Debugging methods and steps for phase contrast microscopy
a. On the basis of adjusting the Kuhler lighting system, use the bright field method to focus the sample clearly;
b. Turn the spotlight to Ph1 and align it with the scale line on the turntable. Select a 10 x phase contrast objective and replace it with the transparent sample to be observed;
c. Remove one of the eyepieces, replace it with a centering telescope, and focus on the two contrast rings in the field of view (the black contrast ring of the objective lens and the transmittance contrast ring of the condenser lens);
d. The two difference rings in the field of view may not necessarily coincide. Adjust the two adjustment devices on the spotlight (adjusting the left and right positions of the difference rings with adjustment rods and friction type knobs for adjusting the front and rear positions), so that the transparent ring moves back and forth to coincide with the black ring;
e. After adjustment, switch back to the observation eyepiece and press the green filter into the optical path to observe the phase difference image of the sample;
f. When observing with 20 x and 40 x objective lenses, the spotlight should be set at position Ph2, and when using a 100 x objective lens, the spotlight should be set at position Ph3.
Scope of application: Suitable for observing transparent, unstained or unstained samples, such as various cells, live tissues, unstained or unstained tissue slices, aquatic organisms, etc.
The basic principle of phase contrast microscope
When light passes through a relatively transparent specimen, there is no significant change in the wavelength (color) and amplitude (brightness) of the light. Therefore, when observing unstained specimens (such as live cells) under a regular optical microscope, their morphology and internal structure are often difficult to distinguish. However, due to the differences in refractive index and thickness of different parts of the cell, there will be differences in the optical path of direct and diffracted light when passing through this specimen. As the optical path increases or decreases, the phase of accelerating or lagging light waves will change (resulting in phase difference). The phase difference of light cannot be felt by the naked eye, but the phase difference microscope can use its special device - a circular aperture and a phase plate, and use the interference phenomenon of light to transform the phase difference of light into an amplitude difference (light and dark difference) that can be detected by the human eye. This makes the originally transparent object show obvious light and dark differences, enhances contrast, and allows us to clearly observe live cells and certain fine structures inside cells that cannot be seen or clearly seen under ordinary optical microscopes and dark field microscopes.
The imaging principle of a phase contrast microscope: The optical source can only pass through a transparent ring of a circular aperture, which is then focused into a beam of light. When this beam of light passes through the object being tested, it undergoes varying degrees of deviation (diffraction) due to the different optical paths of each part. Due to the fact that the image formed by the transparent ring coincides with the conjugate surface on the phase plate and the focal plane behind the objective lens. Therefore, direct light that has not deviated passes through the conjugate surface, while diffracted light that has deviated passes through the compensating surface. Due to the different properties of the conjugate surface and compensation surface on the phase plate, they will respectively generate a certain phase difference and intensity reduction of the light passing through these two parts. The two sets of light will then converge through the rear lens and travel on the same optical path, causing interference between direct and diffracted light, changing the phase difference into amplitude difference. In this way, during phase contrast microscopy, the phase difference that cannot be distinguished by the human eye is converted into an amplitude difference (brightness difference) that can be distinguished by the human eye through the light of a colorless transparent body.
