Page 219 - Fundamentals of Light Microscopy and Electronic Imaging

202 FLUORESCENCE MICROSCOPY

contact the skin, and incubate the cells at 22°C for 60 min. Alternatively, stain
living cells with Hoechst dye 33258 at 0.5 g/mL for 15 min at 22°C. Mount and
examine. Remove the dye and rinse the cells. Look for reticular patterns caused
by euchromatin and heterochromatin in the nuclei. Also look for nucleoli, which
are the sites of RNA processing in the nucleus. What do mitotic chromosomes
look like? Make sketches.

MitoTracker or LysoTracker/mitochondria, lysosomes: Simply add
these potential uptake dyes to 250 nM in the culture medium and incubate the
cells at 37°C for 5–10 min. Mount and examine. Carefully discard the staining
solution. Are the dyes specific for mitochondria and lysosomes? How many
mitochondria/lysosomes are there in a typical cell? What is the distribution of
their lengths/diameters? Determine if you can see mitochondria round up and
migrate toward the center of the cell, a stress response of the cell to non-ideal
environmental conditions and prolonged observation. Make sketches.

Bodipy phalloidin/actin stress fibers: After rinsing in HMEM, fix the
cells in 4 mL 0.5% glutaraldehyde in HMEM for 15 min. Dilute the dye 1:40 in
the culture medium containing 0.1% Triton 100 detergent and incubate the cells
at 37°C for 30 min. Mount and examine after thorough rinsing in HMEM. Are the
filaments you see individual actin filaments or filament bundles? How are stress
fibers oriented with respect to the long axis of the cell, cell borders, and vertices
that represent points of cell attachment? Estimate the amount of fluorescence
associated with stress fibers vs. unorganized filaments subjacent to the plasma
membrane. Prepare careful drawings.

Examination of double stained specimens. If the microscope is
equipped with two different filter sets for the same dye (for example, for
examination of fluorescein, a filter set with a narrow band-pass emission filter
and another set with a long-pass emission filter), it is useful to examine
specimens double stained with fluorescein and rhodamine.
It is possible to directly examine the transmission profiles of the dichroic
mirror-emission filter combination of a given filter set right at the microscope.
Just switch the microscope to transmitted white light, mount a diffraction grating
on the stage, close the condenser diaphragm down to a minimum, and examine
the colors of the first order diffraction spectra with the aid of a Bertrand lens.
Examine all of the filter sets to determine the bands of wavelengths transmitted
by the sets.

(1) Is there any bleed-through of fluorescence of one dye through the filter set
intended for viewing the other dye?
(2) Is the amount of rhodamine bleed-through different for the two types of
fluorescein filter sets? Why? In preparing to answer why bleed-through
occurs, it is useful to compare transmission profiles of the various filters
of the filter sets and to compare them with the excitation and emission