components of phasecontrast microscope
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Answer:The basic principle to making phase changes visible in phase-contrast microscopy is to separate the illuminating (background) light from the specimen-scattered light (which makes up the foreground details) and to manipulate these differently.
The ring-shaped illuminating light (green) that passes the condenser annulus is focused on the specimen by the condenser. Some of the illuminating light is scattered by the specimen (yellow). The remaining light is unaffected by the specimen and forms the background light (red). When observing an unstained biological specimen, the scattered light is weak and typically phase-shifted by −90° (due to both the typical thickness of specimens and the refractive index difference between biological tissue and the surrounding medium) relative to the background light. This leads to the foreground (blue vector) and background (red vector) having nearly the same intensity, resulting in low image contrast.
In a phase-contrast microscope, image contrast is increased in two ways: by generating constructive interference between scattered and background light rays in regions of the field of view that contain the specimen, and by reducing the amount of background light that reaches the image plane. First, the background light is phase-shifted by −90° by passing it through a phase-shift ring, which eliminates the phase difference between the background and the scattered light rays.
Working principle of phase contrast microscopy.gif
When the light is then focused on the image plane (where a camera or eyepiece is placed), this phase shift causes background and scattered light rays originating from regions of the field of view that contain the sample (i.e., the foreground) to constructively interfere, resulting in an increase in the brightness of these areas compared to regions that do not contain the sample. Finally, the background is dimmed ~70-90% by a gray filter ring—this method maximizes the amount of scattered light generated by the illumination (i.e., background) light, while minimizing the amount of illumination light that reaches the image plane. Some of the scattered light (which illuminates the entire surface of the filter) will be phase-shifted and dimmed by the rings, but to a much lesser extent than the background light (which only illuminates the phase-shift and gray filter rings).
The above describes negative phase contrast. In its positive form, the background light is instead phase-shifted by +90°. The background light will thus be 180° out of phase relative to the scattered light. The scattered light will then be subtracted from the background light to form an image with a darker foreground and a lighter background, as shown in the first figure.