophore is in the OFF state,
and the other two fluorophores are in the ON state and separated enough so that they can be localized. At
t3, the fluorophores in the ON state are too close for localization. And finally, at t 4, only the middle
fluorophore is in the ON state, so it can be localized.

Super-resolution microscopy techniques have fundamentally changed the limits offluorescence microscopy. In recognition of this influence, the 2014 Nobel Prize in
Chemistry was awarded to Eric Betzig, Stefan W. Hell, and William E. Moerner for theirrole in developing the super-resolution microscopy techniques mentioned above. Since
its development, super-resolution has enabled the imaging of sub-cellular structures atresolution above the diffraction limit.71,72,75 SOFI is an especially promising technique,given its lower requirements and simple collection protocol. Utilizing fluorescent proteinsas the probes in this technique further enhances its capabilities, minimizing perturbations
to the biological substrates being imaged. While the biliverdin based fluorescent proteins

27

have not been used in super-resolution imaging, better understanding of their dark state
photophysics can be used to extend the wavelength range available for these techniques.

1.5 Thesis Objectives
As outlined in the above sections, the discovery of fluorescent proteins and thedevelopment of super-resolution microscopy have greatly advanced knowledge ofsubcellular biological processes. Despite these advances, deep-tissue imaging is limitedby the low quantum yields and low brightness of near-infrared fluorescent proteins.
Improved understanding of these fluorescent proteins and their dark state photophysics
is necessary to improve their utility. Knowledge of the dark state photophysics of these
proteins would enable application to super-resolution spectroscopy techniques such as
PALM and SOFI and would also allow illumination settings to be controlled to minimize
dark state populations. While near-infrared fluorescent proteins are not currently used forsuper-resolution spectroscopy, a better understanding of the mechanisms controlling
blinking in these FPs can allow them to be further developed for this purpose.
This work seeks to characterize the dark state photophysics of two far-red
fluorescent proteins, smURFP and miRFP670. smURFP has the highest molecularbrightness of any far-red fluorescent protein, and miRFP670 has the highest molecularbrightness of any bacteriophytochrome-based far-red fluorescent protein. To performthese time-resolved fluorescence measurements reproducibly and efficiently, a microwelldevice protocol was developed and characterized.

28

Chapter 2: Design and Fabrication of Microwell Devices

2.1 Introduction
During a time-resolved fluorescence experiment, fluorescent proteins may diffuse
throughout the sample. Diffusion can introduce adverse effects into measurements ofdark-state and photobleaching time constants, since a bleached protein or a protein in
the dark state may diffuse out of the irradiated area, or an unbleached protein may diffuse
in, artificially inflating the measured fluorescence signal. For experiments over a long time
period, it is essential to confine the fluorescent proteins within the irradiated area to collectaccurate measurements. This chapter discusses the methods developed to confine
proteins within a measurement area.
Whether or not a fluorescent protein remains in the irradiated area over the courseof measurements can be evaluated using the Stokes-Einstein equation for diffusion. The
Stokes-Einstein equation (Equation 2.1) describes diffusion for a spherical particle insolution. This equation can be used to obtain approximate values for the diffusion
coefficients of fluorescent proteins in solution, using the hydrodynamic radius calculated
from the molecular weight of each protein.

29

𝐷=

𝑅𝑇
𝑁𝐴 6𝜋𝑛𝑎

Equation 2.1 Stokes-Einstein equation to calculate diffusion coefficient. This equation assumes thatparticles are spherical. R is the gas constant, T is temperature in Kelvin, NA is Avogadro’s number, n is theviscosity of the solution, and a is the hydrodynamic radius of the spherical particle.

Using this equation, the far-red fluorescent proteins smURFP and miRFP670
studied have diffusion coefficients of approximately 107 µm2/s and 102 µm2/s
respectively. To assess this approximation, the diffusion coefficient was calculated for
GFP using the same method, and found a diffusion coefficient of 117 µm2/s. This value
was compared to the experimentally determined diffusion coefficient for GFP in solution,
87 ± 2 µm2/s, indicating the calculation is off by 30%.97
The displacement of the proteins during the timescale of a measurement can becalculated using Brownian dynamics (Equation 2.2). Over the course of a ten second
measurement, smURFP and mCherry will on average travel 65 µm and in the samemeasurement time, miRFP670 will on average diffuse 64 µm. The calculated
displacements were then compared to the full width at half maximum (FWHM) for thelaser beam to determine the extent to which diffusion will affect measurements. For the
beam used for my fluorescence measurements, the FWHM ~30 µm, half the distance thata protein will diffuse within a short measurement. Even assuming the diffusion coefficientsare ~30% lower than calculated, miRFP670 would be expected to diffuse 53 µm within aten second measurement, greater than the 30 µm beam diameter. This indicates thatunless samples are confined, protein diffusion into and out of the irradiated area will occur
during these measurements, affecting the calculated time constants. To prevent such
artifacts from affecting the data, several methods of protein confinement were considered.

30< 𝑥 >⁡= ⁡ √4𝐷𝑡
Equation 2.2 Equation for root mean squared displacement in 2-dimensional space. Describes a molecule
diffusing in 2-dimensional space according to Brownian motion. 2-dimensional motion is a good
approximation for proteins in solution between coverslips.

Initially I utilized octanol microdroplets as a method of protein confinement, atechnique that was first introduced by Patterson and coworkers in 1997 forcharacterization of fluorescent proteins.98 Microdroplets have been used to measure
photoconversion and photobleaching of fluorescent proteins.99-102 The weaknesses of thistechnique include the random distribution of droplets on a coverslip and the wide rangeof droplet sizes produced, making it ineffective for characterizing the photophysics of the
far-red fluorescent proteins within our experimental setup. Due to these concerns
microwell arrays were investigated as an alternative method for protein confinement. This
chapter discusses the drawbacks of using droplets, the challenges related to the
optimization of the design, fabrication, and use of microwell devices, and a discussion of
the evaluation of microwell devices.

2.2 Droplets for Protein Confinement
Droplets are generated using dye or protein in solution and 1-octanol as a meansof confining fluorescent molecules through phase separation between water and 1octanol. To prepare a droplet sample of fluorescent protein a 1:9 ratio of protein in buffer
solution is mixed with 1-octanol and is then deposited on a corner of a coverslip.77 Once
prepared, these samples will have many droplets filled with purified protein solution inside
the imaging area with diameters ranging from 5-200 µm (Figure 2.1A). By adding the31solution to one corner of the coverslip, the droplets within the imaging area are spaced
as far apart as possible. This minimizes the likelihood that fluorescence from nearby
droplets contributes to fluorescence signal.

Figure 2.1 Droplet and Microwell Images (A) Droplet and (B) Microwell samples as viewed under brightfield
illumination and collected by a cMOS camera under 20x magnification. The droplet picture is contrastenhanced for clarity. Circled in red is the portion of the image that will be centered in the laser beam formeasurement. Scale bar is 100 µm.

Within the sample on the coverslip, selected droplets must have the right size andmust not appear to be near any other droplets to be used in a measurement. Distance tothe nearest neighboring droplet is an important consideration when selecting a spot for
fluorescence measurement because proteins in nearby droplets illuminated by scatteredlaser light will contribute to the background fluorescence signal. A portion of the proteins
within that neighboring droplet will be bleached or converted to the dark state, distorting
results if the adjacent droplet is used for a later measurement. To have the right size fora measurement the droplet must be small enough fit within the beam so that diffusion intoand out of the irradiated area does not affect fluorescence signal. Droplets must also be32large enough that fluorescence signal is high at the selected protein concentration. Oncea droplet is selected it is centered within the beam and measured.
While droplets have been useful to confine proteins in many studies,98-102 they
were found to be ineffective for fluorescence measurements of far-red fluorescentproteins. Once a droplet sample is prepared, there is no guarantee how many droplets
within the sample will meet the size and spacing specifications discussed above. The
droplets produced have a range of sizes, with a low percentage of droplets below 30 µm
in diameter. Since the FWHM of the beam at the sample is ~ 30 µm in our setup, thismeans very few droplets will fit within the beam. An additional concern is the random
distribution of droplets within a measurement area. Generating enough data to compare
across multiple types of fluorescent proteins and irradiances requires multiple droplet
samples to be prepared over the course of an experiment. In addition, the process ofscanning the sample for droplets of appropriate size and spacing is very time-consuming.
Since smURFP has been reported to dimerize and bind 1-2 biliverdin chromophores andmiRFP670 possesses two binding sites, it was necessary to devise a more rigorous
technique to control for artifacts and ensure variability in signal is linked to the fluorescentproteins themselves.37,46 This new technique should also have samples arranged
efficiently to minimize the amount of time lost searching the sample for goodmeasurement spots.
After considering alternative options for sample preparation such as functionalizing
the surface of the coverslip or containing samples within liposomes, microwells were
determined as the best means of addressing the challenges encountered with droplets
while not altering protein behavior. Microwells are an array of cylindrical cavities with

33

diameters on the order of microns molded out of polydimethylsiloxane (PDMS). A
microwell device is depicted in Figure 2.2 below. Utilizing microwells instead of droplets
would ideally reduce uncertainty associated with measurements by increasing control
over several variables including measurement volume and spacing between
measurements.

Figure 2.2 Depiction of a single microwell device. Scale bar and outer box makes it easier to locate
microwells under 20x magnification. Each quadrant contains 100 microwells. The tick marks are spaced at
1 mm intervals and the entire box is 13 mm x 13 mm.

2.3 Optimization of Microwell Device Design
The utility of microwell devices has been demonstrated for quantitative study ofreaction products of a single enzyme, single cell analysis, and use in sandwich

34

immunoassays.103-105 The wells on these devices each has identical dimensions on the
micrometer scale and has a volume on picoliter scale. Fabrication of these devices allows
precisely controlled measurement volumes and spacing.
Our research group possessed a microwell master mold that was used by past lab
members and could be used as a starting point for microwell tests. This original design
was an array of 1000 wells, each 8 µm in diameter and spaced 10 µm apart with ~10 µm
well depth. The close spacing of wells on this design permitted multiple microwells to
appear in one field of view on our microscope, unlike the single microwell visible in Figure
2.1 (b). Each device was filled with 2 µL of protein by gently pressing PDMS onto a
coverslip with protein on it.106 These microwell master molds were used to generate
PDMS microwell devices for testing. Initial fluorescence measurements determined thesemicrowells were too close together for my measurements, and fluorescence from nearby
microwells would significantly contribute to signal. I addressed this problem by designingmicrowell templates with wider spacing and with wells in a variety of diameters to use forthe next phase of testing.
In the first stage of the microwell redesign I placed the wells 50 µm apart. Microwelldevices with diameters of 10, 15, 20, 25, and 30 µm were prepared to ascertain the best
diameter to use for fluorescence measurements. Each master had a 100 x 100 array of
microwells, ensuring each device would contain enough wells for repetitive measurement.
After several test measurements using fluorescent proteins to fill the wells, it wasdetermined the wells should be spaced even further apart to minimize the irradianceexposed to nearby wells. A calculation of beam irradiance 50 µm from the center of thebeam determined that nearby wells would be irradiated with 20 W/cm2 when the irradiance35

at the center of the beam is 814 W/cm 2, the lowest power setting used. For the highest
power setting used, the irradiance measured 50 µm away is 540 W/cm 2. To minimize this
irradiance, spacing was increased to 450 µm since this distance is greater than 10-foldhigher than the beam diameter, reducing the irradiance felt by nearby wells by 54%
compared to the previous design. The sparsely arranged wells were generated in a 22 x
22 grid since there was no longer enough space on the template for 1000 wells per device.
Testing of microwells ranging from 10-35 µm in diameter with 450 µm spacing
between them revealed that a diameter of 15 µm was the minimum needed to generate
a fluorescence signal 10-fold above the blank with 30 µM smURFP solution. For the 30µm beam used for these experiments, the maximum microwell size that should be used
is 15 µm. This ensures that the well fits within the full width at half maximum (FWHM) of
the laser profile. The technique for centering the microwell within the beam is shown in
Figure 2.3. Molecules within the FWHM of the beam are irradiated at nearly identical laser
intensity, and for the purposes of these experiments the irradiance within this area canbe treated as constant. By using the largest well diameter that receives a uniform
irradiance, the signal to background ratio is maximized. Based on this analysis, all future
microwell designs were only made for 15 µm. For test measurements, irradiances of 1.4
kW/cm2 were used. Later modification to the setup by replacing the PMT with one withhigher sensitivity in the far-red fluorescent protein emission range enabled the sameprocedure to be done with lower fluorescent protein concentrations (~10 µM protein
concentrations).

36

Figure 2.3 Picture of a single microwell within a laser beam. The full width at half maximum (FWHM) of
the beam is depicted as a white circle with a 30 µm diameter, corresponding to the experimental settings.
The microwell is 15 µm in diameter. Scale bar is 100 µm.

After these microwell masters were used for experiments and evaluated repeatedly
over the course of a year, it was found many of the microwell masters were worn down
and were no longer usable. While this was a setback for the project, it was taken as anopportunity to redesign the microwells for further improvement of the design. This was
the final iteration of the microwell device design, and a depiction of the ultimate device
template is shown in Figure 2.4.

37

Figure 2.4 Microwell master diagram. The blue represents the silicon wafer, and the white represents the
cross-linked photoresist deposited on the wafer. Each wafer has a diameter of 76.2 mm.

This final microwell device design featured PDMS wells with a 15 μm diameter and24 μm depth, each spaced 450 μm apart and arranged in a 400-well array. The array wassurrounded by a box and divided into four quadrants, with each quadrant containing 100
wells. The addition of the box makes it possible to see the edges of each device with the
naked eye, improving the process of cutting microwells out of PDMS. The center of thearray has a set of scale bars marking each millimeter, making microwells easier to locate
under a microscope, accelerating data collection. Each device on the master was labeledwith a number 1-6 to keep track of which devices were fabricated successfully and have
not yet been worn down. The addition of the numbers assists in preparing all microwells

38in the same orientation since the orientation of the numbers is visible with the naked eye,unlike other features on the microwell master. This final design is depicted in Figure 2.2.

2.4 Troubleshooting Microwell Device Fabrication
Microfluidic device protocols produced by our lab were the starting point for
fabrication of microwell devices. A schematic depicting this process is given in Figure 2.5.
The general process begins with production of a microwell master from a template withthe desired design, PDMS is then poured over the design to generate devices based on
the master mold, and finally the devices can be cut out of the PDMS and used formeasurements. This section discusses necessary modifications to the fabrication
protocol, and a detailed protocol for the entire process is included in Appendix B.

Figure 2.5. Fabrication process schematic. Process starts with 75mm silicon wafer and photolithographymask designed in AutoCAD. Output of this process is a microwell master that is used as mold to makemicrofluidic devices, with 6 PDMS microwell devices produced from the master each time.

39

Although a protocol for this process was successfully used for fabrication of otherdesigns, the same protocol had to be extensively modified for fabrication of microwelldevices. Fabrication of the master for the final design of 15 µm diameter wells with wide
spacing between wells required adjustment of UV exposure doses, thoughtfulmodification of heating steps, and careful application of development steps to
successfully produce microwell masters. While in theory the production of masters only
takes a few days, to successfully test a range of settings and modify the procedure fordifferent rounds of design took several months. The result of this process is an updated
protocol that can be applied for improved adhesion for rapid fabrication of new designs
with minimal adjustment. The entire fabrication process is summarized in a flow chart in
Figure 2.6.

