Have you ever understood the basic principles of phase contrast microscopy?
The phase-contrast microscope was invented by the Dutch scientist Zernike in 1935 for observing unstained specimens. For living cells and unstained biological specimens, due to the difference in the refractive index and thickness of the microstructure of each part of the cell, when the light wave passes through, the wavelength and amplitude do not change, only the phase changes (amplitude difference), which is invisible to the human eye. observe. The phase contrast microscope changes the phase difference and uses light diffraction and interference phenomena to change the phase difference into an amplitude difference to observe living cells and unstained specimens. The difference between a phase contrast microscope and an ordinary microscope is that an annular diaphragm is used instead of a variable diaphragm, an objective lens with a phase plate is used instead of an ordinary objective lens, and a telescope for coaxiality is provided.
Basic principles of phase contrast microscopy:
Using the difference in refractive index and thickness between different structural components of the object, the optical path difference passing through different parts of the object is converted into the difference in amplitude (light intensity), and through the condenser lens with an annular diaphragm and the phase difference with a phase plate The objective lens realizes the observation microscope. It is mainly used to observe living cells or unstained tissue sections, and sometimes it can also be used to observe stained samples that lack contrast.
The optical path difference of the visible light passing through the specimen is changed into an amplitude difference, thereby improving the contrast between various structures and making various structures clearly visible. The light is refracted after passing through the specimen, deviates from the original optical path, and is delayed by 1/4λ (wavelength). If it is increased or decreased by 1/4λ, the optical path difference becomes 1/2λ, and the two beams interfere after the axis of light Strengthen, increase or decrease the amplitude, improve the contrast.
The phase contrast microscope has two functions that other microscopes do not have: ① it separates the direct light (background light in the field of view) from the light diffracted by the object; ② it removes about half of the wavelength from the phase so that it cannot interact, resulting in a change in intensity.
