the unique capabilities of the Bioscope™
II combined with an inverted optical
microscope to perform live cell studiesof the cellular effects caused by mutant
huntingtin assembling in intracellular
protein aggregates. We will also
highlight the imaging capabilities of
our tool, especially the combination of
AFM and ﬂuorescence imaging of cells.
In the second paper of this two-partseries on atomic force microscopy ofneurodegenerative diseases, we willemphasize the force spectroscopy
capabilities of the BioScope II.
SAMPLE PREPARATION

CHO-K1 cells were transfectedwith YFP-tagged huntingtin exon 1fragment (HttexQ68-EYFP,AS 1-90;
68 polyglutamines). Transfection was
performed using 2 x105 cells, 1 μg
plasmid DNA, and 3 μl Fugene HD(Roche) per well. Subsequently, cells
were seeded on sterile cover slips(18x18 mm) in 6-well plates. After
24-48h incubation cells were washedtwice with PBS and ﬁxed with 4% PFA
for 10 min.
Brightﬁeld (BF), Differential
Interference Contrast (DIC) and
epiﬂuorescence images were recorded
using a Zeiss Axio Observer equippedwith AxioCam MRc camera andAxioVision software. All AFM images
were recorded on a Veeco Bioscope II
using MLCT and DNP-20 cantilevers.
The system combining AFM and opticalmeasurements was used as a fully
integrated tool. All experiments wereperformed in contact and TappingMode™
in PBS buffer. 3D-rending of the AFM
height images was performed, using
Veeco’s VISION software package.

tip/sample alignment. Starting at
low magniﬁcation — this affords alarger ﬁeld of view of both the sample
and AFM tip position — huntingtin
aggregates are clearly detectable byvarious optical contrasting techniques;in BF, in epiﬂuorescence basedon the strong ﬂuorescent signal
mediated by the YFP-tagged htt, andin DIC based on the aggregates ofhtt which change cell topology.
We start with positioning the AFM tip
in the center of the ﬁeld of view using
the manual X-Y translation stage(important for when switching to higher
magniﬁcation – and hence smaller ﬁeldof view). Then we use the motorizedstage to position a region of interest
relative to tip. This way both the tip andthe region of interest are typically at thecenter of the ﬁeld of view.
In Figure 1, we show an example ofthis situation: Images 1A and 1B show
the BF and the ﬂuorescence imagesof the selected area, respectively, but
the cantilever is not located in thisdisplay window (the shape of the tip
is visible on the right bottom cornerof image 1A). Using successively
manual and motorized stages, it

is extremely easy to move the tip
just above the area of interest.
Positioning both the tip and region ofinterest in the center of the opticalﬁeld of view will also help to minimize
some optical aberrations/warping
effects that could affect the accuracy ofimage registration. These aberrations(chromatic and spherical) are moreprevalent nearer the outer edges of theﬁeld of view for a particular objective
and minimal in/around the optical axis.
The design of the BioScope II’s
motorized stage is extremely stable,and aids in obtaining highly accurate
images. The three motor screws of theBioscope II do not rotate, unlike most
AFM’s; instead, the motor screws movestraight in and out of the head. Thisprovides a “no wobble” approach of theAFM tip, helping to maintain accurate
targeting of a region of interest on thesample during the engage process. TheNanoScope® V Controller’s softwareautomated engage procedure provides
a quick but safe engagement of the tipon the sample surface. This is crucial
when imaging soft samples — forpreservation of both the sample and theAFM tip.

REGISTRATION AND CORRELATION OF
AFM AND OPTICAL DATA

For proper registration and correlationof AFM and optical data it is criticalto have the ability to align the AFM tipwithin the optical ﬁeld of view, as wellas position the tip relative to a region ofinterest on a sample.
The availability of both a manual andmotorized XY-translation stage on
the BioScope II provides an easy andefﬁcient means of tip/optics andPAGE 2

Figure 1: Reaching the area of interest using the “Navigate” function. In A (bright ﬁeld image) and B(ﬂuorescence image), some bright htt aggregates that are of interest are visible but the tip is both far awayfrom the sample and out of the ﬁeld of view (see shadow on the right bottom corner of image A). Using theX, Y motion screws and the joystick allows placement of the tip on top of the area of interest (C and D,brightﬁeld and ﬂuorescence image, respectively.). Optical images obtained with a 20x objective.