40

Figure 2.6. Flow diagram of microwell device fabrication process. Process starts with clean, dry siliconwafers and produces a microwell master mold that can be reused, ending with the production of six PDMS
microwell devices.

The first step in the process to make the microwell master is to design a templatein computer-aided design (CAD) software such as AutoCAD. This template was madeaccording to the specifications in the section 2.3 for each round of microwell template
redesign. The AutoCAD design is then printed on a photolithography mask for later use
in the UV exposure steps.
Silicon wafers with 76.2 mm diameter are used as a substrate for the microwellmasters. The wafers are thoroughly cleaned by rinsing for 5 minutes in acetone, 2 minutes
in methanol, and are then rinsed with isopropanol (>90%), dried with N2 gas, and solventsare evaporated away by placing the wafer on a hot plate at 70 °C for 10 minutes. Starting

41

with clean wafers is essential for proper adhesion between the photoresist and the wafer
in later steps. The wafers are spin-coated with SU-8, a negative photoresist and then soft
baked on a hot plate to evaporate excess solvent. The photolithography mask is used to
expose UV light only to the area designated by the template, producing the acid catalyst.
The acid catalyst promotes the polymerization reaction when the mask is removed andthe wafer is heated during the post-exposure bake, with the design appearing on thewafer after a few minutes of heating. Uncrosslinked photoresist is then washed away with
SU-8 developer, Propylene glycol methyl ether acetate (PGMEA) and the wafer is hard
baked overnight at 150 °C to smooth out any cracks in the photoresist. The wafers arethen silanized with (tridecafluoro-1,1,2,2-tetrahydrooctyl)-1-trichlorosilane to passivate
the surface so that PDMS can be peeled off the master easily.
This wafer with the design is then referred to as the microwell master and is themold from which PDMS devices can be made. PDMS is prepared by mixing the prepolymer and curing agent in a 10:1 ratio. PDMS is poured over the microwell master andis cured overnight in an oven at 70 °C. The microfluidic devices are cut from the PDMS
and the master can be used many times to produce more devices.
While the microwell masters containing closely packed microwells werestraightforward to fabricate, the new microwell masters were considerably more difficult
due to their small size, shallow depth, and sparse arrangement. Several rounds offabrication were needed to optimize each step of the fabrication procedure. For the first
several rounds of fabrication SU-8 2025 was used as the photoresist. Of the photoresists
our group possessed, this one was desirable because it could be used to spin coatphotoresist in thicknesses between 20-80 μm. This coating thickness corresponds to the42depth of the microwells once the photoresist is crosslinked and developed. The spin
speed was set to 4000 rpm to produce well depths of approximately 20 μm. Once theinitial supply of SU-8 2025 ran out I replaced it with SU-8 3025 for improved photoresist
adhesion. The same spin speed settings were used for the new reagent to produce similar
microwell depths based on the product datasheets. 107,108
For the device design with 450 μm microwell spacing (prior to the introduction ofboxes and scale bars), the amount of energy needed for UV exposure was considerably
lower than the guidelines on the SU-8 datasheet. To achieve the recommended 150-160
mJ/cm2 recommended for photoresist with 25-40 micron thickness, 15-17 s of UV light
exposure would be needed based on the 9.5 mW/cm 2 measured power of the UV contactaligner.107 After trying a range of settings, starting with 17 seconds of exposure andgradually lowering UV exposure settings it was determined that 8-9 s of UV exposure
produced 15 μm diameter microwells that successfully formed and remained on thesubstrate following development. Due to the small area of the features, other diametersof microwells required adjustment of the exposure settings for successful fabrication. The
introduction of the boxes and scale bars around the design greatly increased the area of
exposed photoresist and required adjustment of the exposure settings. For the final
microwell device design, UV exposure was increased to 20-22 seconds for each master.
These exposure doses were determined by evaluating microwell formation atseveral stages of master fabrication. Following the post-exposure bake, microwellmasters were removed from the hot plate and viewed under a microscope. If thephotoresist shows cracks on the outside of the design area, that indicates that the crosslinking reactions have spread beyond the area of the design. When this happens, the43wafer has been overexposed and the exposure time should be reduced in future trials.
Similarly, if the photoresist shows cracks on the inside of the design template, thatindicates the plate has been underexposed and the exposure time should be
increased.109 If there is poor contact between the photolithography mask and thephotoresist during UV exposure, a similar effect to overexposure will be observed as thereis less control over the exposed area. Contact between the mask and the photoresist is
improved by adding a few drops of glycerol between them. The glycerol will be rinsed off
later during the development step.
Another issue with master fabrication that made production difficult was poor
adhesion between the photoresist and the wafer. Poor adhesion results in microwells thatwash away during the development step. The most important parameters in the
fabrication process that affect adhesion are exposure time, followed by post-exposurebake settings, and finally with development technique playing the third most importantrole.110 A UV exposure time that is too short results in weak adhesion between crosslinked photoresist and the wafer substrate, increasing the likelihood that wells will wash
away. Exposure doses were evaluated based on the above discussion. If the microwells
appear overexposed following the post-exposure bake but still wash away, the other key
fabrication steps in the process must be evaluated and optimized to improve adhesion.
Since the post-exposure bake is the second most important step for strong
adhesion, it was improved next by starting all heating steps at room temperature andheating at a rate of 6 °C/min. Once the hot plate is warmed to within 10 °C of the desired
temperature, the timer for the heating step is started, and the plate is cooled at the samerate. Once the hot plate is 20 °C below the temperature setting the wafer is removed from44

the heat. This modification results in a gentler heating process. By modifying all heating
steps and not just the post-exposure bake, crosslinking will begin at lower temperaturesand each step will be less abrasive.111
The final fabrication parameter to be optimized was the development technique.
Submerging the wafer in developer induces stress to the photoresist, so care must be
taken to reduce stress during this step.111 To achieve gentler development, the wafer iscompletely cooled prior to submerging in developer, and for the first half of thedevelopment time the solution is left unperturbed. For the second half of the development
time, the solution is gently agitated around the wafer. This produced better resultscompared with the more vigorous rinse that was used before, resulting in a greater
percentage of microwells stuck to the wafer. Whereas before instituting thesemodifications all the microwells washed away in the development step, greater than 90%
of microwells remained adhered to the substrate following development once thesechanges were made.
The lab protocol for silanization with (tridecafluoro-1,1,2,2,-tetrahydrooctyl)-1trichlorosilane called for a few drops of the solution to be placed in a vacuum chamberwith the microwell master for 3 hours. This method resulted in PDMS adhered to themicrowell master that could not be removed without also destroying the photoresist on
the master surface. The protocol was adjusted for the silanization to occur overnight,
guaranteeing that the wafer surface has been sufficiently passivated before removing itfrom the chamber. This alteration yields microwell masters from which PDMS can easily
be peeled off.

45

Protocols for PDMS device cleaning and storage also needed to be adjusted. Oncefabricated, the microwell devices must be kept free of dust so they are ready to be filledwith solution. After cutting out devices in a cleanroom for low dust levels, each side of the
PDMS device would be covered in tape. The tape was left over the wells to prevent dust
from entering them prior to use. After a few trials it was found that the tape residue blocked
the wells. To prevent this issue, tape is removed immediately after cutting out microwells
and is only used to remove PDMS debris immediately after cutting out the devices. N 2
gas is used to blow away dust particles, and the cut devices are rinsed with isopropanolfor 10 s. The devices are dried with N2, and any remaining solvents are evaporated away
by placing the device on a hot plate set to 70°C for 10 minutes.
For device storage, one technique initially used was plasma cleaning the devices
and storing them pressed against a clean coverslip to keep dust out. This technique wasdiscontinued after a few batches of microwell devices became stuck to the glass and were
unusable. Instead, the clean devices are stored in a clean petri dish that is taped closed
and wrapped in aluminum foil. The container is stored in a dust free hood and is only
opened in a clean environment. Microwell devices are only removed immediate before
use to be filled
These adjustments made to the entire fabrication process from microwell masterfabrication through storage of the PDMS devices were necessary to generate a detailed
microwell device generation protocol described in Appendix B. The result was applied for
rapid and successful re-fabrication of the microwell master when it became worn out after
repetitive use. PDMS microwell devices fabricated according to this procedure can be
stored for several weeks and filled with solution without issue.

46

2.5 Microwell Filling Investigation
After optimizing the microwells design and fabrication, the next step is to evaluate
their use in fluorescence experiments using the proteins of interest. After several roundsof measurement of smURFP, I noticed considerable variation in maximum fluorescencesignal. Further examination revealed 5-fold variation between the lowest and highest
signal collected from an individual microwell. Evaluation of the height of individual
microwells, the filling procedure, and whether microwells remain filled over time wasneeded to determine the source of this variation. For accurate data interpretation it isessential that variations in signal are due to smURFP fluorescence and not artifacts
introduced by the data collection method.
The first concern after observing variation in signal levels was whether eachmicrowell contained the same volume of solution. To address this, the microwell depth
distribution for each microwell device on the master was determined using an opticalprofilometer. The optical profilometer measures the height difference between thephotoresist and the wafer in several locations to confirm the photoresist thickness. The
instrument used for these measurements is a white light vertical scanning interferometer.
Using this instrument to evaluate the microwell devices is ideal because it is a non-contact
method so repeated measurement will not damage the microwell masters. The instrument
works by exploiting the low coherence of white light, vertically scanning to vary the optical
path difference, generating an interference function that is used to determine the height
difference between two locations. The height difference was measured in 30 places foreach device to produce a histogram displaying the variation in well depth for each device.
This histogram can then be compared to another histogram that reports initial

47

fluorescence signal for fluorescence measurements made using the same device (Figure2.7). The histogram of initial fluorescence signal showed considerably more variationcompared to the microwell depth distribution, indicating that the initial signal variation
could not be caused by variation in well depth, and must have another cause.

(a)

(b)

Figure 2.7 Comparison of microwell depth distribution to fluorescence signal. (a) Distribution of microwelldepth for a single device measured with an optical profilometer. 50x magnification was used for opticalprofilometer measurements. Microwells with depths above 25 µm were excluded from measurements. (b)Distribution of smURFP fluorescence initial signal from microwells filled with 30 µM smURFP. Unfilled
microwells were excluded (signal with less than a 2-fold increase compared to the blank).

Once well depth variation was ruled out as a cause for variation in initial
fluorescence signal, the next parameter to be evaluated was microwell filling levels acrossthe device. This assessment was needed to confirm that microwells contain the same
volume of solution across the device. A series of experiments were performed todetermine the maximum volume of purified protein that could be used and to determine
the proper technique for filling them. Ideally the microwells would be filled with enoughsolution for a 10-fold signal above the blank and no signal between wells. This ensuresthat all the solution has been pressed into the microwells and expelled from the space
between wells.

48

Pressing the device on top of the coverslip with solution was not sufficient to force
solution into microwells and out of the space between wells, since this technique resulted
in high signal between wells. The procedure was adjusted by placing a magnet on top of
the device after pressing it on top of a coverslip with a drop of fluorescent protein solution,
wicking away any solution that leaks out of the edges of the device with a piece of filter
paper. This technique resulted in signal between microwells comparable to the signal
produced from blank media between two coverslips. The filling volume was optimized
using this procedure. To determine the optimal volume of fluorescent protein solution to
add to the coverslip, a range of volumes from 1 µL to 20 µL were used for testing. 15 µL
was selected as the optimal volume since it is the highest volume that can be used without
increasing signal between microwells, and so maximizes microwell filling and therefore
fluorescence signal that can be produced from a given protein concentration. Higher
volumes of fluorescent protein solution will result in higher signal between wells, indicating
not all solution is removed from between the microwells. Fluorescence signal 10-foldhigher than the blank was successfully produced with 15 µL of 10 µM smURFP solution.
After the microwell filling procedure was optimized, the next step to consider is how
long the microwells can be used for experiments after they are initially filled. An overnighttime lapse experiment was performed to determine how long and to what extent
microwells stay filled once protein is added. In this experiment, a microwell device wasfilled and imaged under brightfield illumination at 40x magnification in three locations
every 10 minutes to determine microwell filling levels for over 8 hours after being filledwith fluorescent protein. The absorbance was averaged across these three microwells
and the results were normalized to yield a plot of absorbance over time (Figure 2.8). One

49hour after the initial measurement the absorbance was 89 % of the initial value. After twohours, the absorbance was 77 % of the initial value. After 8 hours, the absorbance was
66 % of the initial value. From this result it was determined that microwell devices should
be used as soon as possible after filling and microwell experiments should be concluded
within 1-2 hours of filling. For longer experiments, the microwell devices could be sealed
to the coverslip by applying a layer of clear nail polish between the PDMS and thecoverslip.

Figure 2.8 Results of microwell time lapse experiment. This data is the result of imaging three smURFPfilled microwells filled under brightfield illumination. The wells were imaged every 10 minutes over 8 hours
and the results were averaged. Results show a 66% reduction in signal intensity at the end of themeasurement.

50

2.6 Pre-illumination Study
After assessing microwell device well-depth variation, filling procedure, and fillinglevels over time, there was still considerable variation in initial fluorescence signal. Preillumination of microwells prior to imaging was the final source of variation investigated.
Light scattered through the PDMS reaching the nearest neighboring microwell would
cause a portion of molecules within samples to be bleached or converted to the darkstate. The extent of the pre-illumination phenomenon was evaluated by preparing
samples of bulk protein solution and imaging repetitively. Bulk solution was used rather
than microwells for these measurements to quickly assess this occurrence without
requiring additional microwell device fabrication. 10 measurements were made on eachsample, moving the microscope stage in the x-direction 500 µm after each measurement
to mimic microwell spacing. Three samples were prepared at each irradiance and thefluorescence decay profile for the three measurements were averaged to produce plotsof average signal reduction following subsequent measurement. These measurements
were compiled to show how initial fluorescence decreases as number of measurements
increases. (Figure 2.9)

51

(a)

(b)

Figure 2.9 Results of pre-illumination study (a) 100 W/cm2 irradiance. (b) 20 kW/cm2 irradiance. Arrows
show the direction of increasing number of measurements within one sample. The arrows show that for
both irradiances, fluorescence signal decreases with increasing number of measurements.

The compiled fluorescence plots measured at irradiances of 100 W/cm 2 and 20
kW/cm2 show a reduction in signal over the 10 measurements. At lower irradiances, thereduction in signal was less pronounced. To quantify the pre-illumination effects, I
calculated the irradiance 450 µm away from the center of the beam, revealing that at an
irradiance of 20 kW/cm2 within the FWHM, the nearest neighboring well experiences anirradiance of 200 W/cm2. This irradiance experienced by the nearest neighboring
microwell is nonnegligible and must be taken into consideration when analyzing results.
The pre-illumination effect may have been artificially enhanced in these measurements
compared to in actual microwells by using bulk solution to study this effect instead of
microwells. Some of the observed pre-illumination can be attributed to diffusion of
bleached molecules into the measurement area. The large drop off in signal between thefirst and second measurement at high irradiances suggests that future designs should

52

space microwells even further apart to minimize pre-illumination and low irradiances
should be used to minimize the effect.

