|A fluorescence microscope is a microscope that uses ultraviolet light as a light source to illuminate an object to be observed to make it emit fluorescence, and able to observe information on the position, shape, and structure of the fluorescent portion of the object.
Some substances in nature can fluoresce themselves when exposed to ultraviolet light. Some substances cannot fluoresce themselves, but they can also fluoresce after being dyed with fluorescent materials. Fluorescence microscope is an important tool for studying cytology. It is widely used in scientific research and teaching fields such as medicine, polymer structure, and luminescent materials research, and so on.
The most important feature of the fluorescence microscope is that it has an illumination source that can emit ultraviolet light, forming a lighting system with the configured fluorescence condenser and filters that are designed for excitation and acceptance of fluorescence.
Fluorescent light sources generally use mercury or metal halide lamps, as well as high-power LEDs or laser sources. The ultraviolet light emitted by the light source has a shorter wavelength and thus has a higher resolution. Generally, the high-pressure mercury lamp has a continuous spectrum and a large irradiation intensity, making it a relatively ideal fluorescent microscope source; the xenon lamp has stable intensity in the visible light spectrum range and higher intensity in the infrared band than the mercury lamp, but has certain defects in the ultraviolet band. High-power LED light source cannot provide continuous band spectrum, but has advantages in the range of specific wavelengths, there is no need to preheat, start up and use, has long service life and low labor maintenance cost. So when choosing a light source, you need to understand the characteristics of the light source to match the specific application. Halogen lamps have a narrower range of excitation light wavelengths and can only be concentrated in certain specific wavelengths, such as blue light, so applications are less.
Fluorescence microscope can use transmitted light and reflection light for illumination. It can also be installed with a dark field device to become a dark field fluorescence microscope. A phase contrast accessory can be added to form a phase contrast fluorescent microscope, or an interference device can be added to become a fluorescent interference microscope.
Reflection light is also commonly called incidence light fluorescence microscope, also known as the EPI-Fluorescence Microscope. The illumination source and the excitation light are passed through the same objective lens. The excitation light path does not pass through the slide as it is directly irradiated onto the specimen, with small excitation light loss and high fluorescence efficiency.
The illumination path of the transmitted light fluorescence microscope needs to pass through the slide. In order to reduce the loss of the excitation light, the transmissive fluorescence microscope should use quartz glass slides and coverslips. Because, as consumables, quartz slides are expensive, people prefer to use an EPI-fluorescence microscope.
The inverted fluorescence microscope is composed of fluorescent accessories and an inverted microscope. The objective lens and the condenser have a long working distance, and can directly observe and study the object in the culture dish, characterized by microscopic observation in a culture flask or petri dish, mainly used for fluorescence of living tissues such as cells, and are suitable for microscopic observation of tissue culture, in vitro cell culture, plankton, food inspection, etc. in the fields of biology and medicine.
Fluorescence microscope systems require the use of two sets of filters for both excitation and emission (Emission) processes.
1. Excitation: After the light source emits light, it first enters the incident light filter, filters out the visible light, and only retains the wavelength portion for exciting the sample, therefore the incident light filter is called an exciter filter.
2. Then, the excitation light is directed vertically to the objective lens through the dichroic lens or a reflector, and then to the specimen through the objective lens, so that the specimen is excited to generate fluorescence and, at this point, the objective lens directly functions as a condenser.
3. The fluorescence, after excitation, is reflected back through the objective lens to the dichroic lens and the barrier filter to block other light except the wavelength of the emitted light from the sample (i.e., the fluorescent portion), so that the observer observes through the eyepiece or the camera to form image. The barrier filter can also filter out the ultraviolet rays between the eyepiece and the objective lens to protect the observer's eyes.
The reflective layer on the reflector of fluorescent microscopes is generally aluminized. Aluminum absorbs less ultraviolet and visible light in the blue-violet region and reflects more than 90%, while silver reflects only 70%.
