Page 128 - Fundamentals of Light Microscopy and Electronic Imaging

PHASE CONTRAST MICROSCOPY 111

The phase shift of light, , which occurs when light passes through a cell
with refractive index n in a medium with refractive index n , is given by (n
o m o
n ) t/λ, where t is the thickness of the object and λ is the wavelength. The
m
phase contrast microscope converts the phase shift into an observable change in
amplitude. When n n (the case for most cellular structures), objects appear
o m
dark against a gray background (positive contrast). When n n , objects look
m o
bright against a gray background (negative contrast). When n n , no relative
o m
retardation occurs and the object is invisible. If blood cells are placed in a series
of albumin solutions of increasing concentration (made isotonic by adjusting the
concentration of NaCl to prevent shrinking and swelling due to osmotic effects),
positive and negative cells can be counted under the phase contrast microscope
and a curve can be constructed, and the isotonic point can be determined from the
point at which 50% dark and bright cells are observed. This is a sensitive null
method that can be used to obtain the concentration of solids in cells. In this case,
we will calculate the intracellular molarity of hemoglobin in erythrocytes. Note:
For erythrocytes, the intracellular concentration of hemoglobin is so high that
cells look bright against a gray background when examined in normal isotonic
saline. In the following exercise on erythrocytes, positive cells appear bright, and
negative cells look dark.

1. Swab the tip of your finger with 70% ethanol and prick it with a sterile
disposable lancet. Place a small drop of fresh blood (5 L) on a micro-
scope slide. Immediately place one drop ( 60 L) of albumin test solu-
tion on the droplet of blood, cover with a coverslip, and count the number
of positive and negative cells—100 cells total—for each sample. Count
only single cells that are seen face on. Do not count cell aggregates. If
necessary, you can check for “invisible” cells by turning away the con-
denser annulus, but perfect refractive index matching occurs only rarely,
because the biconcave shape of the cells results in optical path differences
through a single cell. Midpoint cells will appear both black and white
simultaneously. It is recommended that you prepare and score one slide at
a time. To recognize typical positive and negative cells, you should test
the extreme albumin concentrations first. You should place your slides in
a moist chamber (a sealed container with a moist paper towel) so that they
do not dry out while you are busy counting other slides. Calculate the
number of negative cells for each solution. Show your work.
2. Plot the % negative cells vs. mg/mL albumin on a piece of graph paper,
and determine the concentration of albumin giving 50% positive and neg-
ative cells.
3. Given that hemoglobin has about the same specific refractive index incre-
ment as albumin and comprises 97% of the cell solids, having determined
the concentration of albumin that is isotonic to the cells, calculate the
molar concentration of hemoglobin in the erythrocytes. The molecular
weight of native hemoglobin tetramer is 64,000 daltons. Treat the con-
centration (mg/mL) of bovine serum albumin (molecular weight, 67,000
daltons) as if it were hemoglobin, and use the molecular weight (of either)
to calculate the molarity of hemoglobin in erythrocytes. How many times