2.7 Conclusion and Future Outlook
The creation of microwells posed several challenges, both in design andfabrication. Several iterations of design were needed to generate microwells that wereappropriate for the collection settings. Initial rounds of master fabrication resulted in
microwell devices where all or most of the wells washed away or the wells were severely
warped from overexposure of the photoresist. Fabrication of microwell masters was
improved by using a slow ramp up of temperature for all heating steps, using gentle
agitation during the development step, and by adjusting the UV exposure time based on
the photoresist thickness and design template. After several rounds of modification to thefabrication procedure, microwells were successfully fabricated with most wells remaining
adhered to the wafer.
Once the microwell devices were successfully fabricated, they had to be evaluatedfor use in fluorescence experiments. Assessing the microwell depth with an opticalprofilometer showed minimal variation across devices. Microwell filling was optimized forfilled microwells with signal between wells comparable to the blank and high signal withinwells. It was determined that microwells can reasonably be imaged for up to 2 hours after
initial filling and could be imaged even longer by sealing the PDMS device to the coverslip.
The pre-illumination study determined the source of variation in initial fluorescence signal
intensity, and yielded recommendations for future improvement to the microwell device

53

design by increasing spacing between adjacent wells. The results of the pre-illumination
study also suggest that use of lower irradiances in future studies would minimize artifacts
due to pre-illumination.
Overall, microwells are a considerable improvement compared to droplets.
Repetitive measurements are faster since the box and scale bar on each device makes
it easier to locate microwells. The arrangement in a 400-well array provided a hugeincrease in number of measurements that could be made in one sample compared todroplets. Every microwell is the same size, eliminating the need to evaluate the size of adroplet before each measurement. Microwells are evenly spaced and far apart, reducing
fluorescence contribution from nearby wells and keeping the minimal fluorescence
contribution from nearby microwells constant throughout the experiments. The spread out
spacing of microwells on the device also reduces pre-illumination effects compared todroplets. For these reasons, fluorescence measurements in later chapters use microwells
alone for fluorescence measurements.

54

Chapter 3: Data Collection and Analysis Methods

3.1 Introduction
This section details the experimental methods used to measure the fluorescencedecay profiles of fluorescent proteins as well as the analysis methods used to extract therate constants for dark state conversion, ground state recovery, and photobleaching fromthe data. The first three sections discuss the experimental methods, with how samples
were prepared for optical measurements described in Section 3.2, followed by theexperimental setup in Section 3.3, and lastly a description of the procedure for collecting
measurements in Section 3.4. The final three sections describe the processes used fordata analysis, with Section 3.5 describing in detail the calculation of irradiance, followed
by a thorough discussion of data analysis of continuous illumination data in Section 3.6.

3.2 Sample Preparation
This section describes how protein samples are prepared for fluorescencemeasurements. Samples for measurements done in microwells or droplets are preparedusing purified fluorescent protein in Tris buffer (150 mM NaCl, 50mM Tris-HCl, pH 7.4) tofill them with protein concentrations in the range of 5-10 µM. Protein is thawed
immediately before use and samples are stored at 4 °C for no more than one week. Small55

aliquots are used so that samples are used within 24 hours after thawing. Protein
concentration is determined by measuring the absorbance of the protein solution using a
cuvette with a UV-Vis spectrometer or using a NanoDrop to measure the absorbance ofsmall volumes of solution (1-2 µL). The stability of smURFP and miRFP670 was assessedby comparing the UV-Vis absorption spectrum of a sample that was frozen and later
thawed to that of a sample of freshly purified sample. The spectra of both proteins was
identical.
Microwells were filled by adding 15 µL of purified protein to a 22 x 22 mm coverslip
and pressing a clean microwell device on top. A magnet is then placed on the device topress the solution into the microwells and press the solution out of the space between
microwells. Filter paper is used to wick solution away from the edges of the device. The
device with the coverslip is flipped over to ensure all excess solution is removed. Prior touse, dust is removed from the coverslips and PDMS devices with an air gun.
For test measurements, droplets are prepared by mixing a 1:9 ratio of protein
solution with 1-octanol. The solution is vortexed for 30 s and is pipetted 5 times to mix
immediately before use. Mixing immediately before use increases the likelihood of
producing a good number of smaller droplets (~15 µm) that are ideal for measurementswith the beam size in the setup. 5 µL of droplet solution is added to a corner of a clean
22 x 22 mm coverslip before placing another coverslip on top. Uniformity of droplets canbe improved by sonicating the droplet solution or by applying a hydrophobic coating to
the glass slides.112
For experiments done in live cells, the sample was prepared by expressing thefluorescent protein in E. coli cell on an agar plate (See protocols in Appendix B). Since E.

56coli cells produce heme but do not produce biliverdin, the proteins were co-expressedwith heme oxygenase (HO-1) to generate biliverdin, as is necessary to produce
fluorescence. After at least 24 hours of growth at 37 °C, the plate was stored at 16 °C forat least 24 hours prior to measurement. Immediately prior to measurement, a single
colony is selected and removed from the agar plate using an inoculum loop. The colony
is dissolved in 250 µL of blank media. The colony is vortexed for 30 s and pipetted 10
times to mix. 20 µL of solution is placed on a clean 22 x 22 coverslip and a second
coverslip is placed on top. This technique resulted in bright fluorescence for E colimeasurements with cells suitable to easily be selected with a 40x objective (Olympus).
An agar plate was used to prepare E. coli cells instead of liquid culture because liquid
culture resulted in much smaller E. coli cells that did not produce fluorescence signal atleast 5-times signal produced from the blank, the standard used to select measurements.

3.3 Experimental Setup
This section describes the features of the optical setup and why the selected optics
and settings were chosen. For fluorescence measurements of near-infrared fluorescentproteins, a 637 nm diode laser (Coherent, OBIS 637LX) is used as the excitation source.
A 650 nm dichroic mirror (Brightline, FF650-Di01) is used to reflect the excitation light intoa custom-built inverted microscope. Laser light is focused onto the sample by a 20x 0.75
NA air-immersion objective (Olympus). A 50:50 beam splitter is used to direct 50% of thescattered light from the sample to be focused onto a CMOS camera (ThorLabs,
DCC1545M) for sample visualization, with the other 50% of the scattered light focused

57

onto the Photo-Multiplier Tube (PMT, Hammamatsu Photonics, R9880U-20). The
combination of the dichroic mirror mentioned above and a pair of identical 731 nm
emission filters (Brightline, FF01-731/137) prevent stray excitation light from passing to
the PMT. Photocurrent from the PMT was converted to voltage using a custom-built ACcoupled linear transimpedance operational amplifier to improve the signal-to-noise ratio
by removing high and low frequency noise components. Noise components outside thebandpass of 160 mHz to 106 kHz bandpass were removed. The resulting voltage levels
were digitized at a sampling frequency of 100 kHz with a PC-based data acquisition board(DAQ, National Instruments) and LabVIEW software (http://ni.com/labview, National
Instruments, 2012 version).
Data collection was operated with custom-built LabVIEW software which controlled
power setting, sampling frequency, and modulation of the 637 nm laser. The laser isoperated in digital modulation mode and interfaces with the computer using the DAQ
board. The irradiance of the laser is tuned by changing the laser power setting of the 637
nm laser and by attenuating the beam using the half-waveplate and polarizer in the setup.
Figure 3.1 shows the diagram of the experimental setup as well as labeled photos of theexperimental setup.

58

PMT voltage is digitized at a 100 kHz
sampling frequency with a DAQ
board

Figure 3.1 Optical setup. (a) Optical diagram of fluorescence microscope. Each M is a silver coated mirror.
L1 and L2 are lenses λ/2 is a half-waveplate. P is a calcite polarizer. L3 is a lens focusing light into themicroscope. DM is the 650 nm dichroic mirror and BS is a 50:50 beamsplitter. L4 is a lens focusing scattered
light onto the PMT. F1 and F2 are the emission filters. (b) Image of beam path. (c) Image of microscope
stage

59

Due to the smaller size of E. coli cells (~2 µm long) compared to droplets andmicrowells, the setup was modified for measurements with E. coli cells by replacing the
20x objective with a 40x 0.60 NA air-immersion objective (Olympus). For experiments
done with red fluorescent proteins, the experiment was modified due to the different
excitation and emission properties of these fluorescent proteins compared to nearinfrared fluorescent proteins. The excitation source is replaced with a 561 nm diode laser(Coherent Genesis MX), The filters (F1 and F2) are replaced with a set of red emission
nm filters and the 650 nm dichroic mirror is replaced with a 573 nm dichroic mirror(Brightline, FF573-Di01). The 561 nm laser pulse profile was obtained with an acoustooptic modulator (AOM, Gooch & Housego, 35210-BR). The RF input to the AOM driver
was delivered by a function generator (Agilent 332050A) controlled by custom MATLAB(http://www.mathworks.com, The Mathworks, Inc., version 2019a) software. For mCherry,an acquisition rate of 1 MHz is used. All other aspects of the experimental setup remained
the same when switching between fluorescent protein type.

3.4 Optical Measurements of Dark State Photophysics
The same procedure for setting up and running fluorescence measurements wasfollowed to ensure measurement reproducibility, as described in this section. The firststep of performing these measurements is checking the alignment with the laser power
set to 1 mW. The microwells are co-focused with the beam to ensure that the best
microwell focus is also the best focus of the beam. A clean coverslip is placed on the60sample stage to aid in the alignment check. Mirrors are adjusted so that the reflections
from both sides of the coverslip are directly overlapped.
Once the alignment check is complete, the sample is prepared as described in
Section 3.2 and is placed on the sample stage as shown in Figure 3.1c. In the case of the
microwells, the sample is oriented with the coverslip facing down and toward theobjective. The cell, droplet, or microwell to be measured is centered within the beam area
drawn on the computer screen using the CMOS camera and white light illumination.
Samples are centered within the FWHM to ensure uniform irradiance across themeasurement since the irradiance within this area can be assumed constant. Eachmeasurement location is selected within a sample by moving the microscope stage in thex- and y-direction until the cell, droplet, or microwell is centered within the FWHM aslabeled on the computer screen. For the near-infrared fluorescent protein measurements,
data is acquired using LabVIEW program smURFP_v06.vi to control data collection
settings. The sample collection rate is set to 100 kHz. The PMT voltage is set to 500V,
within the linear range of the PMT. Fluorescent protein sample concentrations wereselected to avoid saturation of the PMT. When necessary for high irradiancemeasurements, a neutral density filter was added immediately before the PMT to prevent
saturation.
To gain insight into the light-dependence of the dark state conversion and groundstate recovery processes, experiments were performed under both continuous andpulsed illumination conditions. For the continuous illumination measurements, the laser
is set to turn on for 90 s and turn off for 5 s. These settings were selected to ensure thatthe fluorescence for each trial was collected over one pulse. The 90 seconds setting was

61

the longest possible for the 100 kHz frequency and the LabVIEW memory settings, any
longer and the memory is overloaded. Collecting measurements at longer times would
require a reduction in sampling frequency.

3.5 Calculation of Irradiance
At the beginning of each set of fluorescence measurements, the irradiance must
be characterized to accurately determine how much light is illuminating the sample. Thissection will discuss the steps taken to carefully measure and calculate the irradiances foreach measurement. The beam spot size is measured at the focus by setting the power to
100 mW. The image of the beam is collected on the camera with the exposure set to thelowest setting. Previous characterization of the laser beam determined that the beam area
is constant above a power setting of 20 mW and the beam area decreases by 22% belowthat power. If the camera is saturated at 100 mW, a power of 50 mW is used for thatwaveplate and polarizer setting. This produces a beam size for the laser at the focus
which fluorescence is generated, since for each sample type (cells, droplets, microwells),
the sample is imaged through a coverslip. Within the camera program a circle is placedaround the center of the beam. Samples are then centered within this area to ensure
uniform irradiance across the measurement.
To calculate the laser spot size, ImageJ (http://imagej.net/, ImageJ, version 1.52p)
is used to extract the beam line profile in the horizontal and vertical directions. The
extracted line profiles are then loaded into OriginPro (https://origin.com, OriginLab,version 2019b) and fitted to a Gaussian to calculate the FWHM of the beam. This FWHM

62

is measured in pixels and is converted to centimeters by using a unit conversion
determined from measuring a calibration standard within the setup. The calibration
standard is a set of 5 µm squares with a 5 µm pitch and is used to calculate the numberof µm per pixel. The process of measuring the profile and fitting the line profile wasrepeated three times to determine error associated with the process. The variability of the
FWHM using this technique was determined to be less than 0.01%.
Using the FWHM from both the vertical and horizontal line profiles, the area of the
spot size is calculated by using the equation for the area of an ellipse (Equation 3.1).

𝐴𝑟𝑒𝑎 = 𝜋 ∙

𝐻 𝑉
∙
2 2

Equation 3.1 Area of an ellipse. H is the horizontal FWHM (cm) and V is the vertical FWHM (cm).

The error on the calculation for area is given in Equation 3.2.

𝜕(𝐴𝑟𝑒𝑎) 2
𝜕(𝐴𝑟𝑒𝑎) 22
𝐸𝑟𝑟𝑜𝑟 = √() ∙ 𝑠𝐻 + () ∙ 𝑠𝑉 2
𝜕𝐻
𝜕𝑉
Equation 3.2 Equation for error on the spot size calculation. s H is the standard deviation of H and sv is thestandard deviation of V. All variables have units of centimeters.

Another aspect that must be considered to find the beam irradiance is the laser
power at the sample. The fraction of laser power that reaches the sample is measured
with a power meter immediately above the objective. For powers below 30 mW at the
sample, a silicon sensor (Coherent, OP-2 VIS) is used to measure the power. For powers
above 30 mW, a position-sensing air-cooled thermopile sensor is used (Coherent, LM10).

63

It was concluded from measurements on the 637 nm laser at a range of power settings
that the fraction of light that reaches the sample is constant at multiple laser powers, so
this measurement can be used for all power settings. The power is carefully measured toensure the detector is accurately placed and the maximum power reading is recorded.
The measurement is repeated three times and the average value is used to calculate theirradiance. Power measurements were made at the beginning and end of experiments
and reported a 0.1% change in power from the beginning of measurements to the end.
Using the information on spot size and fraction of power that reaches the sample,
the irradiance can be calculated using Equation 3.3 for each power used for experiments.

𝐼=(𝑃𝑜𝑤𝑒𝑟⁡) ∙ (𝐹)𝐴𝑟𝑒𝑎

Equation 3.3. Equation for Irradiance, I. Power is the laser power setting in Watts. F is the fraction of thelaser power that reaches the sample.

The error of the irradiance calculated is found using Equation 3.4.

2
𝜕𝐼 2
𝜕𝐼
𝐸𝑟𝑟𝑜𝑟 = √( ) ∙ 𝑠𝐹 2 + () ∙ 𝑠𝐴𝑟𝑒𝑎 2𝜕𝐹𝜕𝐴𝑟𝑒𝑎
Equation 3.4. Error calculations for Irradiance. sF is the standard deviation of F and sArea is the standard
deviation of the spot size.