The condenser of the fluorescence microscope is made of quartz glass or glass that can transmit ultraviolet light. It can use bright field condensers and dark field condenser, and there is also the phase contrast fluorescent condensers. The bright field condenser has strong condensing power, convenient to use, and is suitable for low and medium multiple power observation. The dark field condenser can produce a dark background because the excitation light does not directly enter the objective lens, thereby enhancing the brightness and contrast of the fluorescent image, capable of observing the fluorescent fine particles that cannot be distinguished by the bright field. The phase contrast fluorescent condenser needs to be used together with the phase contrast accessories and the phase contrast objective lens to observe the phase contrast and the fluorescence effect at the same time. It can see both the fluorescence image and the phase difference image, which helps the accurate positioning of the fluorescent structure.
Ordinary fluorescence microscopes can adopt various kinds of conventional objective lenses. As the more the number of lenses of the objective lens, the greater the loss of fluorescence, and in particular, the apochromatic objective lens contains quartz and other components, which can spontaneously fluoresce to form interference, therefore, the general use of flat field achromatic objective lens can already achieve very good results. The fluorescence brightness in the field of view of the microscope is proportional to the square of the numerical aperture of the objective lens, and inversely proportional to the magnification. For specimens with insufficient fluorescence, in order to improve the brightness of the fluorescence image, an objective lens with a large numerical aperture should be used, especially when using high power microscope for observation, it is often necessary to use a dedicated high-magnification fluorescent objective to provide excellent chromatic aberration correction and image quality.
Fluorescence Microscope Camera and Image
A fluorescence microscope camera can be connected to a fluorescence microscope to form a fluorescence microscope imaging system. The fluorescence image seen by the fluorescence microscope has both the morphological features, the fluorescent color and brightness and other features, and the two need to be combined for comprehensive judgment.
The brightness of the fluorescence is relatively weak, and it usually requires the camera to have high sensitivity to low light and signal capture ability in order to obtain a good fluorescence image. The camera can adjust the focus with a brighter fluorescent area. Adjust and set the exposure compensation according to the distribution ratio of the fluorescent image in the photometric area and the brightness of the image. Generally, it is necessary to increase the exposure compensation appropriately to obtain a bright and vivid fluorescent image on a dark background. The camera needs to adopt the appropriate fluorescence shooting mode, do the appropriate background subtraction processing, and set the appropriate parameters, such as Binning, Gain, Gamma, etc., so that the exposure time and gain can be maximized by software control.
Fluorescence microscope can also use a fluorescent cold-cooling camera to reduce noise and improve the signal-to-noise ratio of the image. Especially in low light conditions, using dark field illumination, or under chemiluminescence conditions, long time exposure is required. Using a cooling camera can reduce the dark current noise and obtain a clearer image.
In the weaker fluorescence field of view, in order to obtain a better contrast image, appropriate adjustments should be made to reduce the fluorescence aperture diaphragm so as to obtain images with large depth of field, or use a neutral density filter (ND for short).
When shooting with a high power or magnification objective, any vibration should be avoided, and it is best that an anti-vibration table should be configured.
The slides and coverslips used in fluorescence microscope must have a smooth surface, uniform thickness, and no autofluorescence. The thickness of the slide should be between 0.8~1.2mm. The thickness of the standard coverslip is about 0.17mm. If the slide is too thick, it will cause short-wave excitation light and fluorescence loss. If necessary, quartz glass slides can be used to increase the fluorescence transmittance.
The tissue section or other specimen of the fluorescent specimen is usually about ≤10μm, should not be too thick, otherwise it will affect the excitation light penetrating the specimen, and excessive non-essential cell overlap or impurity masking will affect the observation effect of the objective lens on the upper part of the specimen.
The sealant of the specimen must be colorless and transparent, without autofluorescence. The brightness of the fluorescence is brighter at pH 8.5~9.5, and it is not easy to fade quickly. Usually, glycerin can be used. When using dark-field fluorescence microscope or oil lens for observation, lens oil must be used, and it is best to use special non-fluorescent oil. Especially in the U and V-band excitation, the conventional cedar oil will have cyan fluorescence. Glycerin, liquid paraffin, etc. may also be used.
