Glyoblastoma cells: FLIM
AFM & FLIM aligned

Fluorescent nano beads

Application Note #137
Performing Enhanced Multiparameter Cell
Imaging with Combined Fluorescence
Lifetime Imaging Microscopy and Atomic
Force Microscopy
Fluorescence Lifetime Imaging Microscopy (FLIM) provides
optically encoded information about processes in live cells.
Atomic force microscopy, on the other hand, provides
nanometer-resolved surface topography and mechanicalinformation, and has recently been expanded to nanometerresolved live cell mechanical property mapping. The
integration of the two advanced live cell imaging techniques
into one tool, with the capability to acquire simultaneous
nanometer-mechanical and optical/biochemical information
promises to provide new insights into cell mechanics. Thisapplication note describes the integration, demonstrated
combined performance, and overlaid data of a cellular model
system on the MicroTime 200 from PicoQuant and Bruker’s
BioScope Catalyst™ Atomic Force Microscope (AFM), on
the basis of an IX71/81 Inverted Optical Microscope with
PiFoc focus control.

Benefits of Combined Techniques
Characteristics of surfaces on a single molecule levelcan be examined using both confocal fluorescencemicroscopy and atomic force microscopy. The techniques
analyze different aspects of the sample, for example,
one can investigate molecular dynamics with timeresolved fluorescence spectroscopy and characterize

Mechanical dissipation

surface topography with atomic force microscopy. Thus,
a combination of both techniques into a single instrument
enhances the methodology and opens up new investigation
schemes, including:• Live Cell Imaging
Cell growth and differentiation are associated withchanges in cell shape and content. Changes in cellsurface properties, such as membrane elasticity
and protein concentration and localization during cell
development, can be monitored by synchronizedacquisition of atomic force microscopy andfluorescence images. The topographical changes
in cell shapes depend on changes in the cell
cytoskeleton. The reorganization of those proteins canbe associated with changes in protein conformation,
localization, or orientation. The variation in theenvironment and in the protein structure itself can
influence the fluorophore behavior.
• High-Resolution Molecular-Scale Imaging
Atomic force microscopy can reveal the packing
and structure of both native and reconstituted
membranes. Such membranes often consist of aheterogeneous distribution of lipid, sterol, and protein

components. The simultaneous study of atomic
force microscopy with fluorescence techniques
could reveal an association of such structures withprotein compartmentalization and thereby reveal
their function.
• Force and Mechanical Studies
Another well-established atomic force microscopy
technique is single-molecule force spectroscopy,
wherein single molecules or pairs of molecules aremechanically interrogated to measure inter- andintra-molecular forces. By the parallel observationof fluorescence parameters, e.g., FRET processes
induced by the movement, it is possible to determineinter- or intra-molecular distances.

Combined System Description
The MicroTime 200 from PicoQuant is a confocal timeresolved fluorescence microscope with single-moleculesensitivity.1 It is built around an inverted microscope bodyand uses the method of Time-Correlated Single Photon
Counting (TCSPC) for time-resolved data acquisition
along with single photon sensitive detectors andpicosecond pulsed diode lasers for excitation. For image

measurements, such as Fluorescence Lifetime Imaging(FLIM), the system is equipped with a 3D piezo scanningsystem with an effective scan range of 80µm x 80µm x
80µm. In the standard configuration, the sample is placedon the scanner and moved across the stationary excitation
beam (“sample scanning“). In an alternative configuration,
the objective is mounted on the scanner and moved below
the stationary sample (“objective scanning”). This special
configuration allows free access to the sample fromabove and is therefore well suited to applications requiring
special sample compartments or for integration withother techniques requiring patch-clamps, cryostats, or as
demonstrated here, atomic force microscopy (see figure 1).
Imaging living cells with atomic force microscopy in nearphysiological conditions has seen steady advances over thelast two decades.2-4 The BioScope Catalyst™ from Bruker isa high-performance AFM designed specifically for biologicalapplications.5 The AFM uses a flat sample scanner designthat isolates the piezos from damage by fluids. The open
design of the AFM head allows virtually unrestricted
optical and physical access from above the sample. The
scanning range is 150 x 150µm in closed-loop operation,with extremely quiet sensors to ensure high-resolution

Figure 1. Combined setup of the BioScope Catalyst and the MicroTime 200. The AFM with its sample stage is mounted on the inverted
microscope body of the MicroTime 200, which is configured for objective scanning.

