MICROSCOPY: IMAGING OF BIOLOGICAL SPECIMENS - A Guide to Methods in the Biomedical Sciences - page 89

of their very dynamic structural components, the organelles, or to look
within tissues and observe living cells moving within them. The addition
of motorized stages or objectives on the microscope now allows inves-
tigators to look not only at a flat image of a cell, but to progressively
“slice through” the cell from bottom to top. Thus, cells can be visualized
not only in two dimensions, but in three dimensions. The capture of the
information from the third dimension, the so-called z-stack, once ren-
dered by computational software within the computer, now allows for the
reconstruction of a three-dimensional image of the cell. Together with
conventional biochemical techniques, a much deeper understanding of
cell structure and function is emerging from the use of modern light
microscopes.
The following sections outline some of the methodology that is used
to image cells and tissues at high resolution.

B. Light microscopy

Light microscopy is a generic term for a variety of techniques that
all employ a standard compound microscope. Specimens are usually
placed on slides for viewing, and may be living or fixed (and therefore
are not viable). Some microscopes are fitted with stage adaptors that
keep samples warmed to biological temperatures. This allows the activity
of the cells, or organelles within them, to be viewed overtime using time-
lapse photography.

Phase-contrast

The simplest form of light microscopy uses “brightfield” illumination,
in which a beam of light is focused on the object to be viewed. Although
cells and tissues can be seen, many biological specimens are almost
transparent under these conditions. Consequently, contrast is poor, and
details are lacking. Phase-contrast helps to correct this by improving the
resolution of unstained biological specimens. This method was adapted
in the late 1930's and 1940's by Zeiss laboratories. The contrast of
cells is improved because the image is revealed as lighter or darker
depending on the different densities within the cell being viewed; the
greater the density, the longer the path length of the light being viewed,
and therefore the darker they appear.

Differential interference contrast (DIC)

DIC microscopy, also known as Nomarski optics , is a method used to
further increase the surface contrast of specimens in greater detail than