Fluorescence Microscope Operation Precautions
As the fluorescence is relatively dark, so the fluorescence microscope is best placed in a dark room, which also allows the human eye to adapt to the darkness for better observation effect. When lighting required, local lighting can be used. In addition, because some mercury lamps produce a great deal of heat, and some xenon lamps can generate a lot of ozone, and therefore good ventilation is required.
Before use, open the packing list first, check the integrity and status of the parts and accessories, and operate according to the instructions.
Before performing fluorescence observation, first check the condition of the lighting equipment of the fluorescent device, turn on the halogen light for illumination first, place a sample film, and do the lens focusing and centering the filament in advance. Then, check whether the fluorescence excitation filter and the emission filter are installed in the nosepiece, and whether the objective lens configuration is proper. If there is a phase contrast observation of transmitted light in the system, check the convergence axis of the condenser lens in advance, and whether the phase contrast ring plate and objective lens are matched correspondingly.
Inspect the slides, coverslips, and other sample vessels, whether the thickness of the sample is within the range of the calibrated working distance of the objective lens, and whether there are liquid, smudges, dust and other interference.
Because the illumination source contains ultraviolet light, avoid looking directly at the ultraviolet light source when the light source is adjusted. The fluorescence microscope should be equipped with a brown UV protection visor to prevent UV damage to the retina of the eyes.
After the above inspection, place the specimen to be tested and turn on the high-pressure lamp source. When the high-pressure mercury lamp is fully lit, stop the excitation and then observe.
Generally, after the high-pressure mercury lamp is turned on, the excitation light intensity tends to be stable in about 5-10 minutes, and reaches the brightest point in 15 minutes. The working time when the mercury lamp is turned on is preferably 1 hour each time. Exceeding 90 minutes, the excitation light intensity will gradually decrease and the fluorescence will be weakened. After 15 minutes of excitation of the specimen, the fluorescence will also be significantly attenuated.
After the high-pressure mercury lamp is turned off, it cannot be re-opened immediately. I can be started again only after it is completely cooled. Otherwise, the mercury lamp will be unstable and affect the service life.
After the mercury lamp is turned off, it takes at least 10 minutes to start again, so that the mercury vapor is cooled to the original state, otherwise the service life of the bulb will be affected.
The power supply should be equipped with a voltage regulator, which will reduce the service life if the voltage is unstable.
The excitation device of the fluorescence microscope and the high-pressure mercury lamp have a limited life span, and the specimens should be collectively inspected in batches so as to reduce the number and time of mercury lamp activation.
First, observe with a low power objective lens, ensure that the specimen is located in the center of the entire illumination spot, and then gradually changed to high power observation. Under the premise of not affecting the resolution, the adjustment ring of the field diaphragm and of the numerical aperture diaphragm of the objective lens can be minimized as much as possible to reduce the excitation area, avoid the influence of stray light, and improve the depth of field.
When observing the sample, in order to prevent the fluorescence of the sample from quenching due to excessive excitation of light illumination during the process of focusing and searching for the image, it is best to adjust the excitation light to a moderate intensity by first reducing the aperture diaphragm or using the ND filter. After finding the key feature points, adjust the fluorescence to the optimal brightness, and finally observe and take pictures.
Long-time excitation light illumination of the specimen will cause the fluorescence to attenuate and disappear. Therefore, the specimen should be observed immediately after dying. If stored for too long, the fluorescence will gradually weaken until quenching. When not observed for a while, the excitation light path should be blocked by a visor. The dyed specimens can be wrapped in black paper, placed in a polyethylene plastic bag, and stored at a low temperature of about 4 °C, which can delay the quenching time of the fluorescence and prevent the sealing agent from evaporating.
Before and after use, the grease and dust contaminating the lenses in front of the objective lens should be inspected and removed. Gently brush it with a soft brush. In the place where fingerprints and oil stains are present, use a soft, clean absorbent cotton, lens rubbing paper dipped in anhydrous ethanol (or methanol) to gently wipe clean, and wipe the oil stain on the surface of the objective lens with gasoline
For more information on the use of fluorescence microscope, please refer to the Biological Microscope on the BoliOptics website.
|Microscopes and components have two types of optical path design structures.
