Introduction to the classification and use of various optical microscopes
There are many classification methods of optical microscopes: according to the number of eyepieces used, it can be divided into binocular and monocular microscopes; according to whether the image has a stereo effect, it can be divided into stereo microscopes and non-stereo microscopes; according to the observation object, it can be divided into biological microscopes and gold microscopes. microscope. Phase microscope, etc.; according to the optical principle, it can be divided into polarized light microscope, phase contrast microscope and differential interference microscope, etc.; according to the type of light source, it can be divided into ordinary light, fluorescence, ultraviolet light, infrared light and laser microscope, etc.; according to the type of receiver, it can be divided into Vision, digital (camera) microscope, etc. Commonly used microscopes include binocular stereo microscope, metallographic microscope, polarized light microscope, fluorescence microscope, etc.
1. Binocular Stereo Microscope
Binocular stereo microscope, also known as "solid microscope" or "dissecting mirror", is a visual instrument with a positive stereoscopic sense. It is widely used in slice surgery and microsurgery in the biomedical field; in industry, it is used for the observation, assembly and inspection of tiny parts and integrated circuits. It has the following characteristics:
(1) Using a dual-channel optical path, the left and right beams in the binocular tube are not parallel, but have a certain angle - the volume viewing angle (usually 12 degrees -15 degrees), that is, the left and right beams. Both eyes provide a three-dimensional image. It is essentially two single-tube microscopes placed side by side. The viewing angle formed by the optical axes of the two lens barrels is equivalent to the viewing angle formed when a person observes an object with both eyes, thereby forming a three-dimensional visual image in a three-dimensional space.
(2) The image is straight, easy to operate and dissect, because the prism under the eyepiece makes the image upside down.
(3) Although the magnification is not as good as that of a traditional microscope, it has a long working distance.
(4) The focal depth is large, which is convenient for observing the whole layer of the inspected object.
(5) The diameter of the field of view is large.
The optical structure of the current stereoscope is: through an ordinary main objective lens, the two beams of light after imaging the object are separated by two sets of intermediate objective lenses-zoom lenses to form an overall viewing angle and then imaged through the respective eyepieces, by changing the intermediate The distance between the mirror groups to obtain the change of its magnification, so it is also called "Zoom-stereomicroscope". According to the application requirements, the current stereoscope can be equipped with a wealth of optional accessories, such as fluorescence, photography, videography, cold light source, etc.
2. Metallographic microscope
A metallographic microscope is a microscope specially used to observe the metallographic structure of opaque objects such as metals and minerals. These opaque objects cannot be observed with ordinary transmitted light microscopes, so the main difference between metallography and ordinary microscopes is that the former uses reflected light, while the latter uses transmitted light for illumination. In the metallographic microscope, the illumination beam is emitted from the direction of the objective lens to the surface of the observed object, reflected by the surface of the object, and then returned to the objective lens for imaging. This reflective illumination method is also widely used in the inspection of integrated circuit silicon wafers.
3. Polarizing microscope
Polarizing microscopes are microscopes used to study so-called transparent and opaque anisotropic materials. All substances with birefringence can be clearly distinguished under a polarizing microscope. Of course, these substances can also be observed by staining, but some are not possible and polarizing microscopes must be used.
(1) Features of polarizing microscopes
A method of turning ordinary light into polarized light for microscopy to identify whether a substance is monorefringent (in all directions) or birefringent (anisotropic). Birefringence is a fundamental property of crystals. Therefore, polarized light microscopes are widely used in minerals, chemistry and other fields, and also have applications in biology, botany and other fields.
(2) The basic principle of polarized light microscope
The principle of polarized light microscopy is more complicated, so I won't introduce it too much here. The polarizing microscope must have the following accessories: polarizer, analyzer, compensator or phase plate, special stress-free objective lens, rotating stage.
