INTERFEROMETRICSCATTERING MICROSCOPY

Invited Speakers

Stroboscopic quasiparticle tracking with an
iSCAT probe
Naomi S. Ginsberg
University of California, BerkeleyiSCAT has been employed very powerfully to identify and track small particles in awide range of contexts. We describe our recent work to instead track photoexcitedspecies in a wide range of semiconducting and composite materials—electrons andholes, bound electron-hole pairs, heat, and sound—at the nanometer scale. Because
these quasiparticles often travel nanometers over very short time scales, we follow
how these different forms of energy travel through materials stroboscopically. In otherwords, a confocal impulsive light pulse first generates a localized population of
excited species, and an iSCAT probe light pulse is used to interferometrically detect
how these species modify their index of refraction locally, generating differential
contrast. By progressively delaying the probe from the pump, we obtain the meansquared expansion of the initial distribution as a function of time. Doing so enables
measurement of quasiparticle diffusivity and, more importantly, identifies deviations
from bulk diffusive behavior due to material heterogeneity. This heterogeneity can becaused by material morphological features such as polycrystallinity/grain boundaries,
surface specific properties, or structural anisotropy. It can also be due to structuralvariations that affect the energy landscape of the quasiparticles more microscopically,leading to subdiffusion. I will show various recent examples of our approach,stroboSCAT, that illustrate the mapping energy flow in four dimensions of spacetime
with few-nanometer lateral precision and direct correlation to material morphology
via iSCAT on its own.

Watching RNA viruses self-assemble
Rees F. Garmann, Aaron M. Goldfain, Vinothan N.
Manoharan
Harvard John A. Paulson School of Engineering and Applied Sciences,
Harvard University, Cambridge MA 02138
Department of Physics, Harvard University, Cambridge MA 02138
vnm@seas.harvard.edu

Nearly 60 years ago, Caspar and Klug [1] coined the term "self-assembly" to describe theformation of a virus from its constituent parts (Cold Spring Harbor Symposia on Quantitative
Biology 1962). But we still don't understand how this process occurs even in the simplest
viruses, positive-sense RNA viruses. Such viruses consist of proteins that form a highlyordered protective shell (called a capsid) around the viral RNA. Viral particles can selfassemble spontaneously in a mixture of RNA and coat protein in a buffer, in the absence ofany host factors. The yield and fidelity of the assembly is particularly remarkable in viruses
with a triangulation number of 3 or higher, in which case some of the proteins must find their
way to 5-fold coordinated sites and others to 6-fold coordinated sites on the same shell. To
understand how such systems assemble, we use interferometric scattering microscopy [2] tomeasure the kinetics of individual assembling viral particles (MS2 bacteriophage) on timescales ranging from 1 ms to 1000 s. By comparing the scattered intensity to that of the wildtype virus, we infer the mass of the protein shell growing around the RNA as a function oftime [3]. We find that individual particles grow to nearly full size in a short time following amuch longer delay period. The distribution of delay times suggests that the assembly follows anucleation-and-growth pathway. I will discuss how such a pathway might allow the virus toassemble so robustly.
1. Caspar DLD, Klug A (1962) Physical Principles in the Construction of Regular Viruses.
Cold Spring Harbor Symposia on Quantitative Biology, 27:1–24.
https://doi.org/10.1101/SQB.1962.027.001.005
2. Ortega-Arroyo J, Kukura P (2012) Interferometric scattering microscopy (iSCAT): newfrontiers in ultrafast and ultrasensitive optical microscopy. Physical Chemistry Chemical
Physics, 14(45):15625–15636. https://doi.org/10.1039/C2CP41013C
3. Garmann RF, Goldfain AM, Manoharan VN (2019) Measurements of the self-assembly
kinetics of individual viral capsids around their RNA genome. Proceedings of the National
Academy of Sciences, 116(45):22485–22490. https://doi.org/10.1073/pnas.1909223116

Fluctuating dynamics of single disassembling
microtubules
Milan Vala, Łukasz Bujak, Kristýna Holanová, Antonio García Marín, Marek Piliarik
Institute of Photonics and Electronics of the Czech Academy of Sciences, Chaberská 1014/57, 182 51
Prague, Czech Republic

Dynamic instability of microtubules is a stochastic process that underpins a large variety of essential
intracellular motion, including the movements driving cell division, morphogenesis and cell motility. The
tubular shaft of the microtubule consists of laterally-connected chains of tubulin dimers (protofilaments).
Static electron micrographs suggest structural transition of straight protofilaments into curled ones
accompanied by breaking of the lateral tubulin bonds at the tip of a disassembling microtubule. However,the dynamics of the disassembly is not understood, and no method has been able to capture the underlying
structural transitions in real-time.
We combine two optical microscopy techniques to decipher the conformation changes of protein oligomers
preceding the microtubule disassembly. We explore fluctuation in the scattering anisotropy at the very tipof disassembling microtubules and associate its momentary changes with conformation switching at thelevel of single protofilaments. Additionally we track the detailed trajectory of single disassembling tubulins
from the microtubule shaft using scattering labels confirming the fluctuating character of disassembling
microtubules explaining the thermally driven mechanisms of the dynamic instability of microtubules.

Visualizing and understanding bacterial
extracellular structures using iSCATAlexandre Persat
École polytechnique fédérale de Lausanne, Persat Lab

The surface of bacteria is covered with structures that allows single cells to physically interact
with their environments. For example, single cells swim with flagella, and attach to and moveon surfaces with pili. Both flagella and pili are micrometer-long protein polymers only a few
nanometer-thick extending from the cell body. These structures have been traditionally studied
by means of genetic manipulation combined with macroscale motility assays, but little is
known about their dynamics and spatial localization. This is mainly due to the difficulty invisualizing these nanometer-wide structures. We solved this shortcoming by imaging pili andflagella in live cells at high spatial and temporal resolution by iSCAT. iSCAT offers an
unprecedented dynamic view of these extracellular structures, enabling detailed biophysicalcharacterization of their activity in a native environment. We leverage this microscopy
technique in studies of bacterial motility and mechanosensation that depend on the dynamics
of flagella and pili. In particular, we were able to demonstrate that the pathogen Pseudomonas
aeruginosa synchronizes pili extension and retraction with attachment of the tip with a surface.
In summary, iSCAT fills an important technological gap in the field of microbiology.

Dissecting molecular membrane organization
with combined fluorescence and scattering
microscopy
C. Eggeling1,2,3, C. Lagerholm3, F. Reina1,2,
1Friedrich‐Schiller‐University Jena, Institute of Applied Optics and Biophysics, 07743 Jena,
Germany Finland

2

Leibniz Institute of Photonic Technology e.V., Department Biophysical Imaging, 07745 Jena,
Germany

3

University of Oxford, MRC Human Immunology Unit and Wolfson Imaging Center, OX3 9DS
Oxford, United Kingdom
e-mail: christian.eggeling@uni-jena.de

Molecular interactions are key in cellular signaling. They are usually ruled by theorganization and mobility of the involved molecules. We present a combined
fluorescence and scattering microscopy study to determine such information andpotentially extract interaction dynamics. Specifically, the direct and non-invasive
observation of the interactions in the living cell is often impeded by principle limitationsof conventional far-field optical microscopes, for example with respect to limited spatiotemporal resolution. We depict how novel details of molecular membrane dynamics canbe obtained by using advanced microscopy approaches such as the combination ofsuper-resolution STED microscopy with fluorescence correlation spectroscopy (STEDFCS), spectral detection, and interferometric scattering (iSCAT). We highlight how
these approaches can reveal novel aspects of membrane bioactivity such as of theexistence and function of potential lipid rafts.

Coherent brightfield microscopy: iSCAT
microscopy in transmission
Chia-Lung Hsieh
Institute of Atomic and Molecular Sciences (IAMS), Academia Sinica, Taiwan

Coherent brightfield (COBRI) microscopy, the iSCAT microscopy in transmission,
measures the forward scattering signal by recording its interference with the transmitted
non-scattered light. The COBRI microscopy shares many common features with thereflective iSCAT microscopy, including the exquisite sensitivity, the phase retrieval for
3D localization, and the high-speed, long-term observation. In addition, thetransmission geometry of COBRI provides a unique advantage for measurements inlive cells by avoiding the complication of the fluctuating background signal originatedfrom the reflective interfaces, e.g., the cell membrane. In this talk, I shall start with theconcept of the COBRI microscopy and benchmark its performance. I will introduce thepupil function engineering that allows us to tune the imaging sensitivity of COBRI overa wide range. I will present a few examples of biophysical studies of high-speed 3D
tracking of single nano-sized particles in live cells, including the virus particles, cellvesicles, and gold nanoparticles. Finally, the perspective of label-free visualization ofmolecular dynamics in live cells will be discussed.