An example of a typical irradiance calculation from measurements made on
12/04/2018 found the fraction of laser light that reached the sample was 0.806 ± 0.002,
with a horizontal beam diameter of 21.217 ± 0.002 μm and a vertical beam diameter of64

23.750 ± 0.002 μm. This resulted in an area of (3.96 ± 0.01) x 10-6 cm2 and an irradianceof 2.037 ± 0.009 kW/cm2 when the power was set to 10 mW and 14.26 ± 0.06 kW/cm 2
when the power was set to 70 mW.

Section 3.6 Analysis of Continuous Illumination Data
Analysis of continuous illumination fluorescence data for fluorescent proteins tofind the rate constants for photophysical properties required careful development of adata analysis scheme. The data for near-infrared fluorescent proteins had more variability
compared to similar measurements of red fluorescent proteins so a rigorous procedure
was developed to ensure that no biases were introduced in steps taken to remove outliers.
All data received the same treatments in the processing steps. This section discusses theprocedure used for data processing, fitting, and reduction. An overview of the data
analysis scheme is depicted in Figure 3.2.

65

Figure 3.2 Flow chart summary of data analysis scheme. Scheme starts with data processing in
MATLAB, followed by fitting using a sum of exponentials, and reduction through outlier analysis.

Fluorescence data is analyzed in MATLAB using a series of scripts developed forthis purpose. The first phase of this analysis is data processing, which consists of loading
the data, smoothing, baseline correction, and normalization. After loading data into
MATLAB, the data for each trial is trimmed to cut off the portion of the data collected
before the laser is turned on and after the laser is turned off, leaving the laser rise toensure none of the fluorescence decay is erroneously cut while still isolating thefluorescence decay for fitting.
Once the fluorescence decay is isolated for fitting, the baseline is adjusted to zeroby subtracting the minimum value of the decay. The data is then smoothed using a moving
average filter and normalized before averaging across all the trials with the same
experimental settings (protein concentration, irradiance setting, and preparation method).
Data is carefully examined after each step of data processing to ensure no artifacts are

66

introduced by the processing. Preparation of average decays concludes the dataprocessing phase. Data analysis proceeds to the fitting phase.
Fitting begins by decreasing the total number of datapoints for faster fitting speeds
using MATLAB and to weight the fits towards the fastest part of the decay. The first 15
points of the fluorescence decays are retained, and the rest of the points are culled using
a logspace function to select logarithmically spaced points. This process weights the fits
toward the beginning of the measurements, where dark state conversion occurs.
Additional weighting is done by applying a weight to the first 10 data points and the last 5
points to ensure the fit function decays to the baseline. The average fluorescence decays
are initially fit with a two-exponential function. If this results in a poor fit, then a threeexponential fit is tried. This approach leads to the minimal model that fits the data being
selected, since an overly complex model with too many parameters will overfit the data
and an oversimplified model will exclude normal patterns within the data are outliers. The
fits are evaluated using the R2 coefficients and the residuals. Fit parameter quality isevaluated using confidence intervals. A three-exponential fit based on the 3-level model
including dark state conversion discussed in Chapter 1 is given in Equation 3.5.

𝐹 = 𝑎 ∙ 𝑒 −𝑘𝐷 ∙𝑡 + 𝑏 ∙ 𝑒 −𝑘𝐵1∙𝑡 + 𝑐 ∙ 𝑒 −𝑘𝐵2∙𝑡 + 𝑑
Equation 3.5 Three-exponential fit equation for fluorescence. k D is the rate constant associated with thedark state. kB1 and kB2 are the first and second photobleaching rate constants, respectively. a, b, and c are
the fractions of each exponential decay component.

The rate constants for the dark state and for photobleaching are extracted by first
fitting to Equation 3.5, or a two-exponential version based on the best fit. The fastest
component of the three-exponential fit is considered to be the dark state rate constant,

67

and the slower two components are photobleaching rate constants. This assignment ismade because dark state conversion occurs on a much faster timescale compared tophotobleaching. Blinking timescales have been measured for a variety of fluorescentproteins, and have been observed on timescales of hundreds of microseconds tomillesconds,58,65,74,75 while photobleaching has been measured on timescales from tensof milliseconds to seconds for several fluorescent proteins.58,65
In cases where none of the decay components are on the timescale of dark state
conversion and ground state recovery and the fastest decay is on photobleachingtimescales, the fastest decay would be designated kB1 instead of kD. Otherwise for a twoexponential fit the two rate constants would be designated kD and kB. Figure 3.3 shows asample two-exponential fit for miRFP670 fluorescence data.

Figure 3.3 Sample miRFP670 and smURFP fluorescence decays. (a) Sample 3-exponential fit forcontinuous illumination fluorescence data of smURFP in microwells collected with an irradiance of 870
W/cm2 (b) Sample 2-exponential fit for continuous illumination fluorescence data of miRFP670 in microwells
collected with an irradiance of 2 kW/cm2

68

In addition to this initial exponential fit to determine a rate constant associated withthe dark state, the data is further analyzed by simulating data using a numerical to thesystem of kinetic equations describing the populations of these states (Equations 3.6).

𝑑[𝑆0 ]= −𝑘𝑒𝑥 [𝑆0 ] + (𝑘𝑒𝑚 + 𝑘𝐼𝐶 )[𝑆1 ] + 𝑘𝐺𝑆𝑅 [𝐷]𝑑𝑡
𝑑[𝑆1 ]= 𝑘𝑒𝑥 [𝑆0 ] − (𝑘𝑒𝑚 + 𝑘𝐼𝐶 + 𝑘𝐷𝑆𝐶 )[𝑆1 ] + 𝑘𝐷𝑆1 [𝐷]𝑑𝑡
𝑑[𝐷]= 𝑘𝐷𝑆𝐶 [𝑆1 ] − (𝑘𝑆1𝐷 + 𝑘𝐺𝑆𝑅 )[𝐷]𝑑𝑡
Equations 3.6. Kinetic equations for 3-level system. S0, S1, and D are the ground electronic state, firstexcited state, and the dark state, respectively. kem, kIC, and kex represent rate constant for emission, internalconversion, and the excitation rate, respectively.

When modeling the results, several simplifications are made to find the simplest
possible model that fits the data. The simplest model starts with just the two-level model
where the dark state and rate constant leading to the dark state are omitted. If that model
does not describe the data, the three-level model described by Equations 3.6 is used. A
simplified version of this model starts by treating the rate of conversion from the dark state
to the excited state as negligible. This simplification can be made because the dark stateis lower energy compared to the excited state, so this rate constant will be small compared
to

other

processes.

Thenumerical

solutionwasobtainedin

Python(http://www.python.org, Python Software Foundation, version 3.0) and plotted to compare
the results to the experimental data. The kDSC rate constant is estimated based on
experimental results for other fluorescent proteins, and the value of k D from theexponential fit is used for kGSR, since ground state recovery is the rate limiting step of the

69

dark state processes. The values for kem, kex, and kIC are fixed within the fit equation arecalculated based on the lifetime, fluorescence quantum yield, and extinction coefficientfor the fluorescent protein. The equation used to calculate excitation rate is described in
Equation 3.7.

𝑘𝑒𝑥 =l n( 10) ∙ 1000 ∙ 𝜀 ∙ 𝜆 ∙ 𝐼ℎ ∙ 𝑐 ∙ 𝑁𝐴

Equation 3.7. Equation to calculate excitation rate, k ex. kex depends on extinction coefficient (ε), excitation
wavelength (λ), and irradiance (I). h is Planck’s constant, c is the speed of light, and N A is Avogadro’s
number.

Data reduction is the final phase of data analysis. Initial data reduction occurs
during data collection. Trials which showed fluorescence signal no higher than the blankwere eliminated since they can be assumed to contain no fluorescent protein. Microwells
or droplets which are non-fluorescent are assumed to be empty, and non-fluorescent E.
coli cells are assumed to have low protein expression levels. After the data has beenprocessed and fit, a careful evaluation of the fits is done to reduce the data set by
eliminating outliers. An outlier is a measurement that substantially diverges from the othermeasurements made with the same collection settings (irradiance, fluorescent protein
identity and concentration). An outlier could indicate one of two possibilities: the outlier
could be an extreme example of the random variability intrinsic in the data or it could
result from some deviation from the experimental procedure or an error in calculations. In
the first instance the outlying data is an interesting facet of the fluorescent protein
behavior and could indicate a separate mechanism, and in the second instance it is adata point that will skew results and should be excluded from analysis. Outlier analysis

70

involves balancing the need to keep valid observations while discarding flawed ones. If
the source of the deviation is not readily apparent, careful statistical analysis is needed
to determine whether the outlying data occurs from a deviation in the experimental
procedure or an error in calculation. The key to this analysis is minimizing the risk ofincorrectly rejecting a good observation during the data reduction process. 113,114
The first step of data set reduction was to select only fluorescent traces withfluorescence signal at least 5-fold higher than the blank. This process will remove
samples that have been excessively pre-illuminated from the analysis flow. Once this set
is narrowed, the fluorescence decay of each trial is fit in addition to previously fitting the
average decays and extracting the rate constants. The dark state and photobleachingrate constants from this set of measurements are used to calculate the average rate
constant and standard deviation for the distribution of fits. These rate constants are usedin statistical analysis for further data reduction. The z-value is calculated for each rate
constant to give it a score based on how much it deviates (Equation 3.8). 115

𝑧=𝑥𝑖 − ⁡ 𝑥̅𝜎Equation 3.8. Equation for z-value test. xi is the rate constant for the ith trial and 𝑥̅ ⁡is the average rate
constant for all trials at the same collection settings. σ is the standard deviation for rate constant for the setof collection settings.

A higher z-value indicates a higher likelihood that a data point should be rejected.
This method assumes the data follows a normal distribution. When there are higher
numbers of outliers within a data set, there will be a masking effect and the ability to use
the rejection test to identify outliers is reduced.114 Additional testing is used to evaluate
whether more than one outlier should be removed. Each data set is evaluated based on

71

its kurtosis, a measure of the widths of tails of a probability distribution and skewness, ameasure of the asymmetry of the probability distribution of the variable about the mean.
The rate constants extracted from the exponential fits are used in the tests for kurtosis(Equation 3.9) and skewness (Equation 3.10).

𝑛

𝑘 = 𝑛∑
𝑖=1(𝑥𝑖 − 𝑥̅ )(𝑛 − 1)2 𝜎 4Equation 3.9 Equation for test of kurtosis. xi is the rate constant for the ith trial and 𝑥̅ ⁡is the average rate
constant for all trials at the same collection settings. n is the total number of samples. σ is the standard
deviation.
𝑛(𝑥𝑖 − 𝑥̅ )3𝑠=∑(𝑛 − 1)𝜎 3
𝑖=1Equation 3.10 Equation for test of skewness. xi is the rate constant for the ith trial and 𝑥̅ ⁡is the average rate
constant for all trials at the same collection settings. n is the total number of samples. σ is the standard
deviation.

The kurtosis test is less sensitive to masking when there are multiple outliers andmeasures the thickness of tails for the distribution, and skewness measures theasymmetry of the distribution about the mean.116,117 If excess kurtosis is over 2 or if
skewness is greater than 1.5, then the trial with the highest z-value is removed from thedataset.
By removing outliers using these tests, outliers are removed from the dataset to
avoid obscuring the analysis. While there was no trend in dark state rate constants forpre-illuminated samples, photobleaching rate constants would be affected by thisphenomenon since the sample has been partially bleached. Following data reduction, thedata is returned to the data processing phase following the arrow shown in the Figure 3.4

72

flow chart. Outliers are removed and the narrowed data set is used to construct a newaverage fluorescence decay. This average fluorescence decay is then fit and moved
forward to the data analysis stages. The results from these measurements and theconclusions drawn for them are discussed in Chapter 4.

Figure 3.4 Overview of data analysis scheme. This scheme was used to analyze continuous illumination
fluorescence data. This cycle was carried out until no further data reduction was performed, as determinedby the kurtosis and skewness tests.

73

Chapter 4: Results of Dark State Analysis

4.1 Introduction
This section discusses the results of the study of dark state photophysics of nearinfrared fluorescent proteins. These measurements have important implications for using
these proteins for deep tissue imaging and potentially super-resolution microscopy
experiments by predicting blinking and photobleaching rates. In addition to comparing thedark state conversion behavior of these two proteins, measurements were also done at
multiple irradiances to compare the dark state dynamics at excitation rates ranging from
2.5 x 105 to 4 x 107 s-1.
Laser intensity used in these experiments is reported as an excitation rate rather
than irradiance, because the excitation rate is a more accurate way to compare behaviors
between fluorescent proteins with different extinction coefficients. At the same irradianceand excitation wavelength, a protein with a higher extinction coefficient will have a larger
population of molecules in the excited state, resulting in higher rates of reactions that
occur from the excited state, including dark state conversion and photobleaching. By
using the excitation rate to compare the rates of dark state conversion andphotobleaching, a more concrete comparison of the dark state behavior of different
fluorescent proteins can be made.

74

Sections 4.2-4.4 present the results of continuous illumination experiments offluorescent proteins smURFP, miRFP670, and mCherry. Sections 4.5 and 4.6 introduce
the results of mathematical simulations and compare them to experimental results.
Finally, sections 4.7-4.9 discuss the implications of this work and plans for further study.

4.2 smURFP Continuous Illumination StudysmURFP is a far-red fluorescent protein derived from allophycocyanin. It was
selected for study because it has the highest molecular brightness of any biliverdin-based
fluorescent protein. Continuous illumination measurements of smURFP followed theprocedure described in detail in Chapter 3. Samples were loaded into microwells andexamined at a range of irradiances (350 W/cm2-20 kW/cm2), obtained by attenuating thelaser beam with a half waveplate and polarizer. Data was collected at a sampling rate of
100 kHz for 70 seconds with the 637 nm laser set to maximum power (140 mW). The
processed data is shown in Figure 4.1. The data shows a trend of faster fluorescencedecay rates as excitation rate increases. Looking more closely at the data, there aredecays on multiple timescales, with a fast, microsecond timescale decay in addition toslow, millisecond timescale decays. The time axis is displayed on a log-scale to display
and fit the data on both the fast and slow timescales.

Normalized Fluorescence75

Figure 4.1. smURFP experimental data. Experiments are at excitation rates ranging from 1.8 x 10 6 s-1 to
4.1 x 107 s-1. The data shown here is a normalized, baseline-corrected average at each set of experimental
conditions, with the data set selected with the procedure described in Chapter 3. These experiments were
performed for multiple purifications of smURFP

The data was fit using two-exponential, three-exponential, four-exponential, andfive-exponential decays, as shown in Figure 4.2. Assessing the fits in Figure 4.2, clearlyadding exponential terms to the fits improves the quality of the fit. Comparison of theresiduals and confidence intervals for the fits support this claim. To avoid overfitting thedata, the exponential terms from each fit were extracted and evaluated to determine
whether the additional complexity was necessary to fully describe the data.

76

Figure 4.2 smURFP fits. (a-d) Two-, three-, four-, and five-exponential fits of smURFP data with residuals.
Experiments are at excitation rates ranging from 1.8 x 106 s-1 to 4.1 x 107 s-1. The data are fit with a sum of
exponentials in accordance with the labels on each plot.