One type is finite optical structural design, in which light passing through the objective lens is directed at the intermediate image plane (located in the front focal plane of the eyepiece) and converges at that point. The finite structure is an integrated design, with a compact structure, and it is a kind of economical microscope.
Another type is infinite optical structural design, in which the light between the tube lens after passing the objective lens becomes "parallel light". Within this distance, various kinds of optical components necessary such as beam splitters or optical filters call be added, and at the same time, this kind of design has better imaging results. As the design is modular, it is also called modular microscope. The modular structure facilitates the addition of different imaging and lighting accessories in the middle of the system as required.
The main components of infinite and finite, especially objective lens, are usually not interchangeable for use, and even if they can be imaged, the image quality will also have some defects.
The separative two-objective lens structure of the dual-light path of stereo microscope (SZ/FS microscope) is also known as Greenough.
Parallel optical microscope uses a parallel structure (PZ microscope), which is different from the separative two-object lens structure, and because its objective lens is one and the same, it is therefore also known as the CMO common main objective.
|The tube lens focal length is the focal length from the tube lens to the intermediate image plane of the design of infinite microscope, and its typical ranging is from 160 to 200 mm, depending on different manufacturers.
|The magnification of the objective lens refers to the lateral magnification, it is the ratio of the image to the real size after the original image is magnified by the instrument. This multiple refers to the length or width of the magnified object.
System optical magnification is the product of the eyepiece and the objective lens (objective lens zoom set) of the optical imaging part within the system.
Optical magnification = eyepiece multiple X objective lens/objective lens set
The maximum optical magnification of the microscope depends on the wavelength of the light to which the object is illuminated. The size of the object that can be observed must be greater than the wavelength of the light. Otherwise, the light cannot be reflected or transmitted, or recognized by the human eye. The shortest wavelength of ultraviolet light is 0.2 microns, so the resolution of the optical microscope in the visible range does not exceed 0.2 microns, or 200 nanometers. This size is converted to the magnification of the microscope, and it is the optical magnification of 2000X. Usually, the compound microscope can achieve 100X objective lens, the eyepiece is 20X, and the magnification can reach 2000X. If it is bigger, it will be called "invalid magnification", that is, the image is large, but the resolution is no longer increased, and no more details and information can be seen.
|When the instrument is conducting electronic image magnification and observation through a camera or the like, the optically magnified portion may not be the optical path that passes through the "eyepiece-objective lens" of the instrument, at this time, the calculation method of the magnification is related to the third-party photo eyepiece passed.
The trinocular optical magnification is equal to the multiplier product of objective lens (objective lens set) and the photo eyepiece
Trinocular optical magnification = objective lens X photo eyepiece
|Total magnification is the magnification of the observed object finally obtained by the instrument. This magnification is often the product of the optical magnification and the electronic magnification.
When it is only optically magnified, the total magnification will be the optical magnification.
Total magnification = optical magnification X electronic magnification
Total magnification = (objective X photo eyepiece) X (display size / camera sensor target )
|Field of View, is also called FOV.
The field of view, or FOV, refers to the size of the object plane (i.e., the plane of the point of the observed object perpendicular to the optical axis), or of its conjugate plane (i.e., object to primary image distance), represented by a line value.
System field of view is the size of the actual diameter of the image of the terminal display device of the instrument, such as the size of the image in the eyepiece or in the display.
Field of view number refers to the diameter of the field diaphragm of the objective lens, or the diameter of the image plane formed by the field diaphragm.
Field of view number of objective lens = field of view number of eyepiece / (objective magnification / mechanical tube length)
Large field of view makes it easy to observe the full view and more range of the observed object, but the field of view (FOV) is inversely proportional to the magnification and inversely proportional to the resolution, that is, the larger the field of view, the smaller the magnification, and also the lower the resolution of the object to be observed.
There are usually two ways to increase the field of view, one is to replace with an objective lens of a smaller multiple, or to replace with an eyepiece of a smaller multiple.
|Working distance, also referred to as WD, is usually the vertical distance from the foremost surface end of the objective lens of the microscope to the surface of the observed object.
When the working distance or WD is large, the space between the objective lens and the object to be observed is also large, which can facilitate operation and the use of corresponding lighting conditions.
