Chapter 3 – Making Light Work in Biology  75

relationship by comparing the speed of light in water to that inside a cell or tissue on the basis

of differences in refractive indices:

(3.22)

∆z

n

n

n

w

t

w

=

−

(

)

λ

4

An annulus aperture in the front focal plane of the condenser lens, similar to that used for

dark-​field forward scatter microscopy in blocking out the central aperture of illumination,

generates a cone of collimated light onto the sample. Emergent light transmitted through

the sample is collected by an objective lens consisting of both undeviated light (since the

angle of the cone of light is not as oblique as that used in dark-​field microscopy) that has

not encountered any biological material and diffracted (forwarded scattered) light that has

exhibited a relative phase retardation to the undeviated light.

A phase ring in the back focal plane of the objective lens, in a conjugate image plane to the

condenser annulus, converts this retardation into a half wavelength phase shift, a condition

for destructive interference, either by introducing a half wavelength phase increase in the ring

(positive phase contrast microscopy) by having an extra thickness of glass, for example, in

which case the background appears darker relative to the foreground sample, or more com­

monly by introducing a further half wavelength phase retardation in the ring (negative phase

contrast microscopy) by indenting the glass in that region, in which case the sample appears

brighter relative to the background, or by coating the ring in a thin layer of aluminum.

In other words, this process transforms phase information at the sample into amplitude

contrast in the intensity of the final image. The length scale of a few microns over which the

retardation of the light is typically a quarter of a wavelength is comparable to some small cells

in tissues, as well as cellular organelle features such as the nucleus and mitochondria. It is

therefore ideal for enhancing the image contrast of cellular components.

Polarized light microscopy can increase the relative contrast of birefringent samples.

Birefringence, as discussed for polarization spectroscopy techniques in Section 3.2.4, occurs

when a sample has a refractive index which is dependent upon the orientation of the polar­

ization E-​field vector of the incident light. This is often due to repeating structural features in

a sample, which have a spatial periodicity over a length scale comparable to, or less than, the

wavelength of the light, which is true for several biological structures. In other words, this is

a characteristic of certain crystals or more relevant for biological samples due to the fluidity

of the water-​solvent environment and other fluidic structures such as phospholipid bilayers,

liquid crystals.

There are several examples of birefringent biological liquid crystals. These include fibrous

proteins with well-​defined spatial periodicity between bundles of smaller fibrils such as col­

lagen in the extracellular matrix, cell membranes and certain proteins in the cell membranes,

cytoskeletal proteins, structural proteins in the cell walls of plants (e.g., cellulose) and cer­

tain bacteria (e.g., proteoglycans), and the highly periodic protein capsid coats of viruses.

Polarization microscopy is an excellent tool for generating images of these biological liquid

crystal features, and there are also examples of nonliquid crystalline biomolecule samples

that can be investigated similarly (e.g., crystalline arrays of certain vitamins).

For polarization microscopy, a polarizer is positioned in the illumination path between the

VIS light source and a condenser lens, before the sample, and a second polarizer described

as an analyzer is positioned after the transmitted light has emerged from the sample, close to

the back aperture of the objective lens. The transmitted light through a birefringent sample

can be split into two orthogonally polarized light components of p and s, which are either

parallel to the plane of the optic axis or perpendicular to it, respectively. The speed of the light

in each of these separate components is different due to the polarization dependence of the

refractive index in the sample. These components therefore become out of phase with each

other but are recombined with various combinations of constructive and destructive inter­

ference during their passage through the analyzer, depending upon the relative position on

the sample, which is then imaged onto a camera (or viewed through eyepieces) in the normal

way for basic light microscopy. Polarized light microscopy can quantify the precise amount of