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Introduction

This document describes the proper procedures to set

up an Olympus BH-2 microscope for phase contrast

viewing.

Scope of this Document

The procedure described here was performed using an

Olympus BHT microscope, but this procedure also

applies to the BHS, BHSP, BHSU, BHTP, and BHTU

models as well.

What is Phase Contrast?

The technique of phase contrast microscopy was

developed in the 1930s by Dutch physicist Frits Zernike

and began to be broadly used by 1942. Zernike was

awarded the Nobel Prize in Physics for his achievement

in 1953. Phase contrast techniques are most useful for

studying living, non-stained specimens, since live

specimens cannot typically be stained without affecting

their behavior or killing them outright. For these types

of unstained specimens, phase contrast provides

significantly increased contrast as compared to

conventional brightfield microscopy.

phase contrast optics exaggerate the differences in the

phase relationships between the light waves in the

background illumination and the light waves passing

through the specimen, so that they can constructively

or de-constructively interfere with each other at the

intermediate image plane, thereby converting invisible

phase differences into visible image contrast.

How Does Phase Contrast Work?

In conventional brightfield microscopy, a visible image is

formed by wave interference at the intermediate image

plane of the background illumination (the background

illumination is the light that does not pass through the

specimen, which is also known as the "S", or surround

wave-front) and of the diffracted light (the diffracted

light is the light that passes through the specimen under

observation, which is also known as the "D", or

diffracted wave-front). The image of the specimen is

visible to the observer due to the diffraction,

absorption, and phase-shifting that occurs as the D

wave-front passes through the specimen under

observation. Diffraction in the D wave-front occurs as a

result of detail in the specimen. Absorption occurs as a

result

of the

specimen being not

transparent.

Phase shift occurs as a result of

differences in the refractive index of the specimen, as

compared to the surrounding medium.

When viewing live, unstained specimens in brightfield,

the specimen is often difficult to see since light

absorption

can

constructive/destructive interference that occurs as a

Phase Contrast on the Olympus BH-2 Microscopes

In a nutshell,

completely

minimal

and

since

result of the phase-shifted D wave-front is minimal as

well. Phase contrast microscopy utilizes special optics

(both in the condenser and in the objectives) to

accomplish two things: 1) The S wave-front (i.e., the

background illumination) is decreased in amplitude by

the phase ring in the objective so that the intensity of

the D wave-front will not be swamped by the otherwise

bright background lighting. 2) The S wave-front is phase

shifted by a quarter wavelength by the phase ring in the

objective, thereby exaggerating the constructive or

destructive interference that occurs between the S and

D wave-fronts at the intermediate image plane. The

result of these two things is that images of live,

unstained specimens have significantly higher contrast

than could otherwise be obtained using convention

brightfield microscopy.

Phase Contrast Types

Olympus phase contrast optics for the BH-2 are

available in two basic types: Positive and Negative, as

indicated by the markings on the objective barrel.

Positive phase contrast objectives are marked with a

"P" (such as "PL" or "PLL") and negative objectives are

marked with an "N" (such as "NH" or "NM").

In positive phase contrast, the phase ring in the

objective advances the S wave-front by a quarter

wavelength, relative to the D wave-front, whereas in

negative phase contrast, the phase ring retards the S

wave-front by a quarter wavelength, relative to the D

wave-front. In both cases, the phase of the D wave-

front is retarded by areas of the specimen which have a

higher refractive index than the surrounding medium,

and advanced by the areas of the specimen which have

a lower refractive index than the surrounding medium.

In positive phase contrast, the advanced S wave and the

retarded D wave destructively interfere at the

intermediate image plane, resulting in the areas of the

specimen with a higher refractive index than the

surrounding medium appearing darker than the neutral-

gray background. In negative phase contrast, the exact

opposite occurs. The retarded S wave and the retarded

D wave constructively interfere at the intermediate

image plane, resulting in the areas of the specimen with

a higher refractive index than the surrounding medium

appearing lighter than the neutral-gray background.

Setting Up for Phase Contrast Microscopy

The following equipment will be needed to utilize phase

contrast on Olympus BH-2 microscopes.

• Olympus BH-2 microscope

• BH2-PC or BH2-PCD Zernike-style phase contrast condenser

• Olympus CT-5 or CT-30 phase-centering telescope (as

the

appropriate)

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