In general, system working distance is the working distance of the objective lens. When some other equipment, such as a light source etc., is used below the objective lens, the working distance (i.e., space) will become smaller.
Working distance or WD is related to the design of the working distance of the objective lens. Generally speaking, the bigger the magnification of the objective lens, the smaller the working distance. Conversely, the smaller the magnification of the objective lens, the greater the working distance.
When it is necessary to change the working distance requirement, it can be realized by changing the magnification of the objective lens.
|For siedentopf eyetube, when changing the interpupillary distance, it requires two hands pushing or pulling the two eyetubes left and right simultaneously, and the two eyepiece tubes or eyetubes will change their position at the same time.
|Usually the Microscope Eyetube is 45°, some is 30°, Tiltable Eyetube Angle design of a microscope is also known as the ergonomics microscope.
0-30° or 0-45° is an ergonomic design. When the mechanical tube length / focal length of the tube of the microscope is relatively big, the microscope is relatively high, and the user's height or the seat of the work desk is not suitable, long-term use of microscope may cause sitting discomfort.
Eyepiece tube with variable angle can freely adjust the angle without lowering the head. Especially when it is close to 0 degree and the human eye is close to horizontal viewing, long-time or long-term use can avoid fatigue damage to the cervical vertebra.
|After imaging through a set of objective lenses, the object observed and the image seen by the human eye is inverted. When the observed object is manipulated, move the specimen or object, the image will move in the opposite direction in the field of view. Most of the biological microscopes are reversed-phase designs.
When needing to operate works with accurate direction, it is necessary to design it into a forward microscope. Generally stereo microscopes and metallurgical microscopes are all of erect image design.
When observing through the camera and display, the erect and inverted image can be changed by the orientation of the camera.
|The eyepiece of the microscope can have different viewing or observing directions. When the position of the microscope is uncomfortable, the direction of the eyepiece tube of the microscope can be adjusted, to facilitate observation and operation.
Placement method of different viewing angles of the microscope:
General direction: the support column is behind the object to be observed
Reverse direction: the support column is in front of the object to be observed
Lateral direction: the support column is on the side of the object to be observed
Rotating eyepiece tube, different microscopes may have different methods, for some, the direction is confirmed when installing the eyepiece tube of the microscope, for some, by rotating the body of the microscope, and for some, by rotating the support member on the support or holder of the microscope.
|The distance between the two pupils of the human eye is different. When the image of exit pupil of the two eyepieces of the microscope are not aligned with the entry pupil of the eye, the two eyes will see different images, which can cause discomfort.
Adjust the distance between the two eyepieces, to accommodate or adapt to the pupil distance of the observer's eyes. The adjustment range is generally between 55-75mm.
|For most people, their two eyes, the left and the right, have different vision; for the eyepiece tube, the eyepoint height of the eyepiece can be adjusted to compensate for the difference in vision between the two eyes, so that the imaging in the two eyes is clear and consistent.
The range of adjustment of the eyepiece tube is generally diopter plus or minus 5 degrees, and the maximum differential value between the two eyepieces can reach 10 degrees.
Monocular adjustable and binocular adjustable: some microscopes have one eyepiece tube adjustable, and some have two eyepiece tubes adjustable. First, adjust one eyepiece tube to the 0 degree position, adjust the microscope focusing knob, and find the clear image of this eyepiece (when the monocular adjustable is used, first adjust the focusing knob to make this eyepiece image clear), then adjust the image of another eyepiece tube (do not adjust the focusing knob again at this time), repeatedly adjust to find the clear position, then the two images are clear at the same time. For this particular user, do not adjust this device anymore in the future.
As some microscopes do not have the vision adjustment mechanism for the eyepiece tube, the vision of the two eyes are adjusted through the eyepiece adjustable.
|The third eyepiece splitting in the trinocular microscope is to borrow one of the two sets of eyepiece optical paths as the photographic light path. The beam split prism or beam splitter can reflect part of the image light to the eyepiece, and part passes through to the third eyepiece photographic light path, such a trinocular microscope is called trinocular simultaneous imaging microscope, or true-trinocular.
