Thesis-PDF - IAP/TU Wien

Figure 5.6: If the user menu AFM_TUVIENNA is loaded, the user can
chose one of four custom functions to apply to the available scan data
liquid medium. At some point of the drying process the cells outer walls break
and the inner cell parts are spilled out. The cells lose shape and material attaches
to the outer cell membranes. This hinders imaging of intact cell surfaces. In
addition enzymatic activity damages biological membranes that get in contact
with it, altering their properties or slowly dissolve them altogether.
Dried whole cells allowing for AFM investigation in air were prepared according
to the following protocol (see Fig. 5.7): 100 µl of cell suspension were pipetted
onto a glass slide and covered with a coverslip. Finger-tight force was applied
onto the coverslip, removing excess solution and air bubbles. Slow evaporation
of the solvent at room temperature resulted in a concentration gradient of nutrient
embedding whole unscathed cells especially at the edges. After 5 minutes the
coverslip was carefully removed by dragging it horizontally over the glass slide,
and the samples were investigated with AFM. Such preparations were good AFM
specimens for several days.
Crystalline cell parts for AFM investigation were prepared according to the
preparation method described in 5.1.2. Before AFM imaging, one milliliter of the
solution was centrifuged with a home-made centrifuge (see Fig. 5.8) at 800 g for
10 minutes (to remove small suspended particles). Then the fraction of the solid
precipitate containing the heaviest particles was resuspended in one milliliter of
HEPES solution, spread on a glass slide and dried.
A number of variations of this protocol was tried as well, including the use of
specially coated slides (e.g. poly-l-lysine or gelatin coated slides) to improve the
adhesion of the algal cells to the slide.
5.3.3 Preparation for AFM imaging under liquid
Plastic petridishes were used for imaging under liquid. The petridish was attached
to the scanning stage of the AFM. Then a piece of glass slide was attached at its
bottom. A few drops of liquid medium containing the cells or cell parts was spread
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