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imaging and precise force measurements. The tip can be
moved independently with a maximum range of morethan 20µm (in Z-direction), which enables measurementsof large samples, such as living cells. The complete AFM
also includes a sample stage with tip-sample alignment
and tip-optics alignment, suited for the IX71/81 inverted
microscope body from Olympus. The AFM control softwareis optimized for exceptional ease of use, including Bruker’s
exclusive ScanAsyst ® AFM imaging mode that automatically
optimizes atomic force microscopy imaging parameters.
It also includes microscope image registration and overlay(MIRO ®) software, which enables direct calibration andcorrelation of AFM images with light microscopy images.
Finally, using the new PeakForce QNM ® mode, one canadditionally access nanomechanical information such as
sample modulus, tip-sample adhesion, and dissipation
by measuring and analyzing force curves at every image
point while simultaneously imaging sample topography athigh resolution.
The BioScope Catalyst AFM is optimally suited for
combination with the MicroTime 200 as it fulfills allnecessary requirements:
1. The BioScope Catalyst is a sample-scanning AFM,
2. The controller of the BioScope Catalyst provides ascan synchronization signal,

Synchronization of the Two Systems
The combination of the MicroTime 200 with the BioScope
Catalyst allows the parallel acquisition of fluorescence
lifetime and AFM images. The key for this procedure is
the special time-tagged time resolved (TTTR) mode of
the PicoQuant TCSPC unit, which allows the insertionof external synchronization signals (“markers”) in thecontinuously recorded data stream of photon arrival times.
In case of the AFM scanning systems, these markers are
signals at the beginning and end of each scanning line,
which basically divide the acquired photon stream into thescan lines and further allow the assignment of photons
into image pixels.6 The required “line start” and “linestop” synchronization signals are readily available from theAFM controller.

Optical alignment
Before starting measurements, the AFM cantilever tip
and the laser focus of the microscope must be aligned.
The cantilever is observable through the eyepieces of theMicroTime 200. In one eyepiece, a cross hair is visible,
which is aligned to the optical axis of the microscope (seefigure 3A). The first coarse alignment can be done bymoving the AFM sample baseplate until the cantilever tip isplaced directly under the cross hair (see figure 3B).

3. The scan head of the BioScope Catalyst fitsmechanically onto the Olympus IX71/81 microscope
body without interfering with normal light microscopy
operation,

Sample scanning AFM

Fluorescent specimen

4. The sample baseplate of the BioScope Catalyst
allows adjustment of the AFM tip in x, y and z relative
to the confocal beam.
One of the key goals in the combination of the MicroTime
200 with the BioScope Catalyst is to enable simultaneousrecording of AFM and optical images of the same sample
region. It is therefore necessary to design a setup where
the confocal volume of the MicroTime 200 can be preciselyaligned with the AFM tip. This can be achieved by using
the objective scanning mode of the MicroTime 200 toprecisely locate the AFM tip within the optical scan range.
The sample baseplate itself, and thus the complete AFM,
can be moved in x and y directions, which is useful for the
pre-alignment of the cantilever tip relative to the opticalaxis of the MicroTime 200. The sample is then placed onto
the scan stage of the AFM (see figure 2). All scanning
procedures are directly controlled by the AFM software, andtherefore the functionality of the AFM is not compromisedin any way.

AFM
controller

Objective
3D piezo
positioning

Laser

Start/Stop line signals

TCSPC
electronics
SPAD

Figure 2. Schematic of the combined setup: The sample is placed on
the scanner of the AFM and the data acquisition of the MicroTime
200 is synchronized with the scanner movement by including
corresponding electronic marker signals into the collected photon
data stream.

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Figure 3. Cantilever with tip in the eyepiece of MicroTime 200 aligned to the cross hair (left image). The coarse positioning can be done byusing the three micrometer screws at the sample stage (red arrows in right image).