Interferometric Scattering Microscopy toCharacterize Nanometric Objects and Subcellular
Structures.
I.-B. Lee1†, J.-S. Park1†, H.-M. Moon1, K. Zambochova1,2, K.-H. Kim1,3, J.-H. Joo1,4,J.-S. Ryu5, S.-Y. Kong6, S.-C. Hong1,3*, M. Cho1,4*
1Center for Molecular Spectroscopy and Dynamics, Institute for Basic Science, Seoul, Korea
2Department of Natural Sciences, Faculty of Biomedical Engineering, Czech Technical University in Prague, Kladno, Czech

Republic.
3Department of Physics, Korea University, Seoul, Korea
4Department of Chemistry, Korea University, Seoul, Korea
5Center for Breast Cancer, National Cancer Center, Goyang, 10408, Korea
6Division of Translational Science, National Cancer Center, Goyang, 10408, Korea*email: hongsc@korea.ac.kr and mcho@korea.ac.kr; †: equal contribution

It is of great interest to visualize nano-scale objects in bioscience. Fluorescence microscopy, mostpowerful bioimaging tool to date, bears limitations in measurement time, time resolution, and sample
preparation. To circumvent such problems, a new scattering-based method named iSCAT(interferometric scattering microscopy) has been recently developed. Since this technique relies onscattering signal from a target nanoparticle and detects the interference of the signal with a constantreference, it is less prone to the aforementioned limitations. Here, we report our recent achievements:
First, utilizing polarized scattering from an anisotropic scatterer, we determine the orientation of gold
nanorods without sacrificing the bright-field image of the entire view-field, in contrast to dark-fieldimaging [1]. Second, we successfully track the position of nanoparticles along the axial dimension andimage the sample at different depths by adopting the remote-focusing (RF) technique. Our RF-iSCAT
approach would significantly expand the utility of iSCAT [2]. Lastly, we show our iSCAT imaging of
biological cells and multi-protein focal adhesion complexes. The iSCAT technique enables us tovisualize subcellular structures with remarkable spatial, temporal details and sharp contrast even
without labeling [3]. In summary, the iSCAT technique would be an indispensable tool in visualizing
the nanoscopic biological world.
This work was supported by IBS-R023-D1 and NRF-2019R1H1A2077487.
References[1] J.-S. Park, I.-B. Lee, H.-M. Moon, J.-H. Joo, K.-H. Kim, S.-C. Hong*, and M. Cho*, Chem. Sci.,
9, 2690 (2018). J.-S. Park, I.-B. Lee, H.-M. Moon, J.-S. Ryu, S.-Y. Kong, S.-C. Hong*, and M.
Cho*, submitted.
[2] I.-B. Lee, H.-M. Moon, J.-H. Joo, K.-H. Kim, S.-C. Hong*, and M. Cho*, ACS Photonics, 5, 797(2017).
[3] I.-B. Lee, H.-M. Moon, J.-S. Park, K. Zambochova, S.-C. Hong*, and M. Cho*, under revision.

Label-free deep optical imaging using a time-gatedreflection matrix
Wonshik Choi
Center for Molecular Spectroscopy and Dynamics, Institute for Basic Science (IBS), Seoul
02841, South Korea
Department of Physics, Korea University, Seoul 02841, South Korea
wonshik@korea.ac.kr
Optical microscopy suffers from the loss of resolving power when imaging target objects
embedded deep within complex scattering media. With the increase of target depth, the signal
wave is attenuated exponentially due to multiple light scattering, and strong multiple scattering
noise obscures the signal wave. In this talk, I will present imaging methods based on the recordingof a time-gated reflection matrix, which goes beyond the conventional confocal detection scheme.
In essence, we record the amplitude and phase of the backscattered waves not only at the confocal
position but also at other non-confocal positions.
In the image reconstruction process, we made use of the non-confocal signals to enhance theimaging depth beyond the conventional limit. Specifically, we developed a unique algorithm
termed ‘closed-loop accumulation of single scattering (CLASS)’ that makes the preferable choiceof the non-confocal signals containing the object information and coherently add them to theconfocal signals. We could correct the sample-induced aberrations in illumination and imaging
paths separately without the need for guide stars and even in the presence of multiple lightscattering. We performed in vivo and label-free volumetric imaging underneath an intact mouseskull and visualized myelinated axons with the ideal diffraction-limited spatial resolution.

Mind the gap
Kheya Sengupta
Aix-Marseille University, CINaM, CNRS, Campus Luminy, Marseille, France.
sengupta@cinam.univ-mrs.fr

The boundary between a cell and its environment or another cell is important - chemical talk goeson across this boundary, and forces are exerted. But the gap between cell boundaries is difficult toimage, especially because it is dynamic. The cell membranes that line this gap are known to ‘jiggle’and ‘twitch’, that is to say fluctuate, with at least some energy consumption. The twitching impacts
the process of formation of bonds between the two surfaces and across the gap. In fact, this process
is already complicated due to the fact that reaction across two 2D surfaces involving
mechanosensitive biological bonds may not follow familiar laws of physical-chemistry such as thelaw of mass action. However, adhesion mediated by such bond formation is a very important
process in biology – all multicellular organisms for example, depend on membrane-to-membraneadhesion via bio-chemical bonds for structural integrity. While the physics of a free, soft, thermallyfluctuating membrane is well known, combining this with chemistry in two dimensions ischallenging. Another challenge is measuring and describing the fluctuations especially when they
are cell-driven and active. I will present new techniques to measure membrane shape fluctuations[1] and show that these may be a means for living cells to modulate the interaction with theirenvironment [2].
[1] C. Monzel, D. Schmidt, C. Kleusch, D. Kirchenbuechler, U. Seifert, A.-S. Smith, K. Sengupta
and R. Merkel. Nature communications. 6, 8162 (2015).
[2] Susanne Fenz, Timo Bihr, Daniel Schmidt, Rudolf Merkel, Udo Seifert, Kheya Sengupta, andAna-Suncana Smith. Nature Physics, 13, 906–913 (2017).

Four-wave mixing interferometry: A new tool for
imaging and tracking single plasmonic nanoparticles
in 3D background-free
Paola Borri
School of Biosciences, School of Physics and Astronomy, Cardiff University

Imaging and tracking single nanoparticles using optical microscopy are powerful techniqueswith many applications in biology, chemistry, and material sciences. Despite significant
advances, imaging single small nanoparticles in a scattering environment as well as localizing
objects with nanometric position precision in 3D remains challenging.
Here, I will present a four-wave mixing interferometry technique recently developed by us,
whereby single gold nanoparticles in the 10-40nm diameter range are imaged background-free
inside highly heterogeneous biological environments, owing to their specific nonlinear
plasmonic response. The set-up enables correlative four-wave mixing/confocal fluorescenceimaging, opening the prospect to study the fate of nanoparticle-biomolecule-fluorophore
conjugates and their integrity inside cells [1].
Beyond imaging, the technique enables to track single particles with nanometric position
localisation precision in 3D from rapid single-point measurements at 1 ms acquisition time, by
exploiting the optical vortex field pattern in the focal plane of a high numerical aperture
objective lens [2]. The localisation precision in plane is found to be about 15nm, consistent withthe photon shot-noise, while axially it is limited to about 3nm by the nano-positioning sample
stage, with an estimated photon shot-noise limit of below 1 nm. Through phase-sensitive
polarisation-resolved detection, the technique can measure particle asymmetries of only 0.5%
ellipticity, corresponding to a single atomic layer of gold, as well as particle orientations. Thismethod opens new ways of unravelling single-particle trafficking within complex 3Darchitectures.
[1] Giannakopoulou et al., Nanoscale 12, 4622 (2020). DOI: 10.1039/c9nr08512b[2] Zoriniants et al., Phys. Rev. X 7, 041022 (2017). DOI: 10.1103/PhysRevX.7.041022

Rotating coherent scattering (ROCS) microscopy –100 Hz live cell imaging at 150nm resolution.
Alexander RohrbachCentre for Biological Signalling Studies, University of Freiburg

Thermal and active cellular forces drive biological structures. These movements become thefaster the smaller the biological structures because of less friction and steric hindrance. This is
an important aspect for superresolution microscopy, since unfortunately the resolution ofsmaller structures requires more photons and time. Therefore, novel concepts enabling smart
trade-offs between temporal and spatial resolution have to be developed.
Here, we present a variant of an oblique illumination super-resolution microscopy based on
rotating coherent scattering (ROCS). The technique generates thousands of high contrast
images without post-processing at frame rates of more than 100 Hertz and without labeling.
The sample is scanned over all azimuthal illumination angles within a single camera exposure
time and allows for variant illumination and detection modes such as bright-field, dark-field or
total internal reflection (TIR). Thus, structures as small as 150 nm become separable throughlocal destructive interferences. Within one rotation, multiple speckles do not disappear, butchange to the object itself thereby improving contrast and resolution. ROCS is applied todifferent cells revealing unexpected dynamic biological processes.

