The Microscopy Series: Phase Contrast Microscope

When light travels through a medium other than #vacuum, interaction with the medium causes the wave amplitude and phase to change in a manner dependent on properties of the medium. #Camera and human eye are only sensitive to amplitude variations not phase changes, which #PhaseContrastMicroscope promises to show us!

Image Courtesy: Photometrics.com

Components:

The common components of the microscopes like #OcularLens, #ObjectiveLens, #Condenser, #SpecimenStage etc. are not described here for the sake of keeping the article brief.

1. Annular diaphragm: This consists of a circular disc with a circular annular groove, with light rays allowed to pass through it and is placed below the #Condenser. The illuminating rays fall on the object through the #AnnularGroove and an image of this illuminated opening is formed at the back #FocalPlane of objective.

2. Annular Phase Plate (Phase Shift Ring): Placed at the back #FocalPlane of objective, it consists of a #transparent disc with an #AnnularGroove. This groove is made to coincide with the image of the aperture of the annular diaphragm. All the light rays directly pass through the groove in the phase plate whereas the diffracted light pass mainly outside the groove. This eliminates the phase difference between the background and the scattered light, leading to an increased intensity difference between #foreground and #background.

3. Gray Filter Ring: This dims the background to further increase the #Contrast. Some of the scattered light is phase-shifted and dimmed by these rings.

Merits

1. Cells can be observed in #living #natural state.

2. #HighContrast #HighResolution images are produced.

3. Ideal for studying #thin specimens.

4. Optical construction of image is relatively simple.

5. It can be combined with #Fluorescence microscope.

6. Latest instruments with #CCD or #CMOS devices can record #video also.

Demerits

1. #Annuli or #Rings limit the aperture to some extent, compromising on #resolution.

2. Not ideal for #thick specimen.

3. #ShadeOff occurs for larger particles resulting in steady reduction of contrast.

4. #HaloEffect obscures details along the #perimeter of the field.

5. Images may appear #Grey, #Green or #White resulting in poor #Photomicrography.

Applications

1. Study of #Mitosis and #Meiosis in living cells.

2. Study of #permeability, #phagocytosis and #pinocytosis of cell membrane.

3. Study of behaviour of living #Protozoa towards physical and chemical factors.

4. Determination of cell cycle time with #Timelapse microscopy.

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