Multidimensional microscopy on living cells
Introduction
An ongoing trend in scientific research is the development
of techniques that can address multiple components of a
system at a single time. DNA microarray analysis provides
a snap shot of the set of genes expressed within a cell at a
given time [1]. Multidimensional protein identification
technology is being developed to determine the contents of
heterogenous samples of proteins [2]. Force spectroscopy
analysis can address the complexity of cell adhesion by
distinguishing between the contribution of individual
elements to the binding process [3]. Advancements in
microscopy techniques and sample preparations are
extending this type of multi-component approach to the
imaging of cells.
The information generated about cellular structure and
function using various microscopy techniques will differ
depending on how contrast is generated. By combining
techniques, information about different properties of the
one sample can be monitored simultaneously. The JPK
Nanowizard® atomic force microscope is designed to be
installed on an inverted light microscope, enabling
simultaneous imaging of a sample by atomic force
microscopy (AFM) and various optical microscopy
techniques from phase contrast [4] to epi-flourescence [5],
total internal reflection fluorescence (TIRF) microscopy and
laser scanning confocal microscopy (CLSM) [6] to name a
few. This combination of AFM and other microscopy
methods has, thus far, been mainly utilised for structure®
function studies. However, the JPK Nanowizard can be
used to obtain superior images of living cells,
simultaneously with additional microscopic techniques
(figure 1). This makes the JPK Nanowizard® a powerful
10 µm
1 µm
and versatile tool for multidimensional microscopy studies.
Multidimensional microscopy refers to the generation of
multiple images of different properties of a sample over
time. As an example, multidimensional microscopy has
been used to great effect in the study of the composition
and dynamics of focal adhesion structures [7].
500 nm
Fig. 1 Successive error signal images of a living cell at
progressively smaller scan sizes. All images taken in low force,
constant contact mode
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In this case, multiple fluorescent channels were used to
characterise the components of focal adhesions and their
distribution in relation to each other and to actin within the
cell. Advances in available fluorophores and sample
preparation techniques mean that multiple channel
fluorescence can be conducted on both living and fixed
cells.
This simultaneous imaging of multiple fluorescent channels
generates information about the localisation of multiple
components in the cell, however such an approach could
be further extended by combining AFM with fluorescence
microscopy and phase contrast microscopy. In such a
manner, not only can one investigate the location of
particular proteins but also the dynamics of surface
structures and subsurface cytoskeleton in addition to the
overall cell morphology, simultaneously. Here we have
imaged REF52 cells, expressing paxillin-GFP, with phase
contrast, epi-fluorescence and AFM.
Experimental setup
In order to acquire multiple microscopy images on living
cells, the cells were grown on coverslips, which were then
mounted into the JPK Biocell™ for imaging. The Biocell™
(figure 2) is designed to optimise acquisition of
simultaneous optical and AFM images, while maintaining
an environment reflective of physiological conditions.
Fig. 2 The JPK Biocell™. The Biocell is designed to enable
optimal imaging conditions for both AFM and optical methods
while allow rapid and precise temperature control from 20-60°C.
The cells were imaged at 37°C in media containing
HEPES. The JPK Nanowizard® was installed on a Zeiss
Axiovert 200M inverted light microscope. Cells were
imaged in low force constant contact mode, with a flexible,
unsharpened cantilever. At the beginning of each scan a
phase contrast and fluorescence images were obtained.
Living cell images
As can be seen above, in figure 1, contact mode AFM
imaging can be used to obtain overview images of a whole
cell, or can be operated using small scan sizes to resolve
surface structures beyond the resolution of conventional
light microscopy. While the resolution and signal to noise
ratio of AFM are major advantages of this imaging
technique the information generated in images of larger
scan sizes can also be useful, and complementary to other
imaging techniques. AFM imaging is a mechanical
process, based on the interaction of the very flexible AFM
probe with the surface. Consequently, the information
generated is structural and mechanical. Additionally, AFM
is a surface technique, so AFM images are “focussed” on
the surface of the cell and the mechanical structures, such
as the actin cytoskeleton, that underlie it.
The combination of imaging the surface using AFM and
fluorescence imaging of focal adhesions allows the
investigator to compare the dynamics of focal adhesion
structures at the base of the cell with the actin structures at
the surface of the cell. The additional phase contrast
images give an overall impression cell morphology and can
help to determine whether structures observed at the cell
surface are due to vesicles that are clearly visualised in the
phase contrast image. As such, images are generated of
dynamics happening at the base, in the body and at the
surface of the cell.
In figure 3 a series of images is presented – each row
shows phase contrast, epifluorescence and contact mode
error signal images taken at 15 minute intervals. The cells
were dense on the coverslip, to inhibit cell motility, such
that smaller scale movement could be investigated. Larger
changes in cell structure can be seen in the phase contrast
images (circled in A,D,G). Vesicles within the cell can be
seen to have moved positions. A comparison of the three
epi-fluorescent images of GFP-labelled paxillin show little
change between each image.