(3) Polarizing microscope method
A sort of. Orthoscope: Also known as a distortion-free microscope, it is characterized by using a low-magnification objective lens instead of a Bertrand lens to study the subject. Direct study with polarized light. At the same time, in order to make the illumination aperture smaller, the upper lens of the condenser is pushed apart. A normal phase microscope is used to examine the birefringence of an object.
b. Conoscope: Also known as an interference microscope, it studies the interference patterns created when polarized light interferes. This method is used to observe the uniaxiality or biaxiality of an object. In this method, a strongly converging polarized beam of light is used for illumination.
(4) Requirements for polarizing microscopes
A sort of. Light source: It is best to use monochromatic light, because the speed of light, refractive index, and interference phenomena vary with wavelengths. General microscopes can use ordinary light.
b. Eyepieces: Eyepieces with crosshairs.
C. Condenser: In order to obtain parallel polarized light, a swing out condenser that can push out the upper lens should be used.
d. Bertrand lens: an auxiliary element in the optical path of the condenser, which is an auxiliary lens that amplifies the primary phase caused by the object into the secondary phase. It guarantees observation with the eyepiece of a planar interference pattern formed at the rear focal plane of the objective.
(5) Requirements for polarizing microscopes
A sort of. The center of the stage is coaxial with the optical axis.
b. The polarizer and analyzer should be in quadrature positions.
C. Shooting should not be too thin.
4. Fluorescence Microscopy
Fluorescence microscopy uses short-wavelength light to irradiate a fluorescein-stained object to excite and generate long-wavelength fluorescence, and then observe. Fluorescence microscopy is widely used in biology, medicine and other fields.
(1) Fluorescence microscopes are generally divided into two types: transmission type and epi-illumination type.
A sort of. Transmission type: The excitation light is emitted from the bottom surface of the inspected object, and the condenser is a dark-field condenser, so that the excitation light does not enter the objective lens, and the fluorescence enters the objective lens. It's bright at low magnification and dark at high magnification. Oil immersion and neutralization operations are difficult, especially the low magnification illumination range is difficult to determine, but very dark backgrounds can be obtained. The transmissive type is not used for opaque inspection objects.
Transmission type is currently almost eliminated. Most new fluorescence microscopes are epitaxial. The light source comes from above the test object, and there is a beam splitter in the optical path, which is suitable for transparent and opaque test objects. Since the objective lens acts as a condenser, it is not only easy to operate, but also can achieve uniform illumination of the entire field of view from low magnification to high magnification.
(2) Precautions for fluorescence microscopy
A sort of. Long-term exposure to excitation light will cause fluorescence decay and quenching, so the observation time should be shortened as much as possible. .
b. For oil viewing, use "non-fluorescent oil".
C. Fluorescence is almost always weak and should be performed in a darker room.
d. It is best to install a voltage stabilizer in the power supply, otherwise the voltage instability will not only reduce the life of the mercury lamp, but also affect the effect of the microscope.
At present, many emerging biological research fields are applied to fluorescence microscopy techniques, such as gene in situ hybridization (FISH).
5. Phase contrast microscope
In the development of optical microscope, the successful invention of phase contrast microscope is an important achievement of modern microscope technology. We know that the human eye can only distinguish the wavelength (color) and amplitude (brightness) of light waves. For colorless and transparent biological specimens, when the light passes through, the wavelength and amplitude do not change much, so it is difficult to observe the specimen in bright field. .
Phase contrast microscope is to use the optical path difference of the inspected object to perform microscopic detection, that is, to effectively use the interference phenomenon of light to change the phase difference that cannot be distinguished by the human eye into a distinguishable amplitude difference, even if it is colorless and transparent. Matter can also become clearly visible. This greatly facilitates the observation of living cells, so phase contrast microscopy is widely used for inverted microscopes.