77

Comparison of the four and five-exponential fits for smURFP showed averagevariation in fit parameters of 5% across the five irradiances between the two fits.
Comparing the three and four-exponential fit showed variation in fit parameters of 20%
between the two fits. Closer inspection of the three-exponential fit in Figure 4.1 reveals
that the region of the decay around 10-3 s is not fit very well, and so the four-exponential
fit should be used to determine the most accurate determination of the fastest rate
constant.
While the fastest rate constant can be accurately determined with the fourexponential fit, the remaining three rate constants extracted from the decay may be
associated with irreversible photobleaching, and as such should reflect the appropriate
trend with irradiance. This portion of the decay is overfit since the photobleaching is
expected to increase with excitation rate, while none of the extracted rate constants forphotobleaching show a clear trend for the four-exponential fit.
These fits could be improved by extending the time frame for each measurement.
Although the higher irradiance measurements decayed to a stable baseline, collecting
more points along this baseline would improve the quality of the fit. Many trials were
performed to collect average decay behavior so collecting longer decays would limit the
number that could be collected before the sample dried out. The other consideration withtaking longer measurements is the limitation of the LabVIEW software memory
capabilities. Collecting longer decays would require lowering the sample frequency fromthe 100 kHz used for the measurements and would reduce the quality of the fit for the
fastest phase of the decay because the signal in this regime would be under sampled.

78

To ensure a more accurate fit of the fastest decay component given these
constraints, the data was fit with a two-exponential fit up to the steady-state plateau. This
period was chosen to include the fastest decay component, while exluding the decays atlonger times. This corresponds to fitting up to 10 ms for the 1.8 x 106 s-1 excitation rate,
0.7 ms for the 1.0 x 107 s-1 excitation rate, and 0.3 ms for excitation rates from 1.4 to 4.1
x 107 s-1. A fit from 10 µs up to 10 ms is shown at all excitation rates is shown in Figure
4.3 to illustrate this technique.

Figure 4.3 Two-exponential fit to the first 10 ms of the smURFP fluorescence decays. Excitation rates range
from 1.8 x 106 s-1 to 4.1 x 107 s-1. The fit is performed for the first 10 ms to illustrate the extraction of the
microsecond-scale time constant.

The data in Figure 4.3 shows an intial, fast decay on the order of tens of
microseconds. To extract fit constants on the millisecond-scale decays, fits wereconstructed from the steady-state plateau onwards. This was done using the same cut-

79

off times described when fitting the microsecond timescale component, fitting from 10 ms
onward for the lowest excitation rate, 0.7 ms onward for the 1.0 x 10 7 s-1 excitation rate,
and 0.3 ms onward for the three highest excitation rates. There is a clear trend withexcitation rate, with faster fluorescence decays for both the fast and the slower
components as excitation rate increases. The three-exponential fit equation that
describes results on this timescale is given in Equation 4.1.

𝐹 = 𝑎 ∙ 𝑒 −𝑘1∙𝑡 + 𝑏 ∙ 𝑒 −𝑘2∙𝑡 + 𝑐 ∙ 𝑒 −𝑘3 ∙𝑡 + 𝑑
Equation 4.1 Equation for smURFP fluorescence decay. k 1, k2, and k3 are the rate constants extracted fromthe fits, with k1 > k2 > k3. a, b, and c are the fractions of each exponential decay component.

The time constants determined from the smURFP exponential fits are listed inTable 4.1. The fastest time constant (t1) is hypothesized to be related to dark state
photophysics while the slower time constants (t2 and t3) are thought to be associated withirreversible photobleaching. This assignment is made based on comparison of these time
constants to values reported from comparing multiple studies on FPs discussed in
Chapter 1. 58,65,74,80

80

Excitation Rate (s-1) t1 (µs)

t2 (ms)t3 (s)

1.8 x 106

128

390

5.0

1.0 x 107

70

36

0.89

1.4 x 107

72

30

0.74

1.9 x 107

55

15

0.32

4.1 x 107

43

8.1

0.20

Table 4.1 Time constants obtained from fitting smURFP continuous illumination data. The time constants
are obtained by taking the reciprocal of each rate constant. t 1, t2, and t3 are the time constants extracted
from the smURFP exponential fits, in order of increasing time.

4.3 miRFP670 Continuous Illumination StudymiRFP670 was selected for study because it has the highest cellular brightness ofany biliverdin-based fluorescent protein. miRFP670 is a useful far-red fluorescent protein
to study because it was evolved from bacteriophytochromes and is part of a family of farred fluorescent proteins that has been extensively studied. 41-46,118,119 The miRFP670
continuous illumination study followed the same procedure used for the smURFP
experiments. Data was collected at a sampling rate of 100 kHz for 90 seconds with the
637 nm laser set to maximum power. The beam was attenuated with a half waveplate
and polarizer to obtain a range of excitation rates. Figure 4.4 is a plot of the miRFP670
data. The data shows a trend of faster fluorescence decay rates as excitation rateincreases. Unlike the data for smURFP, the miRFP670 data does not appear to show any
fast, microsecond scale decays. The fastest decay is on the order of milliseconds.

Normalized Fluorescence81

Figure 4.4. miRFP670 experimental data. Experiments are at excitation rates ranging from 1.2 x 106 s-1 to
1.8 x 107 s-1. The data shown here is a normalized, baseline-corrected average at each set of experimental
conditions, with the data set selected with the procedure described in Chapter 3. These experiments were
performed for multiple purifications of miRFP670.

Initial fits of the fluorescence decay from two-exponential fit up to a fourexponential fit are given in Figure 4.5. As with fits for smURFP, adding additional terms
to the exponential fit increases the quality of the fit, with the four-exponential fit mostclosely following the data, as assessed through examination of residuals and confidence
intervals. To determine which fit best describes the data, a careful look was taken at therate constants extracted from each fit. The four-exponential fit has multiple fit components
with amplitudes that are much smaller than 1% of the decay, and while a trend ofincreasing photobleaching with irradiance is expected, the fit parameters do not show the82trend. While the three-exponential fit follows the data most closely, the third time-constantis much slower than the expected timescales of the processes being investigated, making
this fit an unreliable descriptor of the data.

83

Figure 4.5 miRFP670 fits. (a-c) Two-, Three-, and Four-exponential fits of miRFP670 fluorescence datawith residuals. Experiments are at excitation rates ranging from 1.2 x 106 s-1 to 1.8 x 107 s-1.

84

Based on the considerations outlined above, the two-exponential fit is the best to
describe the data, and outputs reasonable fit constants. Just as was done for smURFP,
to ensure a more accurate fit of the millisecond decay components, the data was fit witha two-exponential fit from the steady-state plateau onward. This period was chosen to
include the portion of the decay responsible for decay on a millisecond timescale. This
corresponds to fitting from 10 ms onward for the 1.2 x 10 6 s-1 to 5.5 x 106 s-1 excitation
rates, and 1 ms onward for the 1.8 x 107 s-1 excitation rate. A fit from 10 ms onward isshown at all excitation rates is shown in Figure 4.6 to illustrate this technique.

Figure 4.6 Two-exponential fit to miRFP670 fluorescence decays. Excitation rates ranging from 1.2 x 106
s-1 to 1.8 x 107 s-1. The fit is from 10 ms onward to characterize the millisecond-scale fluorescence decays.

From the results shown in Figure 4.6 it appears that miRFP670 lacks the fast decay
component that is attributed to dark state conversion in smURFP. Instead, the fastest
decay component observed in the data for miRFP670 is on the timescale of ms. Based85

on previous studies of dark state conversion and photobleaching in fluorescent proteins,at these irradiances fluorescence decay on this timescale can be assigned tophotobleaching and not dark state conversion.58,65,74,80 The two-exponential equation that
describes the results on this timescale is given in Equation 4.2.

𝐹 = 𝑎 ∙ 𝑒 −𝑘1∙𝑡 + 𝑏 ∙ 𝑒 −𝑘2∙𝑡 + 𝑐
Equation 4.2 Two-exponential fit equation for miRFP670 fluorescence. k 1 and k2 are the rate constants
extracted from the fits, with k1 > k2. a and b are the fractions of each exponential decay component.

The time constants obtained from this fit are listed in Table 4.2. Both fluorescencedecay timeconstants are conjectured to be associated with irreversible photobleaching.
This assignment is made based on comparison to time constants reported in previous
studies on various FPs. 58,65,74,80

Excitation Rate (s-1) t1 (ms)t2 (s)

1.19 x 106

1020

14

1.61 x 106

640

9.2

3.16 x 106

1020

11.4

5.51 x 106

190

3.0

1.80 x 107

37

0.89

Table 4.2 Time constants obtained from fitting miRFP670 continuous illumination data. Time constants are
obtained by taking the reciprocal of each rate constant. t 1 and t2 are the time constants extracted from the
smURFP exponential fits, in order of increasing time.

86

The reliability of both the model and parameters of the fit constants were assessedby comparing the data to mathematical modeling in Section 4.5. The mathematicalmodeling in that section relies on the rate constants extracted from the fit to generate
simulated results.

4.4 mCherry Continuous Illumination Study
Continuous illumination experiments using mCherry were performed as acomparison to the experiments performed on smURFP and miRFP670. The experimental
setup to investigate dark state photophysics in mCherry involves a 561nm CW laser
source modulated externally by an Acuosto-Optical Modulator (AOM). The details of thisexperimental setup have been described in Chapter 3. Experimental data for mCherry is
shown in Figure 4.7.

87

Figure 4.7. mCherry experimental data. Experiments are at excitation rates ranging from 1.8 x 105 s-1 to 8.4
x 105 s-1. The data shown here is a normalized, baseline-corrected average at each set of experimental
conditions, with the data set selected with the procedure described in Chapter 3.

Just as was done for smURFP and miRFP670, the mCherry data was fit withseveral sum of exponential functions and residuals and confidence intervals were used
to assess fit quality. Two-, three-, and four-exponential fits are shown for mCherry in
Figure 4.8 below.

88

Figure 4.8. mCherry fits. (a-c) Two-, Three-, and Four-Exponential fits of mCherry fluorescence data withresiduals. Experiments are at excitation rates ranging from 1.8 x 105 s-1 to 8.4 x 105 s-1.

89

As before, the four-exponential fit decribes the data well, as determined byconfidence intervals and residuals, but is more than is necessary to fit the data. The
extracted bleaching rate constants for the two-exponential plot are in agreement withpublished mCherry data at higher irradiances.65 Similar to the method used for smURFP,
a two-exponential fit up to the steady-state plateau at 1 ms was selected to fit the darkstate part of the curve (microsecond timescale), and a separate fit was done from 1 ms
onward to fit the bleaching part of the curve (millisecond timescale). Figure 4.9 shows a
two exponential fit up to 10 ms to illustrate.

Excitation Rate (x 105 s-1)

Figure 4.9 Two-exponential fit of mCherry fluorescence decays. Fit over first 0.1 s of the decay. Excitation
rates ranging from 1.8x105 s-1 to 8.4x105 s-1

90

The initial, fast fluorescence decay in Figure 4.9 is on the order of hundreds ofmicroseconds, while the second decay component is on the order of hundreds ofmilliseconds. Based on these timescales, although the fit was two-exponential like
miRFP670, the fastest component is a result of the ground state recovery, while theslower component is attributed to photobleaching.58,65 The fit equation describing thisbehavior is given in Equation 4.3.

𝐹 = 𝑎 ∙ 𝑒 −𝑘1∙𝑡 + 𝑏 ∙ 𝑒 −𝑘2∙𝑡 + 𝑐
Equation 4.3 Two-exponential fit equation for mCherry fluorescence. k 1 and k2 are the rate constants
extracted from the fits, with k1 > k2. a and b are the fractions of each exponential decay component.

The time constants obtained from this fit are listed in Table 4.3

Excitation Rate (s-1) t1 (µs)

t2 (ms)1.8 x 105

140

360

2.3 x 10576

130

8.4 x 105

78

70

Table 4.3 Time constants obtained from fitting mCherry continuous illumination data. Time constants are
obtained by taking the reciprocal of each rate constant. t1 and t2 are the time constants extracted from the
smURFP exponential fits, in order of increasing time.

Comparing these measurements to a higher irradiance study on mCherry in ourlab, the microsecond time constant from these measurements is over eight-fold fastercompared to the previously determined ground state recovery time constant. In this study,
the rate of dark state conversion in mCherry was found to be 2-fold faster at an excitation

91

rate of 2.4 x 106 s-1, compared to our lower excitation rate of 8.4 x 10 5 s-1.58 The
differences could be attributed to the differences in collection methods, with the previous
study using frequency domain measurements to determine the rate of dark state
conversion, while the ground state recovery rate constants were determined using pulsed
excitation. This discrepancy indicates that a straightforward exponential alone cannot fully
extract the rate constants from time domain data.

4.5 Comparison of smURFP and miRFP670 to RFPs
Rate constants determined from fits to smURFP, miRFP670, and mCherry data
are compared in Figure 4.10 below. This figure illustrates that the fast, microsecond scaledecay component (k1) for smURFP is slower than dark state conversion for TagRFP-T
and mCherry, while faster than ground state recovery for TagRFP-T and mCherry. This
supports the hypothesis that the microsecond-scale component for smURFP is attributed
to dark state photophysics. Comparing the k2 and k3 rate constants for smURFP to thereported bleaching rate constants for mCherry and TagRFP-T shows that the millisecond
decays are on the same timescale as bleaching for these RFPs. This finding supports the
hypothesis that the two millisecond decay components are due to photobleaching effects.
Both rate constants for miRFP670 data (k1 and k2) are on slower timescales compared tothe bleaching of mCherry and TagRFP-T.58 This supports the hypothesis that both decay
components for the miRFP670 decays are attributed to irreversible photobleaching.

92

(a)

(b)

(c)

(d)

Figure 4.10 Comparison of rate constants (a) smURFP and (b) miRFP670, and (c) mCherry. Determinedfrom exponential fits to experimental data. (d) Rate constants for dark state conversion (k DSC), ground state
recovery (kGSR), and bleaching (kB) for TagRFP-T and mCherry using reported values from Ref. 65.

93

4.6 Kinetic Modeling of Continuous Illumination Results
The exponential fits alone were not sufficient to interpret the fluorescence data.
Kinetic modeling was needed to assess the assignment of rate constants to irreversible
photobleaching or dark state photophysics. After generating the initial fits for smURFP,
miRFP670, and mCherry described in sections 4.2-4.5, the extracted rate constants wereused to simulate results by generating a numerical solution to the differential equations
describing the system. The simplest model was chosen as a starting point and thesimulated data was compared to the experimental data to assess whether that model
accurately described the data. The simplest model that can be used is a two-level systemwith no dark state. The model is described by the two level energy diagram in Figure 4.11.

𝑑[𝑆0 ]= −𝑘𝐸𝑥 [𝑆0 ] + (𝑘𝐸𝑚 + 𝑘𝐼𝐶 )[𝑆1 ]𝑑𝑡
𝑑[𝑆1 ]= 𝑘𝐸𝑥 [𝑆0 ] − (𝑘𝐸𝑚 + 𝑘𝐼𝐶 + 𝑘𝐵 )[𝑆1 ]𝑑𝑡

Figure 4.11. Two-level model diagram and equations. Two-level energy diagram (left) and rate equations(rightdefining kinetics of the states. S0 is the ground state, S1 is the excited state, kEx, kEm, kIC, and kB are
the excitation, emission, internal conversion, and photobleaching rate constants, respectively.