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NanoWizard, CellHesion, BioMAT, NanoTracker and ForceRobot are
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A
B
C
DD
E E
F F
GG
HH
10 µm
I
Fig. 3 Multiple channel time-lapse images of REF52 fibroblast cells. Living fibroblast cells were imaged in phase contrast
(A,D,G) epi-fluorescence (B,E,H) and contact mode with the atomic force microscope (C,F,I) at t = 0 (A,B,C), t = 15 (D,E,F)
and t = 30 (G,H,I).
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 USA  China  Japan  Europe & other regions
NanoWizard, CellHesion, BioMAT, NanoTracker and ForceRobot are
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The white arrows in the fluorescent images (B,E,H)
highlight focal adhesion structures that do not seem to
change over the course of the scans. Interestingly, when
compared to the actin structure in the AFM images, the
focal adhesions do not seem to change, whereas there is a
distinct alignment of the actin fibres along the body of the
cell. In the original image a network is visible in the submembranous cytoskeleton. With each successive image
the cytoskeletal fibres become more aligned (black arrows)
and the cell body higher. In addition, there are many small,
flexible, highly dynamic protrusions on the flat areas of the
cell (circled in black). From these images the subsurfacecytoskeleton seems to be considerably more dynamic over
this time scale than the focal adhesions at the interface
between the cell and the support.
While here only one fluorophore is used, obviously the
fluorescence microscopy could be extended to include
multiple fluorescence channels. The laser in the JPK
Nanowizard® is of a wavelength beyond the visible
spectrum such that red channel fluorescence is not
disrupted by the presence of the AFM laser.
At smaller scan sizes, the degree of dynamism at the cell
surface can be more clearly seen. Two successive images
of the cell surface are presented in figure 4 as three
dimensional projections of topographic data. The two
images were taken successively, 15 minutes apart. The
large cytoskeletal structures at the top of both images are
similar, however much of the rest of the structure has
changed between images. A flexible ridge (black arrow)
seen in the first image has disappeared in the second.
Finer sub-surface structure can also be seen to have
changes considerable between the two images.
Conclusion
The AFM can be used to image living cells at
unprecedented resolution and on a time scale that is
suitable for addressing a number of biological processes.
The design of the JPK Nanowizard® enables
multidimensional microscopy combining AFM with optical
imaging techniques.
Fig. 4 Successive topographic images of the same region of the
cell surface. In both cases the scan size is 5 x 5 µm and the z
range 0-200 nm.
Such a setup can be used to investigate, simultaneously,
events occurring in different regions of the
cell.Alternatively, information gleaned from the different
contrast methods could be used to generate information on
structure-function relationships [8]. While AFM can
generate interesting and unique information about cells as
a stand-alone imaging technique, the integration of the
JPK Nanowizard® into a fully functional, inverted light
microscope extends the potential for AFM for cell imaging.
References:
[1] Schena, M., Shalon, D., Davis, R.W. & Brown, P.O. (1995)
Quantitative monitoring of gene expression patterns with a
complementary DNA microarray. Science 270:467–470.
[2] Kislinger T, Emili A. (2005) Multidimensional protein
identification technology: current status and future prospects.
Expert Rev Proteomics. 2(1):27-39.
[3] Zhang X, Chen A, De Leon D, Li H, Noiri E, Moy VT,
Goligorsky MS. Atomic force microscopy measurement of
leukocyte-endothelial interaction. Am J Physiol Heart Circ Physiol.
286:H359-67. (2004)
page 4/5
© JPK Instruments AG - all rights reserved – www.jpk.com
This material shall not be used for an offer in:
 USA  China  Japan  Europe & other regions
NanoWizard, CellHesion, BioMAT, NanoTracker and ForceRobot are
trademarks or registered trademarks of JPK Instruments AG
[4] Poole K, Khairy K, Friedrichs J, Franz C, Cisneros DA,
Howard J, Mueller D. (2005) Molecular-scale topographic cues
induce the orientation and directional movement of fibroblasts on
two-dimensional collagen surfaces. J Mol Biol. 349(2):380-6.
[5] Sharma A, Anderson KI, Muller DJ. (2005) Actin microridges
characterized by laser scanning confocal and atomic force
microscopy FEBS Lett. 579(9):2001-8.
[6] Chiantia S, Kahya N, Schwille P. (2005) Dehydration damage
of domain-exhibiting supported bilayers: an AFM study on the
protective effects of disaccharides and other stabilizing
substances. Langmuir. 21(14):6317-23.
[7] Kam Z, Zamir E, Geiger B. (2001) Probing molecular
processes in live cells by quantitative multidimensional
microscopy. Trends Cell Biol. 11(8):329-34.
[8] Poole K, Muller D. (2005) Flexible, actin-based ridges
colocalise with the beta1 integrin on the surface of melanoma
cells. Br J Cancer. 92(8):1499-505.
page 5/5
© JPK Instruments AG - all rights reserved – www.jpk.com
This material shall not be used for an offer in:
 USA  China  Japan  Europe & other regions
NanoWizard, CellHesion, BioMAT, NanoTracker and ForceRobot are
trademarks or registered trademarks of JPK Instruments AG