Computational Interferometric Microscopy for High
Resolution Visualization of Biological Nanoparticles
M. Selim Ünlü*, C. Yurdakul, A. Matlock, and L. Tian
Boston University, Department of Electrical and Computer Engineering, Boston, MA, USA

Biological nanoparticles such as viruses and exosomes are important biomarkers for a range of
medical conditions, from infectious disease to cancer. Biological sensors that detect whole viruses
and exosomes with high specificity, yet without chemical labeling, are promising because theygenerally reduce the amount and complexity of sample preparation and may improve measurement
quality by retaining information about nanoscale biological structure. However, label-free opticalbiological nanoparticle imaging often experiences two specific challenges: (i) the weak contrast
due to low refractive index difference and exceptionally small size and (ii) the inability to resolve
nanoscale features. Advances in interferometric scattering microscopy have successfully
overcome weak signal limitations and enabled direct detection of low-index biological
nanoparticles. We have demonstrated label-free identification and visualization of various virusesin multiplexed format in complex samples in a disposable cartridge and detection of exosomes.
However, interferometric scattering microscopy falls short of providing sufficient spatial
resolution to study the morphology of individual nanoparticles, especially in a high-throughput
manner. Recently, we combined wide-field interferometric microscopy with computational
imaging to demonstrate a two-fold resolution improvement. With computational imaging
combingin multiple images acquired under asymmetric illumination, we clearly resolve ~150 nm
weakly-scattering features in visible light (420 nm) over a wide field-of-view of >100m, enabling
sub-wavelength imaging of more than 104 nanoparticles at once. Therefore, high-throughput andhigh-resolution imaging and characterization of a broad size range of biological nanoparticles will
be possible without any labelling.

Phase imaging with computational specificity (PICS)
Gabriel Popescu
Quantitative Light Imaging Laboratory, Department of Electrical and Computer Engineering,
Beckman Institute of Advanced Science and Technology, University of Illinois at UrbanaChampaign, Urbana, IL 61801, USA
gpopescu@illinois.edu

Quantitative phase imaging, has gained significant interest, especially in the past
decade, because of its ability to study unlabeled cells and tissues. As a result, QPI can extract
structure and dynamics information from live cells without photodamage or photobleaching.
However, in the absence of labels, QPI cannot identify easily particular structures in the cell,i.e., it lacks specificity. This represents the major limitation of QPI when applied tobiomedicine.
Recently, deep learning techniques have been translating from consumer to scientificapplications. For example, it has been shown that AI can map one form of contrast into another.
Significantly, it has been demonstrated that the neural network can learn from label-free (brightfield, phase contrast, DIC) and ground-truth fluorescence images to predict where specificfluorophores would bind in an unlabeled specimen.
Inspired by this prior work, we applied deep learning to QPI data, generated by SLIM
and GLIM. These methods are white-light and common-path and, thus, provide high spatial
and temporal sensitivity. Because they are add-on to existing microscopes and compatible withthe fluorescence channels, these methods provide simultaneous phase and fluorescence fromthe same field of view. As a result, the training data necessary for deep learning is generated
automatically.
We present a new microscopy concept, where the process of retrieving computational
specificity is part of the acquisition software, performed in real-time. We demonstrate this ideawith various fluorescence tags and operation on live cells as well as tissue pathology. This new
type of microscopy can potentially replace some commonly used tags and stains and eliminate
the inconveniences associated with phototoxicity and photobleaching. Phase imaging withcomputational specificity (PICS) has an enormous potential for biomedicine.
References (complete list at: http://light.ece.illinois.edu/index.html/publications/articles)
1.

2.
3.
4.

Kandel, M. E., C. Hu, G. Naseri Kouzehgarani, E. Min, K. M. Sullivan, H. Kong, J. M. Li, D. N. Robson,
M. U. Gillette, C. Best-Popescu and G. Popescu (2019). "Epi-illumination gradient light interference
microscopy for imaging opaque structures." Nature Communications 10(1): 4691.
Park, Y., C. Depeursinge and G. Popescu (2018). "Quantitative phase imaging in biomedicine." Nature
Photonics 12(10): 578.
Nguyen, T. H., M. E. Kandel, M. Rubessa, M. B. Wheeler and G. Popescu (2017). "Gradient lightinterference microscopy for 3D imaging of unlabeled specimens." Nat Commun 8(1): 210.
Kim, T., R. J. Zhou, M. Mir, S. D. Babacan, P. S. Carney, L. L. Goddard and G. Popescu (2014). "Whitelight diffraction tomography of unlabeled live cells." Nature Photonics 8(3): 256-263.

5. Popescu, G. (2011). Quantitative phase imaging of cells and tissues. New York, McGraw-Hill.

Sparsity-based techniques for solving the inverse
scattering problem
Michael Unser
École polytechnique fédérale de Lausanne, Biomedical Imaging Group

Inverse-scattering is a challenging problem encountered in many fields such as coherent
microscopy. While classical reconstruction methods were relying on linear approximation of
the forward model, recent works have shown the benefit of combining advances physics(nonlinear models) and sparsity. In this talk, we present a reconstruction algorithm that deploys
the nonlinear Lippmann-Schwinger model together with sparsity-based regularizations. Then,
we show the ability of the method to provide high-quality reconstructions for difficult
configurations in coherent light microscopy (high contrasts, few illuminations).

INTERFEROMETRICSCATTERING MICROSCOPY

Contributed Talks
May 27th

REVEALING THE DYNAMICS OF CYTOSKELETAL NON-MOTOR PROTEINSUSING INTERFEROMETRIC SCATTERING MICROSCOPY
Ł. Bujak*, K. Holanová, A. García Marín, M. Piliarik
Institute of Photonics and Electronics of the CAS, Chaberská 1014/57,
182 51 Prague, Czech Republic
E-mail: bujak@ufe.cz
KEYWORDS: iSCAT, interferometric scattering microscopy, single-particle tracking,single-molecule detection, high-fidelity tracking
Advances in fluorescence-based super-resolution microscopy and nanoscopic tracking
techniques helped to reach a spatial resolution below 10 nm with temporal resolution limited
to milliseconds timescales [1]. To push the spatiotemporal resolution of fluorescent-based
techniques further, we are employing interferometric scattering microscopy (iSCAT). The
sensitivity of iSCAT was previously proven in detection experiments of small scattering
nanoparticles as well as unlabeled single proteins with sub-nanometer spatial and submillisecond time resolution [2, 3].
Here we use a high frame rate of iSCAT microscopy in combination with a novel labelingmethod to track the motion of a single labeled anaphase spindle elongation protein 1 (ASE1)
on a microtubule. We achieved a spatial
resolution better than 2 nm in all three
dimensions, and a temporal resolution of
22 µs. With such spatiotemporal
resolution, we discover that the ASE1
movement is not, as often believed,
directedalongamicrotubule
protofilament and features a morestochastic pattern on the microtubule
lattice (Fig 1). We distinguish different
diffusion characteristics of the axial
Figure 1 A three-dimensional trajectory of the labeled ASE1 dimer motion and the perpendicular motion of
reveals microtubule shape and tubulins position. Time is color-coded
ASE1. We reveal clear statisticalfrom yellow to blue.
signatures of confined motion and areable to detect sub-millisecond confinements in the trajectories, having the periodicity
corresponding to the size of a single tubulin dimer. Our results of high-fidelity ultrafast tracking
shade new light not only on the unknown choreography and mechanisms of diffusive motionof microtubule-associated proteins but also on the interaction of the ASE1 protein with themicrotubule lattice. We were able to reconstruct the energetic landscape of microtubule.
[1] J. Schnitzbauer, M. T. Strauss, T. Schlichthaerle, F. Schueder, and R. Jungmann, “Superresolution microscopy with DNA-PAINT,” Nat. Protoc., 12(6), 1198–1228 (2017).
[2] M. Piliarik and V. Sandoghdar, “Direct optical sensing of single unlabelled proteins andsuper-resolution imaging of their binding sites,” Nat. Commun., 5, 4495 (2014).
[3] S. Spindler et al., “Visualization of lipids and proteins at high spatial and temporal
resolution via interferometric scattering (iSCAT) microscopy,” J. Phys. Appl. Phys., 49(27),274002 (2016).