94

The numerical solution to the system of differential equations described in Figure
4.10 is obtained by using the differential equation solver “odeint” in Python with the initialnormalized population of the ground state set to 1 and the initial population of the excitedstate set to 0. Values of kIC and kEm are set based on measured lifetimes and reported
quantum yields for the fluorescent proteins. kEx is set based on the experimental
conditions and kB is the largest millisecond scale decay extracted from the exponential
fits. Since the experimental results discussed here report on the ground state of thefluorescent protein, the resulting numerical solution for the S0 state is plotted with respect
to time and are compared to the fluorescence data generated experimentally. The
simulated results for the S0 state for miRFP670 are compared to the experimental resultsin Figure 4.12.

(a)

(b)

Figure 4.12. Comparison of miRFP670 two-level model to experimental results. (a) S0(t) simulated data for
miRFP670 fluorescence decay based on two level model. (b) miRFP670 experimental data. k IC was set 6.3
x 108 s-1 and kEm was set to 9 x 107 s-1. kEx values of 1.2 x 106 s-1, 1.6 x 106 s-1, 3.2 x 106 s-1, 5.5 x 106 s-1, and
1.8 x 107 s-1 were used with kB values of 0.68, 1.6, 1.2, 52, and 778 s-1, respectively.

95

Comparing the simulation to the experimental data the miRFP670 data seems to
be well described by the two-level system and supports the hypothesis that miRFP670
does not exhibit dark state conversion. The simulated data shows slower overall decay,
but qualitatively follows a similar shape to experimental data. This indicates that while thetwo-level model can be used to describe the miRFP670 data, more information about thesystem is needed to adjust the simulation parameters to more accurately describe thedata. Since two bleaching rate constants were determined from the miRFP670 data, asecond mechanism for bleaching should be incorporated into the model. One way toincorporate this feature would be to treat this as a three-state model in whichphotobleaching occurs from a dark state. While miRFP670 decays were slower than thetimescales observed for fluorescent protein blinking in other studies, a fast conversion to
the dark state with a high rate of bleaching would not be detected with these experimental
conditions. This possibility is discussed below when assessing the smURFP simulated
data. Additionally, while the fits for miRFP670 were restricted to 10 ms onward, a slightdecay on the microsecond timescale can be observed for the data collected with a 1.8 x
107 s-1, suggesting excitation-dependent miRFP670 dark state photophysics. Comparison
to experiments done with other FPs support the possibility of an excitation-dependent
dark state.67
After assessing the ability to describe miRFP670 data with a two-level model, thetwo-level model shown in Figure 4.11 was used to simulate smURFP data, using theexcitation rates from experiments and the faster millisecond scale decay as the bleaching
term, kB. Data produced from the two-level model is compared to smURFP experimental
data in Figure 4.13.

96

(b)

(a)

Figure 4.13 Comparison of two-level simulation to smURFP experimental data. (a) S0(t) simulated data for
smURFP fluorescence decay. (b) smURFP experimental data. To simulate this data, k IC was set 6 x 108 s1 and k
8 -1
6 -1
7 -1
7 -1
7 -1
Em was set 1 x 10 s . kEx values of 1.8 x 10 s , 1.0 x 10 s , 1.4 x 10 s , 1.9 x 10 s , and 4.1 x
7-1-1
10 s were used with kB values of 3.0, 19, 30, 56, and 102 s , respectively.

Comparing the curves generated in Figure 4.13, the fluorescence decay of
smURFP is not accurately described by a two-level system and the dark state is needed
to fully describe the fluorescence decay. The simulated two-level data lacks the
microsecond scale decay observed in the smURFP experimental data. For this reason, athree-level diagram was applied next to incorporate the dark state into the simulation. The
differential equations and the three-level energy diagram used to simulate the data are
given in Figure 4.14. For simplicity, all irreversible photobleaching was assumed to occur
from the dark state in this model.

97𝑑[𝑆0 ]= −𝑘𝐸𝑥 [𝑆0 ] + (𝑘𝐸𝑚 + 𝑘𝐼𝐶 )[𝑆1 ] + 𝑘𝐺𝑆𝑅 [𝐷]𝑑𝑡

kB

𝑑[𝑆1 ]= 𝑘𝐸𝑥 [𝑆0 ] − (𝑘𝐸𝑚 + 𝑘𝐼𝐶 + 𝑘𝐷𝑆𝐶 )[𝑆1 ]𝑑𝑡𝑑[𝐷]= 𝑘𝐷𝑆𝐶 [𝑆1 ] − (𝑘𝐺𝑆𝑅 + 𝑘𝐵 )[𝐷]𝑑𝑡

Figure 4.14 Three-level model diagram and equations. Three-level energy diagram (left) and differential
equations (right) defining the kinetics of the states. S 0 is the ground state, S1 is the excited state, and D isthe dark state. kEx, kEm, kIC, and kB are the excitation, emission, internal conversion, and bleaching rate
constants, respectively. kDSC and kGSR are the rate constants for dark state conversion and ground state
recovery, respectively.

As before, the numerical solution to the system of differential equations describedin Figure 4.14 is obtained by using “odeint” in Python. The initial population of thenormalized ground state is set to 1 and the initial populations of the excited and darkstates are set to 0. Values of kIC, kEm, kex, and kB are set the same as used for the twolevel model. The value of kDSC is unknown and was estimated 7 x 104 s-1 based on theassumption that kDSC occurs faster than kGSR as observed in dark state studies conductedon RFPs.65,68 The value of kGSR is set to the microsecond scale rate constant extracted
from the fits. The resulting numerical solution for the S0 state is then plotted and comparedto the fluorescence data generated experimentally. The simulated results for the threelevel model are compared to the smURFP experimental data in Figure 4.15.

98

(a)

(b)

Figure 4.15. Comparison of smURFP simulated results using a three-level model (a) Simulated data for
smURFP fluorescence decay based on three-level model. (b) smURFP experimental data. To simulate thisdata, kIC was set 6 x 108 s-1, kEm was set to 1 x 108 s-1, and kDSC was set to 7 x 104 s-1. kEx values of 1.8 x
106 s-1, 1.0 x 107 s-1, 1.4 x 107 s-1, 1.9 x 107 s-1, and 4.1 x 107 s-1 were used with kB values of 3.0, 19, 30, 56,
and 102 s-1 and kGSR values of 2.3 x 103 s-1, 1.17 x 104 s-1, 1.2 x 104 s-1, 1.6 x 104 s-1, and 1.77 x 104 s-1,
respectively.

Both simulated and experimental data show a fast, microsecond decay and slower,
millisecond decay. The microsecond decay component observed in the simulation isslower compared to the experiment, while the simulated decay component on the
millisecond scale are faster compared with experiments. Qualitative comparison of thesimulated data to the experimental data indicates that the three-level model generally
describes the data, but deeper examination of the parameters is needed to understandthe divergence between simulation and experiment.
As mentioned above the dark state conversion rate constant used in simulations
was an estimate chosen based on comparison to studies on dark state photophysics
observed in RFPs.65 The estimate was selected based on the assumption that groundstate recovery is the rate-limiting process, as observed for other FPs.68 To interrogate this99

assumption, the dark state conversion rate constant was varied while all other rate
constants remained unchanged. The simulated data resulting from dark state conversion
much slower than ground state recovery (kDSC << kGSR), about the same magnitude as
ground state recovery (kDSC ~ kGSR), and much faster than ground state recovery (kDSC >>kGSR) is shown in Figure 4.16.

100

(a)

kDSC << kGSR

(c)

kDSC >> kGSR(b)

kDSC ~ kGSR

Figure 4.16. Effect of kDSC on simulated data. Parameters: kIC = 6 x 108 s-1, kEm = 1 x 108 s-1. kEx values of
1.8 x 106 s-1, 1.0 x 107, 1.4 x 107, 1.9 x 107, and 4.1 x 107 s-1 were used with kB values of 3.0, 19, 30, 56,
and 102 s-1. kGSR values were 2.3 x 103, 1.17 x 104, 1.2 x 104, 1.6 x 104, and 1.77 x 104, respectively. (a)kDSC = 7.2 x 102 s-1, an order of magnitude slower than the slowest k GSR value. (b) kDSC = 1.5 x 104 s-1, themedian value for kGSR. (c) kDSC = 2.2 x 105 s-1, an order of magnitude faster than the fastest kGSR value.

101

The results of the simulations in Figure 4.16 support the assumption that dark state
conversion occurs much faster than ground state recovery. If dark state conversion isslower, as in Figure 4.16 (a), no microsecond scale decay component is observablebecause there is not accumulation of dark state population. If dark state conversion is on
the same order of magnitude as ground state recovery, as in Figure 4.16 (b), a slightmicrosecond decay component can be observed, but it is so slight that it may be lost in
the experimental noise if the dark state conversion of a protein with these properties were
measured. This small microsecond decay component can be attributed to a small
accumulation of population in the dark state. The last scenario, where dark state
conversion is faster than ground state recovery (Figure 4.16 (c)), most closely resembles
smURFP experimental data, showing a sharp microsecond decay component,suggesting higher accumulation of population in the dark state. Careful comparison ofexperimental and simulated data narrowed the range of values for dark state conversion
to between 7.4 x 104 and 2.2 x 105 s-1.
Although the simulations in Figure 4.16 were based on smURFP experimental rate
constants, they can also inform interpretation of the miRFP670 data. The scenarios
observed in Figure 4.16 (a) and (b) could describe miRFP670, in which the dark state
conversion effects could be classified as either not observed as in (a) or very slight as in(b). While miRFP670 data was described by a two-level system, dark state photophysics
could describe the slight microsecond decay observed for the 1.8 x 10 7 s-1 excited state
and the multiple timescales for photobleaching.
Following assessment of the dark state conversion parameter, the three-level
model was adjusted by adding a second photobleaching decay term. The 3-level diagram

102

and kinetic equations describing this adjustment are shown in Figure 4.17. According tothis model, bleaching from the dark state (kB) is greater than bleaching from the excitedstate (kB2). Addition of photobleaching from the excited state had a negligible effect on
the simulated data and resulting simulations were indistinguishable from the previous
model.

kB2
𝑑[𝑆0 ]= −𝑘𝐸𝑥 [𝑆0 ] + (𝑘𝐸𝑚 + 𝑘𝐼𝐶 )[𝑆1 ] + 𝑘𝐺𝑆𝑅 [𝐷]𝑑𝑡

kB

𝑑[𝑆1 ]= 𝑘𝐸𝑥 [𝑆0 ] − (𝑘𝐸𝑚 + 𝑘𝐼𝐶 + 𝑘𝐷𝑆𝐶 + 𝑘𝐵2 )[𝑆1 ]𝑑𝑡𝑑[𝐷]= 𝑘𝐷𝑆𝐶 [𝑆1 ] − (𝑘𝐺𝑆𝑅 + 𝑘𝐵 )[𝐷]𝑑𝑡

Figure 4.17 Three-level model with excited-state bleaching. Three-level energy diagram (left) anddifferential equations (right) defining the kinetics of the states. S0 is the ground state, S1 is the excited state,
and D is the dark state. kEx, kEm, kIC, kB, and kB2 are the excitation, emission, internal conversion, dark statebleaching, and excited state bleaching rate constants, respectively. k DSC and kGSR are the rate constants fordark state conversion and ground state recovery, respectively.

The effect of irreversible photobleaching on the relative rate of the millisecond
fluorescence decay was further investigated by switching the bleaching values so that thelarger bleaching rate constant was assigned to the excited state. The effects of this switch
are shown in Figure 4.18. This plot shows that when the larger bleaching component is
assigned to the dark state rather than the excited state, there is a shorter dwell time insteady state, and faster overall fluorescence decay. This supports the initial assignment

103of the faster millisecond bleaching rate constant as associated with irreversible
photobleaching from the dark state.

Effect of Switching Bleaching Terms

Normalized FluorescenceS1 bleach > D bleachS1 bleach < D bleachFigure 4.18 Effect of switching bleaching terms on simulated data. The kB and kB2 terms determined
experimentally are switched within the model to predict source of bleaching. kIC was set 6 x 108 s-1, kEm was
set to 1 x 108 s-1, and kDSC was set to 7 x 104 s-1. kEx values of 1.8 x 106 s-1, 1.0 x 107 s-1, 1.4 x 107 s-1, 1.9 x
107 s-1, and 4.1 x 107 s-1 were used with kB values of 3.0, 19, 30, 56, and 102 s-1 , kB2 values of 0.20, 0.55,
0.69, 3.4, and 6.0 s-1, and kGSR values of 2.3 x 103 s-1, 1.17 x 104 s-1, 1.2 x 104 s-1, 1.6 x 104 s-1, and 1.77 x
104 s-1, respectively.

Comparison of the models developed to experimental data revealed that in thecase of miRFP670, the two-level model qualitatively represents the experimental results,
but the fluorescence decay in the simulation is too slow compared to the experiment. A
careful look at the investigation into the effects of adjustments to the dark state conversion
rate constant (kDSC) on the simulated results revealed that miRFP670 could also be
described by a three-level model in the scenario where kDSC ≤ kGSR.

104

In the case of smURFP, the three-level model best represented the experimental
results, and the simulation of this model allowed the kDSC parameter to be estimated. The
value of kDSC over the irradiance range studied was found to be between 7.4 x 10 4 and2.2 x 105 s-1. This estimate allowed the single molecule fluorescence blinking times to bepredicted, using Equations 4.4 and 4.5 below. Calculated τ on values were between 60 µs
and 5 ms and τoff values between 40 and 130 µs for the excitation rates ranging from 1.8
x 106 s-1 to 4.2 x 107 s-1. Based on these values, smURFP is expected to blink twice as
fast as mCherry, and TagRFP-T blinks 1.2-fold faster than smURFP.65 While brighter
fluorescent proteins are ideal for super-resolution experiments, this fast blinking predicted
for smURFP indicates that further development of smURFP could be advantageous forsuper-resolution imaging.

τon⁡=⁡

𝟏𝒌𝒆𝒙 ∗𝒌𝑫𝑺𝑪 ∗𝝉𝒆𝒎

Equations 4.4 Expression to calculate the on time (τon) for a fluorescence trajectory. kex is the excitation
rate constant, kDSC is the dark sate conversion rate constant, and τem is the fluorescence lifetime.

τoff⁡=⁡𝟏

𝒌𝑮𝑺𝑹

Equations 4.5. Expression to calculate the off time (τoff) for a fluorescence trajectory. kGSR is the groundstate recovery rate constant.