AFM AND NEURODEGENERATIVE DISEASES (PART II)

Images 1C (BF) and 1D (ﬂuorescence)
were taken at the very ﬁnal step of theapproach procedure. Interestingly,
the IR laser used for AFM detection
has no perturbing effects on BF
or epiﬂuorescence images. TheIR superluminescent diode does
not interfere with red biologicalﬂuorophores. At 850 nm, it can also
be easily ﬁltered if a customer hasan IR-sensitive camera and wants toremove the observed spilling of thelaser light around the AFM tip fromtheir optical images.
Figure 2 is a direct example of the typeof studies that are enabled by combined
AFM and ﬂuorescence imaging. Figures
2A, 2B and 2C, which correspond to
BF, DIC, and ﬂuorescence respectively,
show aggregates of mutant huntingtin.
The arrows in the images indicate
the speciﬁc aggregate selected forscanning with the AFM tip whereas
the region outlined in red speciﬁes thescanning area of the AFM tip. Image
2D shows a three dimensional AFM
topography image of the cells within theoutlined region in the corresponding
optical images.
The huntingtin protein aggregate
observed by optical microscopy (yellowarrow) is also easily observed in the
AFM image (red arrow). While thepresence of various aggregates on thesurface of the cells are easily observedby AFM, topography imaging alone does
not allow you to distinguish between
different types of aggregates (besides
the YFP-tagged aggregates othercellular substructures inﬂuencing cell
topology were detected in the AFM scan
[Images 2A, 2B and 2D green arrows]).
By combining AFM with optical
microscopy, the presence/absenceof a ﬂuorescent signal in correlated
ﬂuorescence images allows one toaccurately identify if an aggregate iscomposed of the huntingtin proteins ornot.
Note that some cells exhibit
homogeneous ﬂuorescent cellular
staining that is due to the presence ofsoluble huntingtin protein throughout
the cell cytoplasm. In contrast,
mutant huntingtin proteins alreadyaccumulated in aggregates exhibit

PAGE 3Figure 2: Using AFM to generate additional information to optical microscopy. Mutant YFP-tagged htt
protein forms bright aggregates (images A, bright ﬁeld and B, DIC, image C, epiﬂuorescence, yellowarrows) that are also easily recognized in the AFM topographic channel (image D, red arrow in). In addition,changes in cell topology caused by other cellular substructures similar to that caused by aggregates could
also be detected by the AFM scan (image D, green arrow). Optical images taken with a 20x objective. AFM
scan: 90x90x3μm.

locally very strong ﬂuorescence and arethus easy to identify.
Images were collected using various
objectives with a magniﬁcation
range from 20x to 100x, as well as ahigh numerical aperture 100x TIRF
objective. However, due to the strong
ﬂuorescence intensity of the huntingtin
aggregates, best results were obtainedby epiﬂuorescence with a 20x objective.
RESULTS: HUNTINGTIN AGGREGATESREMARKABLY HETEROGENEOUS

After a complete series of experiments,
cross section analysis performed in
the NanoScope software revealed thehuntingtin aggregates remarkably
heterogeneous in size and shape,from a few hundred nanometers up to
several microns (~4 μm) in diameter.
Friction images (data not shown)
revealed a contrast between theaggregates and the bulk of the cell.
Phase images, collected in
TappingMode (data not shown), also
exhibited an interesting phase contrast
in the region of the aggregates. Relative
phase changes (or contrast) across asample can often indicate differences

AFM AND NEURODEGENERATIVE DISEASES (PART II)

in surface properties, such as
viscoelasticity or electrostatic charge
distribution. Thus, a phase contrast inareas corresponding to the aggregates
would indicate that they are made upof material or components that aredifferent than that of the surrounding
cell surface.
We also investigated cells expressing
wild type form of huntingtin (data notshown). In this case, huntingtin proteinsremain soluble and thus, no changesof cell topology mediated by huntingtin
aggregates were detected.
CONCLUSION

In the present study, the imaging
capabilities of the BioScope II
were integrated with ﬂuorescencemicroscopy to locate and image protein
aggregates of mutant huntingtin, acharacteristic hallmark of Huntington’s
Disease. This application indicatesthe high selectivity and accuracyof the combined system in the
3D identiﬁcation of molecules inbiological systems and investigationof their physical properties throughoptical imaging and topographic,
friction, viscoelastic, and adhesion