Ultrasensitive protein detection
Mahyar Dahmardeh1,2, Reza Gholami Mahmoodabadi1,2, Houman Mirzaalian
Dastjerdi1,2,3 and Vahid Sandoghdar1,2,4
1

Max Planck Institute for the Science of Light, 91058 Erlangen, Germany.

2

Max-Planck-Zentrum für Physik und Medizin, 91058 Erlangen, Germany.

3

Department of Computer Science, Friedrich Alexander University Erlangen-Nuremberg, 91058 Erlangen,Germany.

4

Department of Physics, Friedrich Alexander University Erlangen-Nuremberg, 91058 Erlangen, Germany.
mahyar.dahmardeh@mpl.mpg.deProteins are the physical and biochemical building blocks of the cell and are fundamental to biological
function. Thus, the field of proteomics has emerged over the course of last decades with the aim ofsystematically identifying the entire set of proteins within a given tissue or cell via established methods
such as cryo-EM, and mass spectrometry. In addition, several optical sensing techniques, e.g. using
plasmon resonances or microresonators have been explored.
Here, we present the progress in the use of interferometric detection of scattered light (iSCAT) for
sensing at the single protein level [1]. The iSCAT signal arises from the interference between theprotein’s weak scattered field and a reference field [2]. The amplitude of this interference is determinedby the magnitude of the scattered field and is thus linearly proportional to a protein’s polarizability andmass. In this work we report on measurements on protein samples, spanning the record range of 21-250
kDa [3].

References[1] Piliarik, M., Sandoghdar, V. Direct Optical Sensing of Single Unlabelled Proteins and SuperResolution Imaging of Their Binding Sites. Nat. Commun. 5, 4495 (2014).
[2] Lindfors, K, Kalkbrenner, T. Stoller, P. Sandoghdar, V. Phys. Rev. Lett. 93, 037401 (2004).
[3] Dahmardeh, et al, in preparation.

Electric-Double-Layer-Modulation Microscopy
Sanli Faez,1* Zhu Zhang,1 Kevin Namink1, Xuanhui Meng,2 Philipp Kukura2
1

Nanophotonics, Debye Institute for Nanomaterials Research, Utrecht University, NL
Department of Chemistry, Physical and Theoretical Chemistry Laboratory, Oxford University, UK*s.faez@uu.nl

2

The electric double layer (EDL) formed aroundcharged nanostructures at the liquid-solid interface
determines their electrochemical activity andinfluences their electrical and optical polarizability.
We experimentally demonstrate that restructuring of
the EDL at the nanoscale can be detected by darkfield scattering microscopy. We refer to thisintensity change in the elastic light scattering as thepotentiodynamic optical contrast (PDOC). The
temporal response of the PDOC is influenced mostly
by the physical adsorption of counter-ions with an optical polarizability that is different from the neutral
mixture. We demonstrate this effect by quantifying the temporal relaxation of the PDOC, which is
directly related to the charging time of the EDL. Temporal and spatial characterization of the scattering
signal demonstrates that the PDOC is proportional to the accumulated charge of polarizable ions at theinterface and its time derivative represents the nanoscale ionic current. The material-specificity of the
EDL formation is used in our work as a label-free contrast mechanism to image nanostructures andperform spatially resolved cyclic voltammetry at ion current levels of a few attoamperes, corresponding
to the exchange of only a few hundred ions. This imaging technique provides important additional
information such as spatial resolution, sensitivity to surface heterogeneity, local ion accumulation, andthe possibility of studying deposits, possibly down to single biomolecules.

Publication: Kevin Namink, Xuanhui Meng, Marc T. M. Koper, Philipp Kukura, and Sanli Faez, Phys.
Rev. Applied 13, 044065 (2020) https://doi.org/10.1103/PhysRevApplied.13.044065

Label-free single protein detection with iSCAT:
From purified samples to living cells
Andre Gemeinhardt1, Katharina König1 & Vahid Sandoghdar1,2
1

Max Planck Institute for the Science of Light, Staudtstr. 2, 91058 Erlangen, Germany
Department of Physics, Friedrich Alexander University Erlangen-Nuremberg, Staudstr. 5, 91058 Erlangen,Germany
Corresponding author: andre.gemeinhardt@mpl.mpg.de2

The functionality of the human body is maintained via a complex interplay of various different cell types
that constantly communicate with each other by a range of different signaling molecules. As an example,
the immune response is regulated via proteins like antibodies or cytokines that are secreted by one cell andrecognized by another, which reacts to the signal. In order to understand and potentially treat possible
malfunctions, it is necessary to study the key elements of those processes in more detail, like the secretionof signaling molecules by a cell.
Conventional methods to study cellular secretion mostly rely on the use of large cell ensembles(immunoassays) although new single-cell studies have been reported. Furthermore, established methods
rely on the ensemble analysis of the secreted analytes, often via fluorescence labeling. iSCAT was shown
to be able to detect single small proteins in a completely label-free fashion at high spatio-temporal resolution[1, 2]. Applied to cell secretion, it can reveal important insights into the dynamics of this process at thesingle cell level and in real-time.
In a first application, we demonstrate the suitability of iSCAT to study cell secretion on single LAZ-388
cells, an Epstein-Barr virus-transformed B cell line [3,4]. We find that LAZ-388 cells secrete proteins in awide molecular weight range from 100 to 1100 kDa. By introducing functionalized cover glasses tospecifically bind IgG antibodies, we could identify IgG as one significant part of a LAZ-388 secretome.
We show the capabilities of iSCAT to analyze dynamical changes in the secretional behavior of LAZ-388
cells in real time by introducing stress via the continuous rise of the buffer pH value which causes a distinct
change in the secretion rate. This makes iSCAT a valuable tool for the detailed study of cellular response
to external stimuli, as posed by organisms like antigen-presenting cells or viruses.

References[1] M. Piliarik & V. Sandoghdar, “Direct optical sensing of single unlabelled proteins and super-resolution imaging of their binding sites” Nat.
Comm. 5, 4495 (2014).
[2] Young et al., “Quantitative mass imaging of single biological macromolecules” Science 360, 423-427 (2018)[3] M. P. McDonald, A. Gemeinhardt, K. König, M. Piliarik, S. Schaffer, S. Völkl, M. Aigner, A. Mackensen, and V. Sandoghdar, "Visualizing
Single-Cell Secretion Dynamics with Single-Protein Sensitivity," Nano Lett. 18, (2018).
[4] A. Gemeinhardt, M. P. McDonald, K. König, M. Aigner, A. Mackensen, and V. Sandoghdar, "Label-Free Imaging of Single Proteins Secreted
from Living Cells via iSCAT Microscopy," J. Vis. Exp. (2018).

Implementation of an i-SCAT setup
for imaging dynamic processes in perovskite nanostructures
C. Gruber, A. Mancini, C. Lampe, L. de S. Menezes, S. A. Maier, Y. Li, A. S. Urban,
E. Cortés
Lehrstuhl für Hybride Nanosysteme, Nanoinstitut München, Fakultät für Physik, Ludwig-Maximilians-Universität
München, 80539 München, Germany
ch.gruber@physik.uni-muenchen.de; emiliano.cortes@physik.uni-muenchen.de

Metal halide perovskite nanocrystals have emerged as promising materials for highly efficient optoelectronic devices such as solar cells or LEDs due to their unique opto-electronic properties. To date, thelow stability of these nanophotonic systems hinders them from industrial applications as they are prone todegrade under influence of environmental factors such as moisture, heat and light. Neither their growth nor
their destabilization processes are well understood, limiting the targeted synthesis of stable and application
tailored nanostructures.[1] The advent of interferometric scattering microscopy (i-SCAT) makes in-situ
imaging of these nanoscopic processes accessible.[2]In our group, an i-SCAT microscope (reflection mode; wide-field detection) was implemented following
protocols published by the groups of Kukura[3] and Sandoghdar[4] and first i-SCAT images of an ordinary
cover glass have been taken (Fig. 1a). After conducting several tests to calibrate the system, the smoothnessof the images was improved considerably, as shown in Fig. 1b. The corresponding statistics showed adecrease in standard deviation to 2.210-4 (140 ms effective exposure time; 10.5 ke- full well depth; 55binning; 333 magnification).
Subsequently, first images of polymer micelle protected 27 nm MAPbI3 perovskite nanocrystals[5] intoluene were obtained (Fig. 1c). Here, toluene harbors different challenges for i-SCAT imaging due to arefractive index that nearly matches the index of glass. Following these preliminary experiments, thedynamical assembly and disassembly processes of several perovskite nanostructures including nanocubes
will be investigated.