105

4.7 Discussion
Insight into these dark state conversion results requires looking closely at previous
studies on BV-binding fluorescent protein and their origins. miRFP670 was developed
from bacteriophytochromes, which are composed of the PHY, PAS, and GAF domains,with the GAF domain being the site of most chromophore protein contacts. 117,120-122
Biliverdin binding occurs via a cysteine to the vinyl side chain of the BV A-ring.121-122 Thebiliverdin chromophore sits within its binding pocket, with steric interactions and hydrogenbonding interactions holding it in place. Light activation of the phytochromes involves
isomerization about the C15=C16 double bond in biliverdin, resulting in a flip of the Dring.117,120 In solution, the preferred conformation of biliverdin is mostly helical withextended forms present in smaller proportion, but is extended when bound to protein. 18,40
Brighter, blue-shifted mutants of a bacteriphytochrome were generated byincorporating cysteine residues for BV binding in the GAF and the PAS domain rather
than the PAS domain alone. This group of blue-shifted mutants includes iRFP670, andthe study found that the blue-shift is attributed to the sp3 hybridization of the biliverdin
carbon that binds the cysteine residue instead of forming a double bond, reducing theextent of π-electron conjugation to exclude the BV A-ring.118 The same blue shift is also
observed in miRFP670. The increase in fluorescence lifetime for these blue-shiftedmutants is linked to the increase in steric constraints about the BV A-ring, reducing A-ringmotion, and the reduction of π-conjugation so that it does not extend to the A-ring,affecting the rate of decay from the excited state.118 The miRFP670 biliverdin-binding
pocket is shown in Figure 4.19

106

Figure 4.19. Protein environment around chromophore for miRFP proteins. From crystal structure. Shows
environment around miRFP670 (blue), miRFP703 (green), and miRFP709 (red). Image from Ref. 81.

In the development of near infrared fluorscent proteins (NIR FPs), the PHY domain
was truncated and mutations of key residues in the GAF domain blocked
photoconversion.34,41,42 A femtoseond transient absorption spectroscopy experiment on
iRFP670, a miRFP670 precursor, confirmed that there was no significant formation ofground-state photoproducts associated with photoconversion.118,119 A resonance Raman
study on iRFP reveals the protein has fewer hydrogen bonding interactions with ring D
and fewer water molecules around ring D compared to the phytochrome from which it isevolved.123 The flexibility of the chromophore is reduced, especially around ring A and D
and there is an overall reduction in conformational heterogeneity compared to wild-type
proteins. The T258F mutation may have strengthened hydrophobc interactions with ringD, packing the chromophore more tightly.123
Time-resolved fluorescence studies in H2O and D2O found significant kinetic
isotope effects in fluorescence lifetimes for red-shifted and blue-shifted mutants. Kinetic
isotope effects of ~1.8 for all lifetimes above 0.8 ns were reported.118,119 This result is
consistent with spectroscopic studies of other iRFPs and with wild-type BphPs that had
similar kinetic isotope effects, indicating that the excited-state proton transfer (ESPT)107

model of proton transferred from the pyrrole to the protein backbone observed in these
proteins is also a potential pathway for non-radiative decay in iRFP670.124-126
An ultrafast spectrscopic study comparing behaviors of two wild-typebacteriophytochromes investigated the kinetic isotope effect in these proteins. The study
found that the rate-limiting process for excited-state deactivation was a proton transfer
reaction. Replacing amino acid residues attributed to hydrogen bonding decreased therate of excited state decay while increasing the rate of isomerization product formation.
From these results, the breaking of hydrogen bonds surrounding the chromophore wasdetermined to be rate-limiting for isomerization about the C15=C16 double bond, andthese hydrogen bonds must be disrupted to form photoproducts in the wild-typebacteriophytochromes.124 Additionally, the biliverdin pyrrole ring deprotonation competes
with isomerization about the C15=C16 as a deactivation pathway. 124-125,127 The process
of excited-state proton transfer is shown in Figure 4.20. QM/MM computations on a wildtype bacteriophytochrome reproduce absorption and emission energies 57 andqualitatively support isomerization around the biliverdin D-ring as a possible pathway of
excited-state nonradiative decay in phytochromes.128
The studies discussed here inform the interpretation of the dark state conversion
results for the smURFP and miRFP670. miRFP670 can be more directly compared to thestudies on bacteriophytochromes discussed here since it was evolved from the blueshifted variants in which motion of the A-ring and D-ring has been constricted,46 whereas
smURFP is based on directed evolution of allophycocyanin (APC) to generate a biliverdinbinding variant. Since only the Y56A mutant of smURFP has been crystallized,
interpretation of its results and comparison to miRFP670 results requires consideration

108of the photophysics of biliverdin to infer the protein environment surrounding thechromophore.
Although the two near-infrared fluorescent proteins studied here both use biliverdin
as a chromophore they exhibited vastly different behavior. While smURFP exhibited fast
dark state conversion, no dark state conversion was measured for miRFP670 at theirradiances studied. The additional exponential terms required to fit smURFP compared
to miRFP670 likely stems from structural heterogeneity of the smURFP molecule,including the tendency to dimerize and ability to bind 1 or 2 BV molecules. 37 One possible
interpretation of the smURFP results is a distinct isomerization rate observed for eachconformation. Based on the mutations and structural studies done on precursors to
miRFP670, dark state conversion may be inhibited by the reduced mobility of the BV Aring and the BV D-ring. No such structural studies were done on smURFP, but studies
discuss above on biliverdin alone and bound to protein indicate that isomerization about
the C15=C16 bond may be a deactivation pathway for smURFP.124-125,127 Figure 4.20 (a)shows the protein environment around biliverdin based on a homology model and Figure
4.20 (b) shows the biliverdin binding pocket of a low quantum yield mutant, smURFP
Y56R.37,129 Additional study on NIR FPs that lack the A-ring and D-ring stabilization would
grant further insights into the mechanisms behind DSC in biliverdin-based fluorescentproteins, in addition to deuterium exchange experiments to assess the role of protontransfer in dark state conversion for these proteins.

109

Figure 4.20. Protein environment of smURFP chromophore. (a) smURFP homology model. Picture from
Ref. 37. (b) Crystal structure of smURFP Y56R. Picture from Ref. 129.

The fluorescence decay component for with the smURFP dark state was found tovary from 46-140 µs across the excitation rates measured (1.8 x 10 6 to 4.1 x 107 s-1).
Based on the above analysis, this decay component is most comparable to the groundstate recovery rate constant, indicating smURFP blinking is 2-fold faster than mCherry
and slower than TagRFP-T blinking.65 The fastest decay component for miRFP670 varied
from 25-670 ms, likely associated with bleaching since it occurred much more slowly thanblinking timescales observed for other fluorescent proteins.58,65,74,80 These results indicatethat smURFP is potentially useful for imaging techniques that require a molecule thatshows fast blinking behavior, such as super-resolution techniques. The apparent absenceof dark state conversion in miRFP670 and higher photostability compared to smURFP,
mCherry, and TagRFP-T65 indicate that this is a useful protein to use for deep-tissue
experiments.

110

4.8 Conclusion
In this chapter I reported the results from measurements of the dark state
photophysics of two near-infrared fluorescent proteins that had not previously beenchracterized. I found that miRFP670 exhibited no dark state conversion over theirradiance range measured, and smURFP exhibited dark state photoswitching ontimescales ranging from tens to hundreds of microseconds. This timescale is consistent
with other fluorescent proteins for which the dark-state photophysics has beencharacterized, and is the first time dark state conversion rates have been measured innear-infrared fluorescent proteins. While the exponential fits shown here can be used to
extract rate constants for ground-state recovery, at the excitation rate used the dark state
conversion rate constants cannot be determined and could only be estimated fromsimulated data. These measurements can be used to illuminate the photophysicalproperties of the near-infrared fluorescent proteins, but complementary measurements
such as single-molecule measurements are needed to fully characterize the dark-state
dynamics of smURFP.

4.9 Future Directions
Further experiments should look at lower irradiances (100 W/cm 2 and below) to
give better predictions of blinking behavior under conditions more relevant to wide-fieldimaging. Expanding the range of irradiances used would give more insight into for which
imaging experiments these proteins are best applied. Single-molecule measurements of

111

smURFP should also be done to compare the predicted on- and off- times to theensemble studies described here.
Other experiments needed are dark state measurements on miRFP670 mutants
with mutations designed disrupt the hydrogen bonding interactions around both ring A
and ring D. This would help determine whether motions of the biliverdin A- or D-ring are
responsible for the dark state conversion observed in smURFP. Dark state photophysicalmeasurements on such proteins would yield insight into the mechanism behind dark state
conversion in smURFP and possibly hindering it in miRFP670.

112

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Appendix A: Steady State Fluorescence Measurements

A.1 Publication Status
This section discusses the work done in preparing Figure S6 for the following
publication:
Konold, P. E., Yoon, E., Lee, J., Allen, S. L., Chapagain, P. P., Gerstman, B. S.,
Regmi, C. K., Piatkevich, K. D., Verkhusha, V. V., Joo, T., Jimenez, R. Fluorescencefrom Multiple Chromophore Hydrogen-Bonding States in the Far-Red Protein
TagRFP675. J. Phys. Chem. Lett. 7, 3046–3051 (2016).

A.2 Introduction
This work was done as part of an investigation into TagRFP675 hydrogenbonding between the N-acylimine carbonyl and the Q41 side chain. While other studies
have associated the large Stokes shift observed in TagRFP675 with this and otherhydrogen bonds, the mechanism required further investigation. This investigation used
spectrally resolved transient grating (SRTG) and femtosecond time-resolved
fluorescence spectra (TRFS) measurements in addition to MD simulations tocharacterize the excited-state dynamics of TagRFP675.

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The Q41 interaction was investigated in part by generating mKate/M41Q andTagRFP675/Q41M, to add glutamine, which has a polar side chain, into mKate andremove it from TagRFP675 by replacing it with methionine, an amino acid with ahydrophobic side chain. SRTG measurements on TagRFP675 and mKate/M41 showedthat peak-shifts in the stimulated emission spectral response suggested the presence ofmultiple spectral forms. Time-resolved area-normalized spectra (TRANES) generated
from TRFS measurements supported the existence of multiple discrete emitting
states.106 In this section I discuss steady-state fluorescence measurements on
TagRFP675 and mKate/M41Q that were done to provide additional information on theexistence of multiple ground states.

A.3 Methods
The fluorescent proteins TagRFP675 and mKate/M41Q were prepared in 15 mM
Tris, 100 mM KCl, pH 8.0 buffer. The fluorescence spectra were collected using a QM-6
steady-state spectrofluorimeter from Photon Technology International (PTI). The
excitation and emission slits were opened to 0.5 mm for a 2 nm bandwidth, and thePMT voltage was set to 1000. The step size was set to 0.25 nm.

A.4 Results
The results of the steady-state fluorescence measurements on TagRFP675 andmKate/M41Q are shown in Figure A.1 below.

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Figure A.1 Excitation-dependent emission spectra. (top) TagRFP675 (bottom) mKate/M41Q. For bothplots, the colors correspond to excitation at red-15310 cm-1, orange-15850 cm-1, magenta-16420 cm-1,
green- 17040 cm-1, blue- 18050 cm-1, and black- 19190 cm-1.

Figure A.1 shows variation in emission peak wavelength as excitation
wavelength is altered. These results support the claim that there are multiple groundstates for these two proteins. From these results in combination with other124

measurements, the hydrogen-bonding interactions with the Q41 position in TagRFP675
and mKate/M41Q leads to a heterogenous system with multiple emitting forms.

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Appendix B: Detailed Protocols

B.1 Microfluidic Master Fabrication
Microwell masters are the molds that are used to produce PDMS microwell deices.
Master fabrication takes two days and once generated the masters can be used manytimes to generate microwell devices.

B.1. 1 Selecting materials and Design
Master templates are designed using computer-aided design (CAD)
software such as AutoCAD. The photolithography mask with the design is printed
with the specification that the emulsion side is down. It is a good idea to order afew variations on the same design to troubleshoot. Silicon wafers with 76.2 mm
diameter are used as a substrate to deposit photoresist. The SU-8 photoresist is
selected based on desired photoresist thickness, and the 3000 series isrecommended for its improved adhesion properties. Datasheets are available ontheMicrochem

websitefor

guidance(http://microchem.com/Prod-

SU8_KMPR.htm). Initial trials for microwells were done using SU-8 2025 for 25 µm

126photoresist thickness. Later fabrications used SU-3025 for the same thickness dueto the improved adhesion properties of the SU-8 3000 series.

B.1.2 Wafer Cleaning
If a used wafer has a layer of cross-linked SU-8 it should be submerged inpiranha solution for at least 30 minutes between 90-100 °C to remove thephotoresist. Piranha should never be left unattended while heating. After, rinse
with isopropanol and dry with N2. All used wafers should be ashed in the reactive
ion etcher (RIE) prior to use. This will remove a few microns from the surface. The
RIE setting for this step is pure O2 with 30 sccm and 300 W for 5 minutes. The RIE
can be purged before use by running the program once with nothing in thechamber.
The next cleaning step is optional but recommended for new wafers. The
wafer is rinsed with acetone and then submerged in a dish containing acetone andswished for 5 minutes. The wafer is then rinsed with methanol and then submerged
in a dish containing methanol and swished for 2 minutes. It is then rinsed for ~10
seconds with isopropanol and thoroughly dried with pressurized N2 and placed ona hot plate at 70 °C for 10 minutes to evaporate any remaining solvent.

B.1.3 Spin-Coating the Wafer
The wafers are next spin-coated with SU-8, a negative epoxy typephotoresist. The clean wafer is centered on the spin coater and the vacuum is127

engaged. Failing to center the wafer may lead to an uneven spin coat. A few
milliliters of photoresist is carefully poured on the wafer. Adding too much will lead
to beading on the edge of the wafer. A 2-step spin coat process is used, with an
initial 500 rpm/s step followed by a higher speed step determined from desired
photoresist thickness. Data for spin speeds for specific photoresists and resulting
thickness is given on the datasheets found on the Microchem website(http://microchem.com/Prod-SU8_KMPR.htm).
Procedure 7 in the Keck Lab spin-coater protocol list corresponds to a 2step process that can be slightly modified based on the desired spin settings. The
procedure used for microwell masters with SU-8 3025 and 25 µm photoresist
thickness is first a 500 rpm step for 10 seconds with 100 rpm/s acceleration
followed by 3000 rpm for 30 seconds with 300 rpm/s acceleration.
Thickness of the spin coat can be adjusted by altering the viscosity of thephotoresist during the spin coat by heating the photoresist or the wafer immediately
before spin-coating. Centrifuging photoresist prior to pouring it can reduce thenumber of air bubbles compared to resist straight from the bottle. If many wafers
are being spin-coated at once, the spin coat chamber should be cleaned after a
few wafers have been spin-coated. Otherwise built up photoresist from previous
spins may drip onto the wafer.

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B.1.4 Soft bake
After spin-coating, the wafer is soft baked on a hot plate to evaporate
solvent. To improve adhesion of the microwell masters, a slow ramp was done forevery heating step. The hot plate was set to increase the temperature at a rate of
5 °C/min. The wafers are placed on a hot plate at room temperature (~20 °C) andraised to 95 °C. The amount of time to soft bake is determined by the Microchem
datasheet based on specific SU-8 type used and photoresist thickness. Once thehot plate reaches 85 °C the timer is started. After the time is up, the hot plate isramped down and the wafer is removed from heat once it has cooled to 65°C. For
the 25 µm thick features with SU-8 3025 a 15 minute soft bake was done. For
improved adhesion, the ramp speed can be lowered further to 1-3 °C/min.