The phase contrast microscope is different from brightfield in equipment and has some special requirements:
a. Installed under the condenser and combined with the condenser - phase contrast condenser. It consists of annular diaphragms of different sizes mounted on a disc, with the words 10X, 20X, 40X, 100X, etc. on the outside, which are used in conjunction with objective lenses with corresponding multiples.
b.Phaseplate: Installed on the back focal plane of the objective lens, it is divided into two parts, one is the part through which the direct light passes, which is a translucent ring called the conjugate plane; the other is the part through which the diffracted light "compensates" . Objectives with phase plates are called "phase contrast objectives", and the word "Ph" is often written on the casing.
Phase contrast microscopy is a relatively complex microscopy method. In order to obtain a good observation effect, the debugging of the microscope is very important. In addition, the following aspects should also be noted:
A sort of. The light source should be strong and all aperture diaphragms should be open;
b. Use color filters to make light waves nearly monochromatic.
6. Differential Interference Contrast Microscopy (Diffe Rent Interference Contrast DIC)
Differential interference contrast microscopy appeared in the 1960s. It can not only observe colorless and transparent objects, but also present strong stereoscopic images, and has some advantages that phase contrast microscopy cannot achieve. , the observation effect is more realistic.
(1) Principles
Differential interference contrast microscopy uses special Wollaston prisms to break up the beam. The vibration directions of the split beams are perpendicular to each other and the intensity is equal. The two points of the beam passing through the object to be inspected are very close to each other, and the phases are slightly different. Since the separation distance between the two beams of light is extremely small, there is no ghosting phenomenon, which makes the image appear three-dimensional.
(2) Special parts required for differential interference contrast microscope:
a. Polarizer
b. Analyzer
C. 2 Wollaston prisms
(3) Precautions in differential interference contrast microscopy
A sort of. Due to the high sensitivity of differential interference, there should be no dirt and dust on the surface of the plate.
b. Substances with birefringence cannot achieve the effect of differential interference contrast microscopy.
C. Plastic petri dishes cannot be used when applying differential interference to an inverted microscope.
7. Inverted microscope (Invertedmicroscope)
The inverted microscope is suitable for microscopic observation of tissue culture, in vitro cell culture, plankton, environmental protection, food inspection, etc. in the biomedical field.
Due to the limitations of the above-mentioned sample characteristics, placing the object to be inspected in a petri dish (or culture bottle) requires a long working distance of the inverted microscope objective and the condenser, and the inspected object in the petri dish can be directly inspected. Microscopic observation and research. Therefore, the positions of the objective lens, condenser lens and light source are all reversed, so it is called "inverted microscope".
Due to working distance limitations, inverted microscope objectives have a maximum magnification of 60X. Generally, inverted microscopes for research are equipped with 4X, 10X, 20X, and 40X phase contrast objectives, because inverted microscopes are mostly used for colorless and transparent in vivo observation. If the user has special needs, other accessories can also be selected to complete the observation of differential interference, fluorescence and simple polarized light.
Inverted microscopes are widely used in patch clamp, transgenic ICSI and other fields.
8. Digital Microscope
A digital microscope is a microscope that uses a camera (ie, a television camera objective or a charge-coupled device) as the receiving element. A camera is installed on the real image surface of the microscope to replace the human eye as a receiver. The optoelectronic device converts the optical image into an electrical signal image, and then performs size detection and particle counting. This type of microscope can be used in conjunction with a computer to facilitate the automation of detection and information processing, and is mostly used in occasions that require a lot of tedious detection work.
2. The use of various optical microscopes
Fluorescence microscopy uses the fluorescence emitted by the specimen to observe objects;
Stereo microscopes can be used to observe three-dimensional images of objects;
The projection microscope can project the image of the object onto the projection screen for multiple people to observe at the same time;
Inverted microscopes for cell culture, tissue culture and microbial research;
Phase contrast microscope is used to observe colorless and transparent specimens;
For example, darkfield microscopy is used to observe bacteria and spirochetes. sporty.
