Phase Contrast Microscopy

Phase Contrast Microscopy is a compound microscope fitted with a phase contrast condenser lens and
phase contrast objective lens. In phase contrast microscope, microscopic objects can be seen in
unstained condition due to the difference in the refractive index of the objects and surrounding
medium. Unstained organelles within the cells can also be observed due to the slight difference in the
refractive index or thickness. PCM are of two types that is
1. Positive PCM where object is brightly illuminated than the background
2. Negative PCM where surrounding or background is brightly illuminated than the objects
The technique is based on the fact that the light passing from one object into another object of a slightly
different refractive index thickness undergoes a change in the phase (wavelength). These phase
differences are translated into the variation in brightness of the image and hence detected by the eye.

Fig. Instrumentation (Ray Diagram) of PCM

The image of the object is formed at the rear focal plane of the objective lens. In this plane there is a
phase shifting element or phase plate. The phase plate also has an annular ring that can increase the
wavelength of light passing through it. Light coming through the condenser passes through the object.
Rays which are not deviated by the object pass through annular rings of the phase plate and acquire
longer wavelength. Those rays which are deviated by the object due to the different refractive index
pass through the phase plate not covered by the annular rings. Thus, their wavelength remains
unchanged. Thus, their wavelength remains unchanged. The difference in the phase (wavelength) gives
the contrast which helps in clear visibility of the object. Magnification of unto 400 times can be achieved
in phase contrast microscope.

Specimen preparation
To prepare cell for examination in the PCM, the cells can be grown in monolayer directly on the
coverslip bathed in the cell culture medium. The monolayer containing the coverslip is mounted upside
down on a microscope slide. The specimen can then be examined in the microscope.
To preserve the morphology of cells or tissue, the specimen has to be fixed by drying, freezing or using
chemical fixatives. Thick specimen such as tissues is sectioned before mounting on a microscopic slide.
Microtome (knife) is used to prepare thin slices of the specimen.

Advantages of PCM
1. Since the cells are examined unstained, natural morphology can be observed.
2. PCM gives high contrast and high resolution image
3. The technique is appropriate for studying and interpreting thin specimen
4. Modern phase contrast microscope with charged coupled device (CCD) or complementary metal
oxide semi-conductor (CMOS) computer devices can capture photo and/or video.

Disadvantages of PCM
1. Annular rings limit the aperture to some extent which decrease the resolution
2. The method is not ideal for the thick specimen
3. Image may appear green or grey if white or green light area used, resulting in poor photo
micrograph
4. Shade off and halo effect may be seen as phase artifacts
Fluorescence Microscopy
Fluorescence microscope is an optical microscope that uses fluorescence to generate an image.
Fluorescence microscopes are of two types:
1. Epifluorescence: it is called epifluorescence because the light source lies above the specimen and
objective lens
2. Confocal microscope: it uses optical sectioning to get better resolution of the fluorescent image

Principle of Fluorescence Microscope:

Instrumentation (Ray Diagram) fluorescence Microscope

The fluorescence microscope is based the principle of fluorescence. Fluorescence is a phenomenon in

which certain fluorescent chemicals absorb the light of particular wavelength and then emits the light of
longer wavelength with lesser content of energy.
The specimen is illuminated with light of specific wavelength which is absorbed by fluorophore
(fluorescent emitting compound) causing them to emit light of longer wavelength. The excited light is
separated from the emitted fluorescence through the use of spectral emission filter. The filters and
dichoric filter are selected to match the spectral ecitation and the emitted fluorescence of the
fluorophore, which is used to label the specimen. In this way the fluorescence of a single fluorophore is
detected in the form of image at a time. Multicolor image of several types of flurophores can also be
detected by using different combination of filters.

Instrumentation:
Light source of the epifluoresence microscope are Xenon arc, Mercury vapor, light emitting diodes
(LEDs), lasers etc. The excited light and the emitted fluorescence pass through the same light path (that
is through the objective lens). The excited light is focused on the specimen through the objective lens.
The fluorescence emitted from the specimen is focused to the detector by the same objective lens.