B.1.5 UV Exposure
UV exposure is used to generate the acid catalyst that promotes the
cross-linking reaction. To do this the photolithography mask is placed on the spincoated wafer with the emulsion side down. The emulsion side will have acetate
bubbles when viewed under a microscope and this orientation results in an image
that is readable on the master (all letters, numbers correctly displayed). Arrange
mask to avoid any bubbles or particles on the resist surface. The wafer is thenplaced on the UV mask aligner stage so that it fills the glass area. Both levers to
the left of the machine should be pushed all the way away from the user. The stage
should be raised by turning the stage knob counterclockwise until one of the levers

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moves backward and the separation button lights up. At this point, the lever ispushed back and the contact button will light up. This ensures the wafer is incontact with the photolithography mask for uniform exposure. For small features in
the center of the wafer deselect soft contact and use hard contact instead so thata burst of N2 will push the chuck up for better contact. A single drop of glycerol canbe placed between the wafer and the mask to improve contact. The wafer is now
ready for UV exposure using the Karl Suss MJB3/MJB4.
The UV exposure settings are determined based on the mask aligner power
measurement and the Microchem datasheet for the SU-8 photoresist selected andfeature thickness. The SU-8 3025 is i-line sensitive and recommends 150-250
mJ/cm2 exposure for 25 µm feature thickness. The i-line power of the UV contactaligner is 9.5 mW/cm2 based on the most recent power measurement. Based on
this information, a range of 16 s-26 seconds is recommended for exposure times.
If a wafer is under-exposed, this will appear as cracks on the inside of the area
exposed by the photolithography mask as not enough exposure energy was used
to crosslink the entire exposure area. If a wafer is over-exposed, it will appear asreacted photoresist outside the area exposed by the photolithography mask,
leading to cracks on the outside of the design. If good contact is not achieved
between the photolithography mask and the spin-coated wafer, this will appear as
exposure in the area outside of the area exposed by the photolithography mask.
Adding a few drops of glycerol to the soft baked photoresist before adding thephotolithography mask will improve the contact.

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With 16 seconds of UV exposure, the design was slightly underexposed but
over half of the microwells stuck to the substrate and were not washed away. With
18 seconds of UV exposure, all of the microwells appear to have stuck and allother features are formed, with a few cracks indicating slight underexposure.
These cracks will likely be smoothed out during the hard bake step. A later repeatof the procedure found that 20-22 s of exposure produced even better results with
no apparent under or overexposure.

B.1.6 Post-Exposure Bake
The post-exposure bake step will initiate the cross-linking reaction in thepresence of the acid catalyst. Following UV exposure, the wafer is placed on a hot
plate at room temperature. The heat is slowly ramped up to 95 °C at a rate of 5
°C/min. Once the temperature reaches 85 °C, the timer is started, similar to thesoft bake step. The amount of time the device spends on the hot plate isdetermined from the Microchem datasheet for the selected SU-8 and for thephotoresist thickness. For SU-8 3025 and 25 µm feature thickness, the postexposure bake time is 5 minutes. After the time is up, the hot plate is slowly ramped
down and the wafer is removed from the hot plate once it has cooled to 65 °C.
The latent image should begin to appear after a few minutes of heating. If it
appears more quickly, the photoresist has likely been overexposed Exposurelevels can be assessed during the post-exposure bake step. If the design takes
longer to appear, this is a sign of under-exposure. Typically, the design will appear

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after a few minutes of heating above 50 °C. Paying attention to this step whendeveloping a protocol for a new design can save time on future steps by assessing
whether the procedure worked as intended or not before continuing. After this step
the cross-linked photoresist should be observed under a microscope to assess
whether it has been underexposed or overexposed. This assessment may not be
possible later if the design washes away in the development step.

B.1.7 Development
Once the wafer is at room temperature, it is submerged in fresh SU-8
developer Propylene glycol methyl ether acetate or PGMEA) to rinse any
unreacted photoresist. The development time is found on the Microchem data
sheet for the SU-8 type selected and feature thickness. For SU-8 3025 and 25 µm
feature thickness, the development time selected was 8 minutes. For the first 3
minutes, the wafer is submerged with no agitation. For the final 5 minutes, thewafer is very gently swished in the developer. After this period the wafer is rinsed
with fresh SU-8 developer for ~10 seconds (~10 mL developer). The wafer is thenrinsed for ~ 10 seconds with isopropanol. The wafer is then carefully dried with N2.
If a white residue appears on the wafer following development, this meansthat some unreacted photoresist remains on the wafer. In this case, the wafer
should be submerged in the developer and swished for an additional 30-60 s toremove the remaining photoresist. If the design is washed away at this step, that

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indicates either underexposure, too short of a post-exposure bake, or that theswishing during development was too vigorous. The key to this step is rinsing
vigorously enough to remove all unreacted photoresist, but gently enough that thecrosslinked photoresist remains. After this stage the designs should be checked
under a microscope to assess whether the process was successful. Slight cracks
in the photoresist will be filled during the hard bake step.

B.1.8 Hard Bake
The hard bake step will smooth out cracks in the photoresist and improveadhesion between the wafer and the photoresist. The wafer is is heated overnight(>6 hrs) on a hot plate set between 150 °C – 200 °C. A slow ramp at a rate of 3
°C/min is done to reach this temperature and ensure the integrity of the design
during heating. This step will reflow cross-linked SU-8 to smooth out minor cracks
in the photoresist. The next day the temperature is ramped down to room
temperature.

B.1.9 Silanization
The silanization step is necessary to passivate the surface of the wafer,
improving hydrophilicity to improve the process of pulling PDMS off of the wafer. If
this step is skipped the PDMS may be bonded to the wafer and impossible to133remove. Silanization is done in a vacuum chamber reserved for chlorosilane use.
A few drops of (tridecafluoro-1,1,2,2,-tetrahydrooctyl)-1-trichlorosilane are placedon a low fiber paper towel and placed with the wafers under vacuum overnight (>6hours). The vacuum is arranged so that the wafers are between the silanes andthe vacuum source. If possible, a wafer that is not in use is placed in front to ensure
the wafers are not directly exposed to silane solution and the wafers are facedaway from the vacuum flow. This is the final step in master fabrication. Following
this step the master is taped into a Petri dish and used as a mold for PDMS
devices. The master can be reused several times to produce several rounds ofdevice fabrication.

B.2 Preparation, Cleaning, and Storage of Microwell Devices
Microwell devices are prepared by pouring PDMS into a pre-made microwellmaster mold, cutting the devices out of PDMS, and cleaning the devices which are thenstored in a low dust environment prior to use. Polydimethylsiloxane (PDMS) is preparedby mixing the pre-polymer and curing agent in a 10:1 ratio. After thoroughly mixing,
several gas bubbles will appear. The mixture is degassed under vacuum for 30 minutes
to remove the majority of the bubbles. The mixture is then poured over the microwellmaster in a Petri dish and again degassed under vacuum for an additional 30 minutes.
The dish is then placed in an over pre-heated to 70 °C and left to cure overnight (>6 hrs).
The oven is turned off and vented to cool before removing the dish from the oven. This

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step is necessary to prevent the plastic Petri dish from deforming due to the rapid
temperature change.
The devices are cut out of the PDMS in a cleanroom to prevent dust from blocking
the small features. Tape is pressed against the devices on each side and quickly
removed. This removes small pieces of PDMS generated from cutting out the devices.
Any remaining debris is blown from the microwell master template and the device using
N2 air. The devices are rinsed with isopropanol for ~10 s. The devices are blown dry with
N2 air and placed on a hot plate set to 70 °C for 10 minutes to evaporate any remaining
solvents. Devices can additionally be plasma cleaned, but care must be taken to not let
the top of the device touch anything (even another clean device) after this step as it may
become permanently bonded.
For device storage, a clean, sterile Petri dish is blown with N2 air to remove any
dust and devices are placed in one layer across the bottom, with microwells facing up.
The Petri dish is taped closed and wrapped in aluminum foil. These precautions are taken
to prevent dust from entering the container and blocking microwells during storage. The
container is stored in a clean hood and is only opened in a clean environment. After a
microwell is removed for use, the dish is immediately re-sealed. Microwell devices are notexposed to ambient air once they have been cut out. Containers holding them are only
opened in the cleanroom or in a clean hood. Exposure to particles in the air increases theodds that some of the wells will be clogged.

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B.3 Bacterial Transformation
Bacterial transformation is the process of introducing plasmid DNA into bacterial
cells for the purposes of growing cells expressing fluorescent protein. This protein can beused for optical measurements, or used to start liquid culture for protein purification or
DNA extraction. 50 µL of E. coli competent cells (Top10) are removed from the Ultralow
freezer and thawed on ice. Competent cells have had membranes made porous toprepare for transformation. Plasmid DNA encoding the fluorescent protein of interest isalso thawed over ice. The plasmid should be His-tagged and have the pBAD expression
system under the araBAD promoter to be used according to the fluorescent protein
expression protocol listed in section B.4. 1 µL of plasmid DNA is added to the E. coli cells.
The tube is mixed by gently tapping and is left on ice for 20 minutes. This allows the DNA
to settle onto the cell walls. A water bath is heated to 42 °C and an incubator is set to 37°C for later steps.
After sitting on ice for 20 minutes the cells are heat shocked by placing them in the
42 °C water bath for 35 s. The cells are then returned to the ice bath for 2 minutes andlysogeny broth (LB) media is preheated in the water bath. 400 µL of preheated LB media
is added to the cells and the sample is incubated at 37 °C, 225 rpm for one hour. During
this time an LB agar plate with ampicillin and arabinose is preheated at 37 °C. Once thesamples are done incubating, 30-100 µL of the transformation sample is added to theagar plate and spread until the solution is completely absorbed using a sterile loop. The
plate is placed in an incubator at 37 °C overnight (~12 hours). After the plate is grown up
with many colonies, it can be placed in the fridge at 4 °C to stop growth. Plates are
discarded after a week.

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B.4 Fluorescent Protein Expression, Purification, and Storage
To generate protein solution for fluorescence measurements, cells containing theprotein must be grown, the protein must be produced by the cells (expression), and theprotein must be extracted from the cells (purification). This section details the protocol to
produce fluorescent protein using E. coli cells.

B.4.1 Expression
A plasmid encoding the fluorescent protein was transformed into E. coli
(Top10) competent cells via the procedure described in section B.3. After the E.
coli have been transformed with the plasmid and grown on a plate overnight, asingle colony is selected with an inoculum loop. Colonies are only used from plates
that are less than three days old. The colony is added to a 50 mL conical tube with
12 mL of lysogeny broth (LB) media containing ampicillin to generate a starter
culture. This culture is incubated at 37 °C, 225 rpm overnight (12-14 hours).
While the culture grows overnight, 2XTY media is prepared and autoclaved.
2XTY is prepared by adding 3.1 g 2XTY to 100 mL nanopure water. The cap isloosened and the solution is autoclaved using the liquid program settings. Oncethe solution is cooled to 55 °C or lower, 100 µL ampicillin is added for an ampicillin
concentration of 0.1 mg/mL. Overnight culture is diluted in 2XTY at a 1:100 dilution.
If larger volumes of expression culture are needed the recipe can be scaled up.
1 mL of the overnight culture is added to 100 mL of the 2XTY solution in a
250 mL sterile Erlenmeyer flask and incubated for 2 hours at 37 °C, 225 rpm.

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Expression is then started by adding 1 mL of arabinose stock solution (20% w/v)
for an arabinose concentration of 0.2 %. The expression culture is incubatedovernight (6-8 hours) at 37 °C, 225 rpm. The incubator heat is turned to 28 °C, with
the incubator shaker still set to 225 rpm. After 24 hours at 28 °C, the culture isremoved from the incubator. The culture is centrifuged for 20 minutes at 6000 rpm
with a centrifuge pre-chilled to 4 °C. The supernatant is poured off and disposedof and the protein pellet is stored in the -30 °C freezer for later purification unlessthe culture will be used immediately.

B.4.2 Purification and Concentration
The first step of protein purification is cell lysis. The centrifuge is chilled to
4 °C while the lysis is prepared. The pellets are weighed, and the weight of anempty centrifuge tube is subtracted. 3 mL of bPER II (Bacterial Protein Extraction
Reagent) is added per 1 g of pellet is measured and combined with 10 µL of EDTAfree protease inhibitor per mL of resuspended pellet. The protease inhibitor isnecessary to prevent degradation of extracted proteins. The volume ofresuspended pellet can be calculated from volume of bPER (mL) added to pellet
weight (g). The mixture is added to the cell pellet and mixed to resuspend. The
resuspended cell mixture is added to a 50 mL conical tube and stirred for 30-45
minutes. A small aliquot (~300 mL) can be centrifuged to test whether lysis iscomplete. The pellet should be pale or white indicating the fluorescent protein has

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been released from the cells. The lysis can be left overnight in the 4 °C fridge toremove protein trapped in inclusion bodies.
Once lysis is complete, TNS buffer (50 mM Tris-HCl, 0.1 mM EDTA, 50 mM
NaCl, pH 8.0) is added at an equal volume of the cell mixture. The cold TNS aidsin the precipitation of the lysate. The lysis mixture is chilled in ice water for 10
minutes and then is spun down in the centrifuge at 11,000 rpm for 15 minutes. The
supernatant is carefully decanted into a clean conical tube and is re-spun for
11,000 rpm for 20 minutes. The supernatant is filter using a syringe with a 0.45 µm
polyethersulfone membrane filter, being careful not to expel the filter.
After the supernatant is filtered, it is added to a column of Ni-NTA resin thathas been pre-calibrated in TNS buffer, with 3-4 mL of resin used per protein pellet.
The resin binds to His-tagged proteins which can then be eluted with a highconcentration of Imidazole. The column is stirred or rocked over ice or in the 4 °C
fridge for 20 minutes or until a spun down aliquot shows little to no color in the
supernatant. To test if more resin is needed, it can be added to a small sample
and tested to see if more color is removed from the supernatant. The column isplaced vertically over a conical vial to gravity pack the column. The column isrinsed with approximately 10 column volumes of TNS buffer, followed by 10column volumes of TNS + 10mM imidazole, followed by 10 column volumes of
TNS + 20 mM imidazole. After these rinses the protein is eluted from the column
with TNS + 250 mM Imidazole into a clean tube. Collection stops once the color
has moved off the resin.

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The protein is concentrated with an Amicon Ultra-4 Centrifugal Devices
following the datasheet for these devices. The protein is concentrated in one ofthese Amicon spin columns for 30 kDa proteins for 15 minutes at 7500 rpm. The
process can be repeated until the desired concentration is reached. Buffer
exchange is done using a PD-10 column (GE Healthcare, 10 mL). The
manufacturer instructions are used for gravity buffer exchange with dialysis buffer(150 µM NaCl, 0.05 M Tris, pH 7.4 buffer) as the exchange buffer. The protein is
re-concentrated if needed and the concentration of the protein is measured using
the Nanodrop instrument and is frozen rapidly using liquid Nitrogen to avoid
denaturation. Protein is frozen in small aliquots to avoid repetitive freezing andthawing that may affect biological activity or damage the protein structure. Proteins
are stored for at most one week at 4 °C before being discarded and efforts are
made to not use sample past 24 hours after thawing.