Figure 1. a) Background before system calibration (effective exposure time = 140 ms). b) Background after systemcalibration (effective exposure time = 140 ms). c) Preliminary results of MAPbI3 NCs in toluene.
References
[1]
[2]
[3]
[4]
[5]

J.-K. Sun et al., J. Am. Chem. Soc. 2018, 140, 11705.
a) M. Delor et al. Nat. Mater 2020, 19, 56; b) P. K. Nayak et al., Nat. Commun. 2016, 7, 13303.
J. Ortega Arroyo et al. Nat. Protoc. 2016, 11, 617.
A. Gemeinhardt et al. JoVE 2018, e58486.
V. A. Hintermayr et al., Nano Lett. 2019, 19, 4928.

Multicolor high-speed tracking of single biomoleculeswith silver, gold, and silver-gold alloy nanoparticles
Ryota Iino
Institute for Molecular Science, National Institutes of Natural Sciences, Japaniino@ims.ac.jp

Gold nanoparticles have been used as imaging probes to track the motions of single
biomolecules. To investigate behaviors of various biomolecules simultaneously, increase of thecolor palette is necessary. Here we developed a multicolor high-speed single-particle tracking
system using silver, gold, and silver-gold alloy (5:5 composition ratio) nanoparticles. The peakwavelengths of the plasmon resonances for 30 nm silver, 30 nm silver-gold alloy, and 40 nm
gold nanoparticles were around 410, 460, and 530 nm, respectively, and we constructed
multicolor total internal reflection dark-field microscope with multiple lasers at 404 nm forsilver, 473 nm for silver-gold alloy, and 561 nm for gold nanoparticles. By the use of aspectrophotometer in the detection optics, scattering images at each wavelength were projected
onto different portions of a single two-dimensional detector. High-contrast images of silver,
silver-gold alloy, and gold nanoparticles were simultaneously obtained in different color
channels. After correction of positional shifts among different color channels by affine
transformation, a maximum shift less than 17 nm was achieved. Furthermore, an additional 649
nm laser enabled the detection of plasmon coupling by transient dimer formation of two
nanoparticles. With this system, diffusional motions of phospholipids in a supported membraneand stepping motions of kinesins along microtubules were successfully observed with alocalization precision of 2 nm and a time resolution of 100 μs at each channel.

Reference
Ando J, Nakamura A, Yamamoto M, Song C, Murata K, Iino R, Multicolor high-speed trackingof single biomolecules with silver, gold, silver-gold alloy nanoparticles, ACS Photonics 6,
2870-2883 (2019) DOI: 10.1021/acsphotonics.9b00953

Multi-pass microscopy for signal to noise
enhancement in interferometric scattering
Prof. Dr. Thomas Juffmann
University of Vienna, Austria

thomas.juffmann@univie.ac.at

Interferometric scattering techniques for the detection of unlabeled nanometric particles operate at the
shot-noise limit. Increasing signal to noise is achieved by averaging more and more frames of arecorded data set. This sets high demands on the stability of the setup, or on the camera well depth
and photostability of the system of interest.
Here I will discuss multi-pass microscopy 1,2 as a means to increase the signal-to-noise ratio atconstant photon budget. I will show the design of a multi-pass iScat system, and will present first data
demonstrating the detection of gold nanoparticles.
I will also present a proof of concept experiment3 that demonstrates how one could in principle deal
with background inhomogeneity using wavefront shaping. Technical limitations will be discussed.

1.

Juffmann, T., Klopfer, B. B., Frankort, T. L. I. I., Haslinger, P. & Kasevich, M. A. Multi-pass
microscopy. Nat. Commun. 7, 12858 (2016).

2.

Nimmrichter, S., Chen, C.-F., Klopfer, B. B., Kasevich, M. A. & Juffmann, T. Full-field cavity
enhanced microscopy techniques. J. Phys. Photonics 1, 015007 (2019).

3.

Juffmann, T., de los Ríos Sommer, A. & Gigan, S. Local Optimization of Wave-fronts for
optimal sensitivity PHase Imaging (LowPhi). Opt. Commun. 454, 124484 (2020).

Tracking lipid diffusion on GUVs with gold
nanoparticles of different size
Martin Kaller, Anna Kashkanova, Susann Spindler, Vahid Sandoghdar
Max Planck Institute for the Science of Light, Erlangen, Germany
martin.kaller@mpl.mpg.de

Single-particle tracking is an established method that can reveal essential information about cellular
structures [1] or the organisation of protein recruitment. Generally, fluorescent molecules are used aslabels; however, then the tracking procedure suffers from photophysical effects. To circumvent
photophysical effects one can use non-fluorescent labels, such as gold nanoparticles (GNPs). Here weuse interferometric scattering microscopy (iSCAT) to track lipids labelled with GNPs on giant
unilamellar vesicles (GUV) [2], and show how the size of the GNP influences the measured diffusion.
[1] Taylor et al. Nature Photonics 13, no. 7 (July 2019): 480–87.
[2] Stein et al. Frontiers in Physiology 8 (2017).

Lipid Bilayer Thickness Measured by Quantitative DIC
Reveals Phase Transitions and Effects of Substrate
Hydrophilicity
David Regan1, Joseph Williams2, Francesco Masia1,2, Paola Borri2, Wolfgang Langbein1
1

School of Physics and Astronomy, Cardiff University, The Parade, Cardiff CF24 3AA, U.K.
2
School of Biosciences, Cardiff University, Museum Avenue, Cardiff CF10 3AX, U.K.
langbeinww@cardiff.ac.uk

Supported lipid bilayers are one of the most prolific model systems used to study the biophysicalproperties of phospholipid membranes [1]. They are commonly imaged using fluorescence microscopy,
which suffers from limitations including fluorophore saturation and photobleaching, and artefacts from
insertion of the label. Alternatively, quantitative differential interference contrast microscopy (qDIC)
is a phase technique that allows label free imaging of lipid bilayers, as well as the extraction ofquantitative information about the layer thickness with high sensitivity [2]. Combining Wiener
deconvolution reconstruction [2] with an energy minimization scheme [3], adapted to layered
structures, we retrieve bilayer images with 0.1nm height resolution and low image reconstruction
artefacts.
We investigate [4] the influence of the substrate on the thickness of fluid-phase 1,2-dioleoyl-snglycero3-phosphocholine (DOPC)-supported lipid bilayers (SLBs) and find a thinning of up to 10%, depending
on substrate hydrophilicity, local bilayer coverage, and ionic strength of the medium. With fluorescently
labeled lipid bilayers, we also observe changes in the bilayer thickness depending on the choice offluorophore. Furthermore, liquid-ordered (Lo) domains in bilayers, formed from DOPC, cholesterol,and sphingomyelin, are measured, and the corresponding thickness change between the liquid-ordered
and liquid-disordered phases is accurately determined (see figure). Again, the thickness difference isfound to be dependent on the presence of the fluorophore label, highlighting the need for quantitative
label-free techniques.
[1] Langmuir 22, 3497 (2006).
[2] Biophysical Journal 105, 1414 (2013)[3] Scientific Reports 6, 30420 (2016)[4] Langmuir 35, 13805 (2019)

DOPC/SM/Chol/H-DOPE SLB with Lo domains excluding the fluorescent labels, shown in (a)background subtracted fluorescence (m =0 pe, M = 109 pe), (b) qDIC contrast (m = -0.02, M =+0.02), (c) qDIC phase (m = 1.6 mrad, M = 34.5 mrad), and (d) a composite image showing thephase (red) and fluorescence (green).

