Sample Preparation - JPK instruments nanowizard afm Handbook

5.2 Sample preparation

Substrate choice and cell attachment
For high resolution imaging of isolated molecules, mica is the support of choice,
because it provides such a flat background. However, for cell imaging, good quality
glass is sufficiently flat and has optical properties far superior to those of mica. For
the combination of light microscopy and AFM imaging, the use of a coverslip as a
support can give the best results, but only if the coverslip is mounted in a
sufficiently stable holder, such as the BioCell™ available from JPK. Otherwise the
thin glass coverslip
fluctuations/vibrations will be seen in the images. When using the BioCell for
simultaneous light microscopy and AFM imaging, the choice of objective should
not affect AFM quality and the use of oil or water immersion lenses should not
introduce vibration into the system.
In many cases cells will naturally adhere to glass or mica and no special surface
treatment is necessary. However, with non-adherent samples such as red blood
cells, yeast, bacteria etc the cells must be attached to a substrate before imaging.
When imaging small structures, such as single molecules, many surface
treatments can generate structures that mask the objects of interest. In the case
of cells, however, this is not a problem as the size of the cells will be considerably
larger than the deposited surface layer. One standard approach is to coat the
desired substrate (i.e. mica or glass) with poly-L-lysine. Poly-L-lysine is a positively
charged polymer which adsorbs very well to negatively charged glass or silicon
dioxide leading to positively charged surfaces. Surface coating with Poly-L-lysine
has been used to attach both cells and proteins to glass or mica.
Living or fixed cells?
The decision on whether or not to fix cells will be influenced by the experimental
question to be asked. There are a number of circumstances under which it is either
recommended or necessary to fix cells before imaging. If high resolution images of
the cell surface are required then fixation of the cells will lead to a stiffening of the
cell surface and an increased attainable resolution, as determined by the nature of
the atomic force microscope. Additionally, in many experiments where AFM is to
be combined with fluorescence microscopy there will be a need for fixation to allow
fluorescent labeling. On the other hand, fixation is not appropriate when in situ
experiments are to be performed, when the object of the experiment is to
investigate some dynamic process or when imaging is to be combined with
elasticity measurements.
An optimal fixation protocol will avoid dehydration of the cells, as this leads to
significant changes in surface structure. Additionally, it is desirable to avoid
background fluorescence from the fixation protocol and to maximize fixation of
structures to enhance AFM imaging. One approach that meets these criteria
involves using a short fixation in glutaraldehyde followed by an extended
incubation in paraformaldehyde. Care must be taken to wash cells thoroughly after
fixation as residual fixant can make the surface sticky and cause problems during
scanning.
will not be mechanically
JPK Instruments
stable enough, and
®
NanoWizard Handbook
Poly-L-lysine coating protocol:
Incubate surface under 10 mg/ml
Poly-L-lysine (Mr 1000-4000) for 1-5
minutes. Wash the surface with water
and dry under nitrogen. Incubate
coated surface with sample of cells in
suspension until cells settle and stick
to surface (5 – 10 minutes), gently
rinse with relevant buffer/media
Cell fixation protocol:
Wash cells with PBS (containing
2+ 2+
Ca /Mg ) and add sufficient
glutaraldehyde (2% in PBS
2+ 2+
containing Ca /Mg ) to cover the
surface. Incubate for 45 seconds,
remove glutaraldehyde and add
paraformaldehyde (4% in PBS
2+ 2+
containing Ca /Mg ), incubate for 20
After removal of the
paraformaldehyde solution, wash
cells thoroughly (at least 5 x) with
PBS. Fixed cells can be stored for a
week (or sometimes more) in PBS,
but cells should not be allowed to dry
out at any stage.
Version 2.2a
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minutes.
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