Fundamentals of Phase Contrast Microscopy
Phase contrast microscope is a special microscope that converts the optical path difference (ie phase difference) generated when light passes through the details of transparent specimens into light intensity difference.
When light passes through a relatively transparent specimen, neither the wavelength (color) nor the amplitude (brightness) of the light changes appreciably. Therefore, when observing unstained specimens (such as living cells) with ordinary light microscopy, their morphology and internal structure are often difficult to distinguish. However, due to the difference in the refractive index and thickness of the various parts of the cell, when light passes through such a specimen, the optical path of direct light and diffracted light will be different. As the optical distance increases or decreases, the phase of the speeding or lagging light waves changes (creating a phase difference). The phase difference of light cannot be felt by the naked eye, but the phase contrast microscope can use the interference phenomenon of light through its special device-annular diaphragm and phase plate, to convert the phase difference of light into an amplitude difference (light and dark) that can be perceived by human eyes Poor), so that the original transparent objects show obvious differences in light and shade, and the contrast is enhanced, so that we can clearly observe living cells and cells in cells that cannot be seen or clearly seen under ordinary light microscopes and dark field microscopes. certain fine structures.
The imaging principle of the phase contrast microscope: During the microscope inspection, the light source can only pass through the transparent ring of the annular diaphragm, and then converges into a beam after passing through the condenser. Degree of deflection (diffraction). The image formed by the transparent ring just falls on the rear focal plane of the objective lens and coincides with the conjugate plane on the phase plate. Therefore, undeflected direct light passes through the conjugate plane, while deflected diffracted light passes through the compensation plane. Due to the different properties of the conjugate surface and the compensation surface on the phase plate, they will produce a certain phase difference and weaken the intensity of the light passing through the two parts respectively, and the two groups of light will converge on the same optical path after the convergence of the rear lens. The direct light and the diffracted light produce light interference, and the phase difference is changed into an amplitude difference. In this way, when the phase contrast microscope is inspected, the light passing through the colorless transparent body converts the phase difference that cannot be distinguished by the human eye into an amplitude difference (light and dark difference) that the human eye can distinguish.
