Biology Reference
In-Depth Information
FIGURE 20.2
Optimization of mitotic spindle and cell structure preservation. Orthogonal sections of
cells fixed with 280, 440, or 1100 mOsm solutions. Representative high-magnification
electron micrographs of the cytosol in each condition are shown below. Scale bars 5 mm
(overview) and 100 nm (bottom).
20.2.2.2
Light microscopy
On the day of sample processing, cells of interest can be identified under the light
microscope with a 20
air objective. The low magnification allows images to be
acquired containing a large field of view, useful for cell relocation during later pro-
cessing. Some cells expressing fluorescent proteins require prefixation imaging as
their fluorescence is obscured by the autofluorescence created by glutaraldehyde.
Once the cell of interest has been located and imaged, add fixative solution for
1 h. It should be noted that microtubules are sensitive to temperature changes
(
Engelborghs, Heremans, Demaeyer, & Hoebeke, 1976
); we, therefore, recommend
that imaging of unfixed cells is carried out using an appropriate live imaging cham-
ber at 37
C.
After fixation, replace the fixative solution with 1-2 ml wash solution with 0.1%
Hoechst-33342 (or similar DNA dye), incubate for
20 min, rinse three times with
wash solution (leaving on 1 ml of the final wash), and return the dish to the micro-
scope. This second round of imaging is an opportunity to acquire high-magnification
images of cells of interest, using 60
oil-immersion objectives (
Fig. 20.1
B,
left). Take fluorescent and white light images of the cell, and also images of the
same field of view focused on the coordinates. These will serve as references later
to pinpoint the cell in the resin block and determine the orientation of the spindle
axis. It helps at this stage to carefully wipe off any immersion oil with ethanol,
or 100