Fine alignment of the laser beam path with the cantilevertip can then be done with the MicroTime 200 using theacquisition of scattered and reflected light. The first coarsealignment is sufficient to direct the cantilever tip into thescan area of the MicroTime 200. A good start position in
the z direction is 80µm, the lowest available position for

the MicroTime 200 PIFOC objective nano-positioner. To
avoid damage of the AFM probe, only small steps in Z
should be performed (~2µm). The resulting image is shownin figure 4. In the end, a bright spot in the image center,
which represents the cantilever tip, should be visible (seefigure 4F).

Figure 4. Backscatter images of AFM cantilever with a SiN tip. The bright spot in the image center is the tip apex. After iteratively zooming inand scanning the AFM tip, the laser focus of the MicroTime 200 can be directly positioned at the tip apex. The sample stage is moved 2µm in
z-direction for every image B-F.
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shown in figure 5. The correct confocal plane can be found,again by using the photon count rate as an indicator. Since
the laser power needed for this calibration procedure is very
low, photobleaching during the alignment is not an issue.

Imaging of Fluorescent Beads
In a first experiment, fluorescent beads (TetraSpeck, 100nm
diameter, Molecular Probes) on a glass cover slide wereimaged with the combined setup of the MicroTime 200 and
the BioScope Catalyst. The results are shown in figure 6.

Figure 5. Backscatter image of a probe tip with marked tip apex (redcross), which is determined by the shape of the tip base.

The laser focus is aligned with the AFM by performing
point measurements on the cantilever tip. Fine alignmentof the focal plane should be done using the photon countrate of the probe as an indicator. This alignment procedure
has been shown to work with bare silicon and with silicon
nitride AFM tips on glass surfaces, e.g., glass bottom
culture dishes. In cases where the tip apex does not givea sharp bright spot in the optical picture, one can use theshape of the tip to determine the position of the tip apex, as

The AFM was used in ScanAsyst mode. The excitation
wavelength was 470nm with an LP510 filter in front of asingle photon avalanche diode (SPAD) from MPD (Olympus
60X 1.2 NA water objective). To acquire a synchronizedimage, the MicroTime 200 was used as slave of the
BioScope Catalyst scanner. After a scan, up to eight datachannels are visible in the AFM software and an intensity
modulated FLIM image in the MicroTime 200 software.
The images clearly show how both techniques allow thebeads to be imaged, but they also show the differences.
Not all features are visible in the AFM or the FLIM image(see figure 6A-C). Only with such a synchronized acquisition
technique is this difference visible. A 3D representation of
AFM height and fluorescence image is displayed in figure 7.
To analyze the quality of synchronization, a series of eightcombined AFM-fluorescence images of a ~300nm highfluorescent bead was acquired. The fluorescence channel ofthe SPAD was introduced into an auxiliary photon counting
input of the AFM controller so that the fluorescenceintensity was acquired simultaneously with the AFM data by
the AFM software. The NanoScope XY drift analysis feature
was used to measure drift rates for the AFM itself andfor the correlation between AFM and optical images. The

Figure 6. Synchronised acquisition of fluorescence beads measuring fluorescence lifetime and topography. (A) intensity modulated FLIM
image (B) AFM image and (C) merged AFM and FLIM image. Not all beads that are visible in the AFM picture also show fluorescence in the
FLIM image. Scale bar is 5µm.

5and fluorescence lifetime of the GFP (see figure 8). The
MicroTime 200 was outfitted with a pulsed laser at 470nm
for GFP excitation, and the fluorescence was detected using
a bandpass filter HC520/35 in front of the SPAD detector.

Figure 7. Overlay of the AFM height image as 3D representation andwith fluorescence intensity in green (Image resolution 10µm x 10µm
x 300nm).

maximum drift rate observed for the AFM was 2.8nm/min.
The maximum drift rate observed for AFM-optical alignment
was 11.5nm/min. This suggests that the alignment between
the AFM tip and a diffraction-limited spot at high NA (e.g.,
300nm) could be held for at least a good fraction of an hour.

