Figure 1. In the MIRO canvas, the fluorescence image is used as a background to target AFM measurements. The overlay is made in real time, pixel by pixel. At the end ofthe scan, the AFM image (height channel, 35x35μm) is automatically integrated into the optical image. The software can also be used to directly navigate the AFM probe
to preferential locations for force curve acquisition (yellow crosses), AFM scanning, or force volume measurements (yellow frame).

U2-OS-Tag-RFP-Tubulin, Marinpharm,Luckenwalde, Germany), respectively.
CHO-K1 cells were labeled with DAPI
for nucleus and phalloidin-Alexa488
for actin. Hela cells were labeled withDAPI for nucleus, phaloidin-RITC foractin and anti-αtubulin conjugated with
FITC for tubulin. Cells were grown on
glass-bottom petri dishes (Willco), andtreated directly inside the dishes witheither nocodazole or latrunculin (50μM)
for 30 minutes at room temperature.
This protocol allowed the skipping of thepermeabilization step, which can rapidly
lead to cell death.
EXPERIMENT SETUP

Imaging and force measurements wereperformed with two separate BioScope™
Catalyst™ Atomic Force Microscope
systems that were fully integrated
with inverted optical microscopes. One
system used a Leica DMI 6000 with aHamamatsu ORCA camera, while theother used a Zeiss Axio Observer with anAxioCam camera. Several AFM probes(Veeco MLCT, SNL, MSNL) were utilized
during the experiments; however, thesame tip was used before and after
drug delivery to avoid the influence oftip shape and spring constant whencomparing the force curves.

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Veeco’s Microscope Image Registration
and Overlay (MIRO) software provided asimple and quick way to navigate the tip
to an area of choice to perform the AFM
measurements. The registration can bedone using either three points or fourthor fifth-order polynomial calibration (fornon-ideal optical microscopy conditions).
Though the primary advantage of MIRO
consists in automatically overlaying
optical and AFM images, in these studies
we also utilized MIRO to target the best
locations for force measurements (seefigure 1). As AFM force measurements
cannot be performed at the center of thecells, where they are too soft, the opticalnavigation allowed the direct selectionof the best locations for force volume
images and single force measurements.
This allowed us also to decrease thetime to data acquisition and preserve
the sensitive samples and probes. In
addition, the software gave us the ability
to zoom, adjust color and contrast, andmodify the opacity of AFM channels in
the regions of interest to more perfectly
match the optical images and observe
before-and-after results. Finally, thevertical laser path in the Catalyst
microscope head enabled much higher
stability, lower noise and increased
detection sensitivity.

EXPERIMENTAL RESULTS

Figure 1 shows the “MIRO canvas”
during a typical experiment withfluorescence and AFM images overlaid,
along with navigation markers for force
curve measurements and force volume.
Nuclei are labeled in blue, the tubulin
network in green, and the actin networkin red. The distribution of the red dye
confirms that actin is located at the cell
periphery, whereas tubulin is distributed
more in the deeper cytosol. Typicalareas of interest for AFM measurements
are indicated by the yellow box (forcevolume) and yellow crosses (single force
measurements). These experiments
were done on fixed Hela andCHO-K1 cells.
Additional experiments investigating
the effects of drug treatments wereconducted on living Hela cells andU2-OS osteosarcoma cells. These celllines stably express red fluorescentactin or tubulin, respectively, and offer
the advantage of avoiding the plasmidtransfection step.
The drug treatments led to different
consequences in the optical images
and AFM data (see table 1). The effects

Characterizing the Effect of Anticytoskeletal Drugs on Living Cells Using MIRO Software and the BioScope Catalyst AFM

of the two drugs could easily be seen, but not differentiatedon the optical images. The green and red fluorescences
totally vanished on all samples 30 minutes after incubation
with nocodazole and latrunculin, respectively. However, theanticytoskeletal agents exhibited measurably different effects
in the AFM images. After nocodazole treatment, the tracking
was still good, whereas after latrunculin injection, the cells
appeared much too soft for being scanned in imaging mode.
This indicated that the treatments led to different changes incell mechanical properties. Tubulin disruption caused only aslight change in cell elasticity, whereas actin disruption gave
rise to a dramatic change in cell elasticity.
Figures 2C and 2F give a representative example of force curves
recorded on osteosarcoma cells. (Very similar effects wereobserved on the Hela cells as well.) Note that the AFM force
measurements were performed on the edge of the cells to
avoid indenting too much in the sample, and to ensure clear,
reproducible force curves. It is very likely that the change inelasticity is even greater in the thickest parts of the cells.
Taken together, the results suggest that the actin cytoskeleton
is playing a much more important role in cell rigidity than thetubulin network.

CONCLUSION

This study demonstrates how the use of a fully integrated
BioScope Catalyst can enable enhanced investigation ofdrug effects on cells by combining results from fluorescencemicroscopy and AFM. The integration also enables better,more efficient control of the AFM to guide imaging andlocalized force measurements. This was clearly shown in theanalysis of the effects of two different drugs on two distinct
cytoskeleton networks in living cells. This type of experimentcan be extended to many research fields involving cell elasticity
measurements, including investigation of cardiovascular andinfectious diseases, cancer research, and drug targeting.
Fluorescence

AFM imaging(tracking)

Change incell elasticity

Control++++++_Nocodazole∅+++

Latrunculin

∅

∅+++

Table 1. Effects of nocodazole and latrunculin treatments in both optical and AFM
experiments.

ABCDEF

Figure 2: Nocodazole and latrunculin were incubated with living Hela (A and D respectively) and osteosarcoma (B and E respectively) cells, and a minimum of
30 force volume images were collected on each type of sample. After drug addition, the red and green fluorescence disappeared (not shown here). C and F show
representative “approaching” force curves recorded before (blue trace) and after (purple trace) drug treatment on osteosarcoma cells (results on Hela cells weresimilar). The two agents showed dramatically different effects on cell elasticity. The tubulin network was only slightly disrupted, showing minimal effect on theglobal cell elasticity (note the two curves are only slightly displaced on C). In contrast, the actin network disruption dramatically altered cell elasticity (purple curvereflects a significant softening effect in F).

Characterizing the Effect of Anticytoskeletal Drugs on Living Cells Using MIRO Software and the BioScope Catalyst AFM

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