Direct optical visualisation of single-particle phasetransformations in lithium ion battery materials
Alice Merryweather1,2, Christoph Schnedermann1, Quentin Jacquet2, Clare Grey2,
Akshay Rao1
1

Department of Physics, Cavendish Laboratory, University of Cambridge, JJ Thomson Avenue, Cambridge, UK.
2
Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, UK.
ajm314@cam.ac.uk

Charging and discharging rates in lithium ion batteries are limited by ion transport into and throughelectrode materials. With a rising demand for high-rate batteries, a detailed understanding of how ion
transport kinetics are aﬀected by material morphology at the nanoscale is essential. Here, we introduce
interferometric scattering microscopy (iSCAT) to the ﬁeld of battery characterisation. iSCAT is an
optical, non-invasive and label-free imaging technique which sensitively measures the polarisability of
individual active particles in an electrode, with the potential for nanometre localisation precision. We
benchmark our technique with micron-sized particles of LiCoO2, a well-studied cathode material.
Operando experiments demonstrate that the iSCAT intensity for a single particle changes dramaticallybut reversibly during (de)lithiation, with substantial variations between individual particles.
Additionally, the solid solution regime, biphasic regime and lithium ordering transition are clearly
distinguished during charging and discharging, allowing us to extract the phase propagation velocities.
During delithiation, we observe shrinking-core behaviour in the biphasic regime, whereas uponlithiation, phase fronts propagate from one nucleation point. This indicates that the rate-limiting process
in each case is ion diffusion and charge transfer respectively. Our results demonstrate that iSCAT canprovide fundamental insights into the ion transport dynamics of battery materials, highlighting the needto transition towards a nanoscopic understanding of battery materials.
References:
1. J. Ortega Arroyo et al., Interferometric scattering microscopy and its combination withsingle-molecule fluorescence imaging, Nature Protocols, 2016, 11, 617-633.
2. J. N. Reimers and J. R. Dahn, Electrochemical and In Situ X‐Ray Diffraction Studies ofLithium Intercalation in LixCoO2, Journal of The Electrochemical Society, 1992, 139, 20912097.
3. Neel Nadkarni et al., Modelling the Metal–Insulator Phase Transition in LixCoO2 for Energy
and Information Storage, Advanced Functional Materials, 2019, 29, 1902821

iPSF: The point spread function
in iSCAT Microscopy
Reza Gholami Mahmoodabadi,1,2 Richard W. Taylor,1,2
Martin Kaller,1,2 , Susann Spindler,1,2 Vahid Sandoghdar1,2
1
2

Max Planck Institute for the Science of Light, Erlangen, Germany
Max-Planck-Zentrum für Physik und Medizin, Erlangen, Germany
Email: reza.gholami@mpl.mpg.deInterferometric scattering (iSCAT) microscopy is a powerful label-free technique well suited for the sensitive detection and tracking of nanoscale mattersuch as individual viruses, colloidal nanoparticles, proteins and single molecules[1]. Recent efforts to track the nanoscale mobility of nanoparticles in challenging
speckle environments and over extended axial ranges [2] has necessitated a quantitative description of the point-spread function in iSCAT (iPSF). We present
a robust vectorial diffraction model for the iPSF in tandem with experimental
measurement and rigorous FDTD simulations and how it differs from conventional intensity-based PSFs in the diffraction-limited focal region [3]. We examine the iPSF under various imaging scenarios to understand how aberrations
due to the experimental configuration render its complex ring patterns which
encode information about the nanoparticle. We show that the lateral featureof the iPSF can be used to achieve precise and nanometric three-dimensional
localization either by means of a fit to the model or calibration-free unsupervised machine learning to break the previous limitation of 100 nm unambiguous
height determination. Furthermore, we demonstrate three-dimensional localization over an extended axial range on the order of 10 µm and envisage PSF
engineering could be employed to push the range further, permitting deep tissue
imaging. We discuss the origin of the remarkably long range and high resolutionof iSCAT particle tracking in three dimensions [3].

References[1] R W Taylor and V Sandoghdar. Interferometric scattering microscopy: seeing single nanoparticles and molecules via rayleigh scattering. Nano Letters,
2019.
[2] R W Taylor, R Gholami M, V Rauschenberger, A Giessl, A Schambony,and V Sandoghdar. Interferometric scattering microscopy reveals microsecond nanoscopic protein motion on a live cell membrane. Nature Photonics,
13(7):480–487, 2019.
[3] R Gholami M, R W Taylor, M Kaller, S Spindler, and V Sandoghdar. The
point spread function in interferometric scattering microscopy (iscat). To be
submitted, 2020.

INTERFEROMETRICSCATTERING MICROSCOPY

Contributed Talks
May 28thPiSCAT: A Python package
for iSCAT image analysis
Houman Mirzaalian Dastjerdi,1,3 Reza Gholami
Mahmoodabadi,1,2 Harald Köstler,3 Vahid Sandoghdar1,2,4
1

Max Planck Institute for the Science of Light, Erlangen, Germany
Max-Planck-Zentrum für Physik und Medizin, Erlangen, Germany
3
Chair for System Simulation, Friedrich-Alexander-Universitt Erlangen-Nrnberg(FAU), Erlangen, Germany
4
Department of Physics, FAU, Erlangen, Germany.
Email: houman.mirzaalian-dastjerdi@mpl.mpg.de2

Detection and tracking of nanoscale objects in interferometric scattering(iSCAT) microscopy [1, 2] offers unique opportunities for research in many
areas such as physics, biophysics, chemistry and material science. Some of
the advantages of iSCAT are the ability to record very long trajectories, very
high three-dimensional spatial and temporal resolution, and a remarkablesensitivity for detecting small nano-objects. While the construction of aniSCAT microscope is fairly simple, the analysis of the data can becomecomplex. Thus far, researchers have used a combination of several libraries
and packages in home-made data acquisition and analysis programs. In
this work we present “PiSCAT”, as a unified package based on open-source
python libraries for efficient and advanced processing of iSCAT images andvideos. The package reads different image formats and offers tools to perform
state-of-the-art analysis in various applications such as unlabeled protein
detection or 3D tracking of nanoparticles [2]. In future, we aim to design a
GUI (Graphical User Interface) for an efficient and user-friendly applicationand to develop PiSCAT further to run on a CPU or GPU in parallel.

References[1] K Lindfors, T Kalkbrenner, P Stoller, and V Sandoghdar. Detection
and spectroscopy of gold nanoparticles using supercontinuum white light
confocal microscopy. Physical Review Letters, 93(3):037401, 2004.
[2] R W Taylor and V Sandoghdar. Interferometric scattering microscopy:
seeing single nanoparticles and molecules via rayleigh scattering. Nano
Letters, 2019.

Picometer-resolved universal single-lens interferometer
unveiling ultraprecise frugal devices
Pooja Munjal, & Kamal P. Singh
Department of Physical Sciences, IISER Mohali, Sec-81, Manauli 140306, Punjab, India
Email: poojamunjal4@gmail.com

Abstract:
The application of precision interferometers is generally restricted to expensive and smooth high-quality
surfaces. Here, we offer a route to ultimate miniaturization of interferometer by integrating beam
splitter, reference mirror and light collector into a single optical element, an interference lens (iLens),
which produces stable high-contrast fringes from in situ surface of paper, wood, plastic, rubber,
unpolished metal, human skin, etc. A self-referencing real-time precision of sub-20 picometer(∼λ/30000) is demonstrated with simple intensity detection under ambient conditions. The principle ofiLens interferometry has been exploited to build a variety of compact devices, such as a paper-based
optical pico-balance, having 1000 times higher sensitivity and speed, when compared with a high-end
seven-digit electronic balance. Furthermore, we used cloth, paper, polymer-films to readily constructbroadband acoustic sensors possessing matched or higher sensitivity when com-pared with piezo andelectromagnetic sensors. Our work opens path for affordable yet ultra-precise frugal photonic devices
and universal micro-interferometers for imaging.

Fig. 1: Schematics of iLens interferometer. An iLens produces high-contrast structured fringes (left image)from a surface using a low-power He-Ne probe laser (10mW, λ= 632nm). The z-position of the sample from theiLens, d(t), is controlled by a piezo-positioner driven by VPZT from a function generator (FG).

