Page 191 - Fundamentals of Light Microscopy and Electronic Imaging

174 DIC MICROSCOPY AND MODULATION CONTRAST MICROSCOPY

scrape the underside of your tongue with the edge of a #1.5 coverslip to col-
lect 10–20 L of clear saliva and surface cells of the stratified squamous
epithelium, and then mount the coverslip on a microscope slide.

1. Examine the preparation by phase contrast and DIC optics using a 40 or
100 oil immersion objective, and compare the quality of the images.
Focus through the specimen during observation and notice the clarity of
optical sectioning using DIC optics.
2. Carefully examine and test the alignment of polarizers and DIC prisms, as
viewed at the back focal plane of the objective lens with a telescope eye-
piece or Bertrand lens.
• Examination of living tissue culture cells. Obtain a coverslip of tissue culture
cells (COS7 cells, U2OS cells, or other flat epithelial cells are ideal). Cul-
tures are prepared 1–2 days beforehand using plastic tissue culture dishes
containing #1.5 coverslips presterilized by dipping in ethanol and holding
briefly over a flame. Cell organelles that are identifiable in DIC include the
nucleus, nucleolus, heterochromatin, lysosomes and peroxysomes, secretion
granules, lipid droplets, mitochondria, rough endoplasmic reticulum, Golgi
apparatus, centrosomes and centrioles, and stress fibers consisting of bundles
of actin filaments. Membrane specializations, including the leading edge,
membrane ruffles, filopodia, and microvilli, should also be visible. DIC
optics provide high sensitivity of detection and the ability to optically section
through the specimen.
1. Carefully remove a coverslip with a pair of fine forceps, making sure not
to damage the forceps or wipe off the surface layer of cells. Next clean
off and wipe dry (carefully!) the back side of the coverslip with a water-
moistened lab tissue, again being careful not to break the coverslip. The
cleaned surface should be immaculate and dry.
2. Mount the coverslip with Vaseline spacers as shown in Figure 10-14.
Clean off the back surface of the coverslip with a moistened lab tissue.
Place the dry coverslip on a clean surface, cell-side-up, and wipe away
cells and medium within 1 mm of two opposite edges of the coverslip. Act
quickly so that the cells do not dry out. Smear a small bead of Vaseline on
the edge of the palm of your hand and drag the edge of the coverslip over
the surface to make a small, even ridge of Vaseline on the two dried edges
of the coverslip (the side facing the cells). Mount the coverslip on a slide
as shown, with the edges containing Vaseline parallel to the long axis of
the slide, and gently tap the coverslip to ensure good attachment to the
glass slide. Place 1 drop of Hepes-buffered Minimal Essential Medium
(HMEM) against one open edge of the coverslip to nearly fill the cham-
ber. Do not overfill, or the coverslip will float loose from the slide and be
difficult to focus. Use the edge of a filter paper to wick away excess
medium beforehand if necessary. It is better to leave a little airspace than
to overfill the chamber. To prevent evaporation, you can seal off the two
exposed edges with small drops of immersion oil.