Fixed Cells
Since the BioScope Catalyst is optimized for biologicalsamples, we also measured fixed glioblastoma cells (brain
cancer cells) stored in PBS buffer (pH 7.2) (Sample Courtesyof Celine Heu, FEMTO, Besancon, France). The transfected
cells expressed a free diffusible GFP that should be visible
in the cytoplasm as well as in the nucleus. The DNA wascounter-stained using DAPI, a DNA intercalating dye, which
is not excited by the laser line used here. The combined
setup of the MicroTime 200 and the BioScope Catalyst
enabled us to examine the combined cell topography

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In figure 8B-H the information taken by the BioScope
Catalyst in PeakForce QNM mode is displayed. Bymeasuring and analyzing a force curve at every image point,
one obtains topography, modulus, adhesion, deformation,and dissipation of the cell. The measurement was madequantitative by prior spring constant calibration of the AFM
cantilever, using the integrated thermal-noise calibration
method. Fluorescence intensity (photon count) wasrecorded pixel-synchronously with the AFM measurement(see figure 8E), by distributing the MicroTime 200’s digital
APD signal into the NanoScope® V AFM controller’s pulsecounting input. Figure 8A shows the intensity modulated
GFP lifetime distribution in the cell taken by the MicroTime
200. An overlay of these images is possible by exporting the
FLIM image and overlaying it in Bruker’s MIRO software.
Both techniques and instruments, the MicroTime 200 timeresolved fluorescence microscope and BioScope Catalyst
AFM, are routinely used individually for live cell studies,
therefore this proof of performance experiment can be fully
extended toward combined AFM-FLIM live cell imaging.
For long term (hours-days) studies of sensitive eukaryotic
cells, without introducing environmental stresses,
combined FLIM-AFM studies can be performed in sterile,
CO2-buffered media at physiological temperatures, using
the Catalyst Heater Stage and Catalyst Perfusing Stage
Incubator (PSI) accessories.

Figure 8. Synchronized acquisition of fixed glioblastoma cells expressing GFP using the MicroTime 200 in combination with the BioScope
Catalyst. (A) Fluorescence lifetime distribution in the cell. (Image size 100 x 100µm) acquired by by MicroTime 200, and aligned using
MIRO within the NanoScope software. (B-H) Data taken by the BioScope Catalyst AFM in PeakForce QNM mode showing topography,
and spatially resolved quantitative mechanical cell properties. (E) Photon count from the MicroTime 200, synchronously recorded by
NanoScope AFM controller.

Conclusions
The combination of AFM and time-resolved confocal
microscopy opens up many new ways of investigating
single-molecule dynamics. The presented combination of
the MicroTime 200 and the BioScope Catalyst has beenshown to be relatively straightforward without need ofmodifications to the systems. An alignment procedure
has been developed and first results demonstrate thecapabilities of such a combined setup. Prospective

applications include live cell imaging and the investigation of
the impact of protein changes on cell shape and structure
and vice versa; high-resolution molecular-scale imaging by
merging of sub-nanometer AFM topographic imaging with
optically encoded functionality; and force and mechanicalstudies that investigate inter- and intramolecular distances
using atomic force spectroscopy and nanomanipulation on
the single-molecule levely.

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References
1. http://www.picoquant.com/products/microtime200/microtime200.htm.
2. Z. Shao et al., Adv Phys 45 (1996) 1–86.
3. H.W. Wu et al., Scanning 20 (1998) 389–97.
4. C. Rotsch and M. Radmacher, Biophys J 78 (2000) 520–35.
5. http://www.bruker-axs.com/bioscope-catalyst-atomic-force-microscope.html
6. http://www.picoquant.com/technotes/technote_lsm_upgrade_kit.pdf –section “Synchronization”

Authors
Johannes H Kindt (Johannes.kindt@bruker-nano.com) and Alexandre Berquand(Bruker Nano Surfaces Division)

Bruker Nano Surfaces Divison
Santa Barbara, CA · USA+1.805.967.1400/800.873.9750
productinfo@bruker-nano.com

www.bruker .com

registered trademarks of Bruker Corporation. All other trademarks are the property of their respective companies. AN137, Rev. A0©2012 Bruker Corporation. All rights reserved. BioScope Catalyst, MIRO, NanoScope, PeakForce QNM and ScanAsyst are trademarks orMarcelle König, Marcus Sackrow, Andrea Bleckmann, Felix Koberling(PicoQuant GmbH)