Keywords: Interferometry, picometer, rough surface, devices
References:[1] P. Munjal, & Kamal P. Singh, “A single-lens universal interferometer: towards a class of frugal opticaldevices”, Applied Physics Letters, 115(11), 2019.
[2] P. Munjal, & Kamal P. Singh, “Optically probing picometer resolved photo-dynamics of solid surfaces”, URSIRadio Science Bulletin, 2019(370), 12-16, 2019.

Point Spread Function Engineering in RealTime for iSCATChristopher Parperis, Vivien Walter, Mark Wallace
Imperial College London, UK

c.parperis16@imperial.ac.uk

Optical processing of images is commonly used in microscopy to optimise the collected signals by
increasing contrast or decreasing noise. Fourier plane processing is a common method to selectspecific image properties and, while powerful, the application of optical processing to iSCAT has
typically been limited to static apodising filters. Adaptive Fourier filtering can be performed using
Spatial Light Modulators (SLM), computer controlled high resolution devices capable of applying any
type of filter in real-time.
We demonstrate here the application of real time SLM processing applied in iSCAT microscopy toobtain a 6-fold increase of the signal-to-noise ratio. We investigate a range of optical processing
methods, and demonstrate that this optical pre-processing reduces the molecular weight threshold ofdetectable label-free proteins and polymers.

Sub-millisecond tracking of single lipids oncell membranes through ISCAT microscopy
F. Reina1,2,5, B.C. Lagerholm3, C. Eggeling1,2,4
1

Leibniz-Institut für Photonische Technologien, Jena, Germany
Insitute of Applied Optics and Biophysics, Friedrich-Schiller Universität, Jena, Germany
3
Wolfson Imaging Centre, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK
4
MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK
5
Presenting
2

francesco.reina@uni-jena.de

The study of lipid dynamics on cellular surfaces is a topic of great interest in membrane
Biophysics. One of the prevalent models for lipid diffusion is the compartmentalized diffusion
model, whereby the cellular membrane is subdivided by the actin cytoskeleton in smallerareas. The diffusion of lipids is therefore heterogeneous, with periods of confinement
alternating with free diffusion. The study of single particle trajectories is particularly suited tohighlight the peculiarity of this diffusion motion. Interferometric Scattering microscopy has
proven particularly successful at these tasks in similar situations, and therefore is ideallysuited to pursue this objective. For this purpose, we have therefore delivered headgroupbiotinylated lipids to an epithelial cell line, PtK2, which has been widely used in the study oflipid membrane diffusion. These lipids have been labelled with gold nanoparticles of two
different sizes (20nm and 40nm diameter), and their diffusion was tracked on the cell surface.
In order to properly analyse the trajectory data thus obtained, we have adopted a novel
analysis approach, in which no prior knowledge of the diffusion mode of the particle isrequired, and that adequately addresses influence of localization errors in the case of highframerate SPT measurements. Our results show that the motion of a majority of the tracked
lipids is compartmentalized. Moreover, we show that the gold nanoparticles strongly affectthe long-range diffusion behaviour of the lipids, by slowing them down, whereas it appears
the compartment diffusion is less affected by the particle size. Finally, we found out that themagnitude of the short-range diffusion is much smaller than that predicted in the literature,most likely due to the way the localization uncertainty is included in the analysis. The work
we present should therefore serve as a starting point to address the issues relating tomembrane lipid diffusion with a fresh approach, with the aid of ISCAT, and also in
conjunction with other single particle tracking-capable techniques.

Correlative iSCAT and fluorescence highlight a positive
feedback between bacterial mechanosensation and motility
Lorenzo Talà1, Adam Fineberg2, Philip Kukura2 and Alexandre Persat1*
1

Global health institute and Institute of bioengineering, School of Life Sciences, École polytechnique fédérale deLausanne, station 19 1015 Lausanne, Switzerland
2
Physical and Theoretical Chemistry, Department of Chemistry, University of Oxford South Parks Road,
Oxford, OX1 3TA (UK)*alexandre.persat@epfl.ch

The bacterial pathogen Pseudomonas aeruginosa senses surface contact using type IV pili. These pili
are thin filaments (5 nm in diameter) composed of polymerized proteins. They extend and retract fromthe cell body thanks to dedicated molecular motors in order to act as active probes. Upon mechanicalcontact, pili activate a two-component signaling pathway called the Chp system. In response,
Pseudomonas upregulates virulence genes thereby infecting upon contact with its host.
Pseudomonas also extends, attaches and retracts pili to power twitching motility, a surface related
motility mechanism important for exploration and taxis toward favorable niches. Thus, pili act as meansfor displacement as well as transducers of mechanical signals to gene expression.
However, it is unclear if, and how, this mechanoresponse feeds back into the regulation of pili activity
and twitching. Indeed, such a feedback would strengthen the commitment for an infectious state of thebacterium.
To answer this question we observed simultaneous pili activity and biological processes. Fluorescencemicroscopy pipelines are gold standards in visualization of intracellular biological process. However,pili visualization is somehow more challenging as their size prevents their observation in conventionallabel-free microscopy.
Here we combined the powerful interferometric scattering microscopy technique to fluorescence inorder to assess simultaneous pili activity and intracellular protein localization in living Pseudomonas
aeruginosa cells. We correlate pili number to fluorescently labeled motors and regulatory proteins
involved in pili biogenesis and mechanosensation. Our findings show that protein localization profiles
change on surfaces, suggesting that segregation of specific proteins to a certain location in time is crucial
in pili dynamics regulation.
Altogether, we provide a powerful tool to visualize both intracellular and extracellular events with hightemporal and spatial resolution in living organisms.

Interferometric scattering microscopy reveals
microsecond nanoscopic protein motion on a live cell
membraneRichard W. Taylor1,2, Cornelia Holler1,2, Reza Gholami Mahmoodabadi1,2, Alexandra
Schambony1,2,3 and Vahid Sandoghdar*1,2,4
1

Max Planck Institute for the Science of Light, Erlangen, Germany
Max Planck Zentrum für Physik und Medizin, Erlangen, Germany
3
Department of Developmental Biology, Friedrich-Alexander University Erlangen-Nuremberg, Erlangen,Germany
4
Department of Physics, Friedrich-Alexander University Erlangen-Nuremberg, Erlangen, Germany*vahid.sandoghdar@mpl.mpg.de2

The plasma membrane of the living cell is organized in an intricate and dynamic manner across and tounderstand how this organization arises and the consequent cellular function it creates is topic ofintense investigation. It is understood that the membrane is compartmentalised into nanoscaledomains and regulated by mechanisms ranging from confinement by the actin cytoskeleton down toprotein-protein interactions. It is of great interest to investigate how these mechanisms lead individual
membrane molecules to inevitable cellular function, but to do so is challenging owing to the need tovisualise the mobilities of individual molecules in the plasma membrane.
Here we present our work wherein we use the high sensitivity of iSCAT microscopy to track themobility of membrane proteins in 3D to nanometric precision and microsecond temporal resolution.
Using the epidermal growth factor receptor protein (EGFR) as a model candidate, we show that one
can investigate to new levels of detail the role of individual proteins in important biological processes
such as membrane diffusion and endocytosis whilst under physiological conditions [1]. We can also
harness the 3D nature of iSCAT tracking over extended axial ranges to visualise the labelling of themembrane in situ as well as reconstruct the membrane topology the probe travelled over were hence
the probe acts as a cellular nano-rover, shown in Figure 1. We will discuss the possibilities for iSCAT
single-particle tracking in the live cell, describe the main challenges, and provide a perspective on
what the future holds for this promising technique.

Figure 1: (a) High-speed tracking of EGFR diffusion on the live cell membrane, recorded at 20,000 fps. (b) Surface
interpolation of a trajectory of confined mobility revealing a bowl-pattern in 3D recalling an endocytotic clathrin pit.

[1] R.W. Taylor, R. G. Mahmoodabadi, V. Rauschenberger, A. Giessl, A. Schambony and V.
Sandoghdar, Interferometric scattering microscopy reveals microsecond nanoscopic protein motion
on a live cell membrane. Nature Photonics, 13, 480–487, 2019.

