Several special optical microscopes and their differences
1 dark field microscope
The dark field microscope does not have the function of observing the fine structure inside the object, but it can distinguish the existence and movement of particles above 0.004 μm. Therefore, it is often used to observe the structure of living cells and the movement of intracellular particles.
The basic principle of dark field microscopy is the Tyndall effect. When a beam of light passes through a dark room, a bright dust "pathway" in the air can be observed from a direction perpendicular to the incident light. This phenomenon is the Tyndall effect.
After the dark field microscope is replaced with a dark field condenser on an ordinary optical microscope, due to the occlusion of the internal parabolic structure of the condenser, the light irradiated on the surface of the object to be inspected cannot directly enter the objective lens and eyepiece, and only scattered light can pass through, so the field of view is dark.
The basic usage of darkfield microscopy is as follows:
1. Install a dark field condenser (or use a thick black paper to make a light shield and place it under the condenser of an ordinary microscope to obtain dark field effects).
2. Choose a strong light source, usually with a microscope light to prevent direct light from entering the objective lens.
3. Add a drop of cedar oil between the condenser and the glass slide to avoid total reflection of the illuminating light on the condenser, failing to reach the object to be inspected and dark field illumination.
4. Perform center adjustment, that is, move the condenser horizontally so that the optical axis of the condenser and the optical axis of the microscope are strictly on a straight line. Lift and lower the condenser, align the focal point of the condenser (the apex of the conical beam in Figure 1-2) to the object to be tested.
5. Select the objective lens corresponding to the condenser, adjust the focal length, and operate according to the method of ordinary microscope.
Stereomicroscope
Stereo microscopes, also known as solid microscopes or dissecting mirrors, image an upright three-dimensional space image, and have the characteristics of strong stereoscopic effect, clear and wide imaging, long working distance (usually 110mm) and continuous magnification viewing. Often used in biology for real-time observation during dissection
The light source of an ordinary optical microscope is parallel light, so it forms a two-dimensional plane image; while a stereo microscope adopts a dual-channel optical path, and the left and right beams in the binocular tube have a certain angle of view (generally 12o15o), so it can A stereoscopic image in three-dimensional space is formed.
Stereo microscopes are used in a similar way to ordinary light microscopes, but are more convenient. The main difference between the two is:
1. The inspection objects of stereo microscopes do not need to be made into slides.
2. The stage of the stereo microscope is directly fixed on the mirror base, and is equipped with black and white double-sided panels or glass panels, and the operator can choose according to the object and requirements of the microscope inspection.
3. The imaging of the stereo microscope is upright, which is convenient for dissection operations.
4. The stereo microscope has only one objective lens, and its magnification can be continuously adjusted by rotating the adjusting screw.
fluorescence microscope
Fluorescence microscopy is an optical tool for qualitative and quantitative research on the fluorescence intensity emitted by intracellular substances.
There are two types of fluorescent substances in cells, one can fluoresce directly after being irradiated by ultraviolet rays, such as chlorophyll, etc.; other substances do not have this property, but if stained with specific fluorescent dyes or fluorescent antibodies, they can be fluoresced by ultraviolet rays. Can also fluoresce after irradiation
Upright Bioluminescence Microscope/Inverted Bioluminescence Microscope
The principle of fluorescence microscope is to use a point light source with high luminous efficiency (such as ultra-high pressure mercury lamp) to emit light of a certain wavelength (such as ultraviolet light 3650λ or purple-blue light 4200λ) through the filter system as excitation light to excite fluorescent substances in the specimen. After the fluorescence of various colors is emitted, it is filtered by the blocking (or suppressing) filter behind the objective lens, and then observed through the magnification of the eyepiece.
The blocking filter has two functions: one is to absorb and block the excitation light from entering the eyepiece so as not to disturb the fluorescence and damage the eyes; the other is to select and let a specific fluorescence pass through, showing a specific fluorescent color.
Fluorescence microscopes can be divided into two types according to the principle of optical path:
1. Transmission Fluorescence Microscopy
In older fluorescence microscopes, the excitation light source is passed through the specimen material through a condenser to excite fluorescence. The advantage is that the fluorescence is strong at low magnification, but the disadvantage is that the fluorescence decreases with the increase of magnification. So it is only suitable for observing larger specimen material.
2. Epi-fluorescence Microscopy
The excitation light falls from the objective lens down to the surface of the specimen, that is, the same objective lens is used as the illumination condenser and the objective lens for collecting fluorescence.
A dichroic beam splitter (dichroic mirror) needs to be added in the optical path, which forms an angle of 45o with the optical axis. The excitation light is reflected into the objective lens and collected on the sample. The excitation light reflected by the slide surface enters the objective lens at the same time, returns to the two-color beam splitter, separates the excitation light from the fluorescence, and the remaining excitation light is absorbed by the blocking filter. If you change to a combination of different excitation filters/two-color beam splitters/blocking filters, the needs of different fluorescent reaction products can be met.
The advantage of this kind of fluorescence microscope is that the illumination of the field of view is uniform, the imaging is clear, and the greater the magnification, the stronger the fluorescence.
phase contrast microscope
A phase contrast microscope is a microscope that can convert the phase difference (or optical path difference) generated when light passes through an object into a change in amplitude (light intensity). It is mainly used to observe living cells, unstained tissue sections or stained specimens lacking contrast.
The human eye can only identify changes in the wavelength (color) and amplitude of visible light, but not phase changes. However, most biological specimens are highly transparent, and the amplitude of the light wave is basically unchanged after passing through, and only the phase changes.
The phase contrast microscope basically changes the optical path difference of the visible light passing through the specimen 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) at the same time. If it is increased or decreased by 1/4λ, the optical path difference becomes 1/2λ, and the two beams interfere after the optical axis Strengthen, increase or decrease the amplitude, improve the contrast.
Structurally, phase contrast microscopes are different from ordinary optical microscopes in that:
1. The annular diaphragm has a diaphragm with a ring opening, which is installed between the light source and the condenser. The function is to make the light passing through the condenser form a hollow light cone and focus on the specimen.
2. Phase plate The phase contrast microscope adds a phase plate coated with magnesium fluoride inside the objective lens to delay the phase of direct light or diffracted light by 1/4λ. There are two regions on the phase plate, the part through which direct light passes is called "conjugate surface", and the part through which diffracted light passes is called "compensation surface". Phase plates are divided into two types according to their working effects:
(1) A+ phase plate: the direct light is delayed by 1/4λ, and the light waves are superimposed after the two sets of light waves combine to increase the amplitude, and the specimen structure is brighter than the surrounding medium, forming a bright contrast (or negative contrast).
(2) B+ phase plate: The diffracted light is delayed by 1/4λ, and the light waves of the two groups of light are subtracted after the axis is aligned, and the amplitude becomes smaller. The structure of the specimen is darker than the surrounding medium, forming a dark contrast (or positive contrast). The objective lens with a phase plate is called a phase contrast objective lens, which is often marked with "Ph" on the objective lens housing.
