Adjustment of imaging optical path system of light microscope and outline of microscopic examination
Adjustment of Imaging Optical Path System and Microscopic Examination Outline
Adjustment of the imaging optical system of a microscope is carried out according to the needs of different microscopy techniques. The so-called microscopy, in a nutshell, is the method of illumination used when observing a sample through a microscope, and the technique and method of obtaining better contrast in the image formed by the sample. The following briefly describes the microscopy has matured in several methods and the corresponding microscope imaging optical system adjustment method.
1. Transmission bright field:
This is the most traditional and common application method since the invention of microscope. Basic components: a. Objective lens: any objective lens can be used for bright field observation; b. Spotting scope: all kinds of spotting scopes can be used, preferably equipped with an aperture diaphragm. Adjustment method: After the above microscope's Kuhler illumination system is adjusted, the bright field method can be applied. Scope of application: all stained tissue sections, blood smears, etc. Precautions: a. When using the bright field method of observation, the Kuhler illumination system must be adjusted; b. The diaphragm of the field of view should not be opened arbitrarily, and the front lens of the condenser mirror should be posed factually out and into the optical path respectively when using the objective lenses of 10×, less than 10×, and more than 10×; c. The aperture diaphragm of the condenser mirror should not be used to adjust the brightness of the field of view, and the height of the condenser mirror should not be adjusted indiscriminately, or the resolution of the microscope will be lowered and the resolution of the stained tissue will be damaged. resolution of the microscope and damage has been adjusted to the Kuhler illumination system; d. for micrographs, every change in the use of a magnification of the objective lens, you have to adjust the aperture diaphragm of the condenser lens, so that its size is exactly equal to the numerical aperture used in the objective lens of 2 / 3.
2. Transmitted light phase contrast method:
This is a modern microscope examination of a contrast enhancement method. Basic components: phase contrast objective lens, bright field of view and phase contrast multi-purpose spotting scope, focusing telescope, green filters.
Adjustment methods:
a. Based on the adjustment of the Kuhler illumination system, focus the sample clearly using the bright field method
b. Turn the spotting scope to Ph1 to align the dial scale position, choose 10 × phase contrast objective lens, and replace the transparent sample to be observed.
c. Unplug one of the eyepieces, replace it with a centred telescope and focus on the two phase contrast rings in the field of view (the black phase contrast ring of the objective lens and the light transmission phase contrast ring of the condenser lens).
d. The two phase contrast rings in the field of view may not necessarily coincide, adjust the two adjusting devices on the spotting scope (adjusting the left and right positions of the phase contrast ring adjustment lever and adjust the front and rear positions of the friction knob), so that the light transmission ring for the front and back to the right and left to coincide with the black ring
e. After adjustment, change back to the eyepiece for observation and press the green filter into the optical path to observe the phase contrast image of the sample.
f. When there are 20× and 40× objective lenses for observation, the spotting lens should be set on the Ph2 position, and when using 100 objective lenses, the spotting lens should be set on the Ph3 position.
Scope of application: It is suitable for observing transparent, unstained or unstainable samples, such as all kinds of cells, living tissues, unstained or unstained tissue sections, aquatic organisms and so on.
3. Differential interference phase contrast method:
In order to overcome the phase contrast method of observation of the sample details around the image accompanied by a halo, will mask out the details that should have been seen, as well as samples or tissue sections require a fairly thin thickness, in principle, can be thicker than 10?m and other limitations, the use of the principle of dual-beam interference to design the sub-differential interference phase contrast method.
Adjustment methods:
a. The DIC method must be adjusted on the basis that the Kulemin system has already been adjusted
b. Using a 10× objective lens, determine the focusing position of the objective lens that can see the sample clearly with a bright field of view.
c. Place the polarizer into the illumination path, noting that it should be orientated in an east-west direction.
d. Turn the condenser dial to the position corresponding to the use of the 10 × objective lens, i.e., DIC 0.3-0.4.
e. Insert the DIC slider for the 10× objective lens at the rear of the objective lens or on the objective converter.
f. Insert the analyser into the imaging optical path, and note that its orientation should be south-north.
g. Replace the transparent sample to be observed, turn on the light source to focus the sample clearly.
h. Adjust the DIC insert so that the differential interference contrast image to achieve the best effect, that is, the most obvious relief effect.
i. At the same time, adjust the aperture diaphragm of the condenser mirror, so that the effect of the contrast is also optimal.
j. Then fine-tune the details of the sample to see the structure of the sample at different levels.
k. If the complementary colour (first order red retardation plate) is inserted and the DIC insert is adjusted at the same time, the ever-changing brilliant colours can be seen in the field of view, with red, orange, yellow, green, blue, purple, pink, pink-violet and golden-yellow. Scope of application: transparent or unstainable tissue sections, thickness up to about 100?m, live tissue and live cells in culture, small living organisms and so on.