Tunable subdiffusion and ergodic-nonergodicity transition ofgold nanoparticles
Yazgan Tuna1,*, Andreas Dechant2, Anna Kashkanova1, Eric Lutz3, Vahid Sandoghdar1
1 Max-Planck-Insitute for the Science of Light, Staudstr 2, 91058 Erlangen, Germany

2 WPI-Advanced Institute for Material Research (WPI-AIMR), Tohoku University, Sendai 980-8577, Japan
3 Institute for Theoretical Physics I, University of Stuttgart, 70569 Stuttgart, Germany* yazgan.tuna@mpl.mpg.de

Anomalous diffusion usually occurs in complex systems, from disordered semiconductors to
turbulence and living biological cells [1, 2, 3]. The complexity of these systems often renders them out ofreach, and the precise mechanism leading to anomalous dynamics, e.g. trapping or viscoelastic medium, iscommonly not known exactly [4]. This is compounded by the fact that anomalous phenomena are generally
not controllable. In the case of anomalous subdiffusion, amplitude fluctuations are described by a nonlinear
mean-square displacement, ⟨∆x2 ⟩∝tα, with α < 1 [5]. Moreover, they prevent time averages to converge tothe corresponding ensemble average, thus ergodicity is (weakly) broken [6], and the system lies beyond thereach of ordinary statistical mechanics. In this regime, an “average” behavior cannot be simply inferred froman individual trajectory, in contrast to the ergodic trapping phase where single particle tracking or ensemble
measurements are interchangeable.
In this work, we experimentally investigate subdiffusion and nonergodicity of single charged gold
nanoparticles (GNPs) diffusing under the effect of an electrostatic potential. We use wide-fieldinterferometric scattering microscopy (iSCAT) [7] to record individual trajectories of the Brownian particles(Fig. 1 a). The analysis of individual trajectories allowed us to determine the potential depth (β=U0/kBT) for
each particle, while the ensemble analysis revealed that the transition from trapping to normal diffusion(Fig.1 b) is not sharp, rather occurs via an intermediate subdiffusive regime. Furthermore, we have also
shown the transient behavior between ergodic and nonergodic dynamics of a system in a simple setting.

Figure 1. (a) Sample experimental trajectories for various potential depths. (b) Ensemble averaged meansquare displacement (MSD) shows the tunability of the subdiffusion on the ensemble level with the
diffusion exponent α = (3 − β)/2. The dashed lines show the values of α = 1, 0.5 and 0 in order ofdecreasing slope.

[1] I. Golding and E. C. Cox, Phys. Rev. Lett. 96, 098102 (2006).
[2] J.-P. Bouchaud and A. Georges, Phys. Rep. 195, 127–293 (1990).
[3] S. Marksteiner, K. Ellinger and P. Zoller, Phys. Rev. A 53, 3409 (1996).
[4] I. M. Sokolov, Soft Matter 8, 9043 (2012).
[5] Y. Meroz, I. M. Sokolov, and J. Klafter, Phys. Rev. Lett. 110, 090601 (2013).
[6] A. Rebenshtok, and E. Barkai, Phys. Rev. Lett. 99, 210601 (2007).
[7] R. Taylor and V. Sandoghdar, Nano Lett. 19, 4827 (2019).

Interferometric plasmonic microscopy
Prof. Hui YU
Shanghai Jiao Tong University

hui.yu@sjtu.edu.cn

Interferometric plasmonic microscopy (iPM) is the extention of interferometric scattering microscopy(iSCAT) into the plasmonic field. Using surface plasmons as the illumination source has provided
unique advantages, including field enhancement in longitudinal directions, easier tuning between
reference and scattering beams, and quantitative phase imaging. We have successfully applied the iPM
technology for imaging single bioparticles and biomolecules, such as exosomes and DNA molecules.

LABEL-FREE SINGLE MOLECULE QUANTIFICATION OF DNA BY MASS
PHOTOMETRY
Author Block: Yiwen Li1, Weston B. Struwe1, Philipp Kukura1.
1

Physical and Theoretical Chemistry Laboratory, University of Oxford, Oxford, United
Kingdom
Abstract:
A primary advantage of label-free detection methods over fluorescent measurements is its
quantitative detection capability, since an absolute measure of material facilitates kinetic
characterisation of biomolecular interactions. Mass photometry based on interferometricscattering microscopy (iSCAT) is a novel, label-free imaging and quantification approach,having demonstrated accurate mass measurement of single biomolecules in solution. Here,
we show that mass photometry is equally applicable to nucleic acid detection and massquantification. Individual ds-DNA molecules binding to a cover glass surface produce strong
interferometric contrast, with a detection limit on the order of 60 base pairs. Characterisationof standard DNA ladders yields mass distributions with well-resolved peaks, exhibiting aclear correlation between interferometric contrast and the number of base pairs resulting in 2
base pair accuracy and 13 base pair precision. Combined with the capabilities of mass
photometry to study soluble proteins, these results furthermore pave the way towards labelfree, single molecule, solution-based quantification of protein-DNA interactions.

Mass Photometry of Membrane Proteins and Membrane Mimetic Systems
Anna Olerinyova1, Adar Sonn-Segev1, Joseph Gault1, Cédric Eichmann2, Johannes Schimpf3, Adrian
H. Kopf4, Lucas S. P. Rudden5, Dzmitry Ashkinadze2, Radoslaw Bomba2, Jason Greenwald2, Matteo
T. Degiacomi5, Ralf Steinhilper2, J. Antoinette Killian4, Thorsten Friedrich3, Roland Riek2, Weston B.
Struwe1, Philipp Kukura1
1

Physical and Theoretical Chemistry Laboratory, Department of Chemistry, University of Oxford, South Parks
Road, Oxford, OX1 3QZ, UK
2
Laboratory of Physical Chemistry, Department of Chemistry and Applied Biosciences, ETH Zürich, Zürich,
Switzerland
3
Institut für Biochemie, Albert-Ludwigs-Universität, Freiburg, Germany
4
Membrane Biochemistry and Biophysics, Bijvoet Center for Biomolecular Research, Utrecht University,
Utrecht, The Netherlands
5
Department of Physics, Durham University, Durham, DH1 3LE, UK

Integral membrane proteins (IMPs) are indispensable in cell function and signalling. However, they are
inherently challenging to study because they require a lipid-like environment, which is difficult tomaintain in vitro. As such, studying IMPs generally requires use of membrane mimetic systems. We
explore the application of mass photometry (MP) – single-molecule mass measurement byinterferometric scattering in solution - to IMPs and membrane mimetic systems at the single particle
level. We apply MP to membrane mimetic systems including amphipathic vehicles, such as detergent
micelles and amphipols, and lipid and native nanodiscs, characterizing the particle size and the sample
purity and heterogeneity. We differentiate between empty and occupied mimetic systems and study
IMP heterogeneity and multimer formation. Using methods established for cryogenic electron
microscopy, we eliminate detergent background, enabling high-resolution studies of membrane protein
structure and interactions in detergents. Demonstrating the power of this approach, we find evidence
that, when extracted from native membranes using styrene-maleic acid nanodiscs, the potassiumchannel KcsA is present as a dimer of tetramers – in contrast to results obtained using detergent
purification and lipid nanodiscs. Finally, using lipid nanodiscs and NMR, we show that MP can help
distinguish between functional and non-functional KcsA nanodisc assemblies, as well as determine thecritical factors for optimal lipid nanodisc formation. This approach thus opens the door to the routine
use of native preparations of membrane proteins for in vitro functional studies, structuralcharacterization by cryoEM, and in vitro studies of purity, structure and interactions more generally.

Quantifying binding affinities, kinetics and stoichiometry of biomolecularcomplexes with mass photometry
Fabian Soltermann1, Veronica Pagnoni1, Eric Foley1, Martin Galpin1, Weston B. Struwe1, Philipp
Kukura1
1
Department of Chemistry, University of Oxford, Chemistry Research Laboratory, 12 Mansfield Road, Oxford
OX1 3TAThe characterisation of biomolecular complexes and their interactions is essential for understanding the function
and regulation of cellular processes. Many of the existing biosensor techniques, despite their maturity, are often
hampered by the requirement for surface immobilisation, introduction of tags and the inability to differentiate andquantify co-existing species. Here, we show that mass photometry based on interferometric scattering microscopy(iSCAT), can determine the binding affinities, kinetics and stoichiometries of biomolecular complexes in a labelfree fashion in solution at the single molecule level. We illustrate these capabilities by quantifiying antibodyantigen and antibody-receptor interactions ranging in strength over 4 orders of magnitude (KD= 0.1 – 1000 nM)
and showing the simplicity of differentitating between co-existing species. The ease and speed of use (< 1 min),low sample consumption (< picomole protein/run), minimal sample and assay preparation in combination withclear avenues to high-throughput and data analysis makes mass photometry a powerful new method forcharacterising biomolecular interactions and dynamics in vitro.