The beam split prism or beam splitter of the trinocular simultaneous imaging microscope or true-trinocular often has different splitting modes, such as 20/80 and 50/50, etc. Usually, the former is the luminous flux ratio of the eyepiece optical path, and the latter is the luminous flux ratio of the photographic optical path.
The advantage of true-trinocular is that, the real three optical paths can be imaged at the same time, and are not affected by the simultaneous use of the eyepiece observation and the photographic optical path (display). The disadvantage is that, because of the reason of the splitting, the image light of the photography is only a part. In theory, the image effect will be affected, and the effect is more obvious in the binocular eyepiece observation. If viewed closely, one will find that the eyepiece of the light path is relatively dark. However, in the current optical design and materials, the impact on the actual work is not very big, especially in the observation of low magnification objective lens, it has basically no effect at all, and therefore used by many people.
|The adjustable eyepiece is between the lens of the eyepiece and the focal plane, with distance adjustable device.
For most people, their two eyes, the left and the right, have different vision. For adjustable eyepieces, the eyepoint height of the eyepiece can be adjusted to compensate for the difference in vision between the two eyes, making the image in the two eyes clear and consistent.
The range of adjustment of the general eyepiece is that the diopter is plus or minus 5 degrees, and the maximum difference between the two eyepieces can reach 10 degrees. Before use, it is generally necessary to adjust both eyepieces to the initial position where the scale is displayed as 0, which is used as a baseline to facilitate up and down adjustment.
The reticle position of the eyepiece is generally 10mm below the fixed position of the eyepiece tube. Because the vision of each person is different, some people may not be able to see the reticle clearly. For adjustable eyepiece, the height of the reticle position can be adjusted to make the reticle and the observed object clear at the same time, this is the advantage of adjustable eyepiece that mounts the diopter adjustment on the eyepiece tube compared with non-adjustable eyepiece.
When non-adjustable eyepiece is equipped with a reticle, if the diopter is adjusted, the reticle will rotate accordingly, thereby affecting the position of the measurement. For adjustable eyepiece, when its diopter is adjusted, its reticle does not rotate.
|Eyepiece optical magnification is the visual magnification of the virtual image after initial imaging through the eyepiece. When the human eye observes through the eyepiece, the ratio of the tangent of the angle of view of the image and the tangent of the angle of view of the human eye when viewing or observing the object directly at the reference viewing distance is usually calculated according to 250 mm/focal length of eyepiece.
The standard configuration of a general microscope is a 10X eyepiece.
Usually, the magnification of the eyepiece of compound microscope is 5X, 8X, 10X, 12.5X, 16X, 20X.
As stereo microscope has a low total magnification, its eyepiece magnification generally does not use 5X, but can achieve 25X, 30X and other much bigger magnification.
|The eyepiece field of view is the diameter of the field diaphragm of the eyepiece, or the diameter of the image plane of the field diaphragm imaged by the field diaphragm.
The diameter of a large field of view can increase the viewing range, and see more detail in the field of view. However, if the field of view is too large, the spherical aberration and distortion around the eyepiece will increase, and the stray light around the field of view will affect the imaging effect.
|Eye point refers to the axial distance between the upper end of the metal frame of the eyepiece and the exit of pupil.
The exit of pupil distance of high eyepoint eyepiece is farther than that of the eye lens of the ordinary eyepiece. When this distance is greater than or equal to 18mm, it is a high eyepoint eyepiece. When observing, one does not need to be too close to the eyepiece lens, making it comfort to observe, and it can also be viewed with glasses. Generally, there is a glasses logo on the eyepiece, indicating that it is a high eyepoint eyepiece.
|The finite objective is the lateral magnification of the primary image formed by the objective at a prescribed distance.
Infinite objective is the lateral magnification of the real image produced by the combination of the objective and the tube lens.
Infinite objective magnification = tube lens focal length (mm) / objective focal length (mm)
Lateral magnification of the image, that is, the ratio of the size of the image to the size of the object.
The larger the magnification of the objective, the higher the resolution, the smaller the corresponding field of view, and the shorter the working distance.
|In the case of polychromatic light imaging, the aberration caused by the light of different wavelengths becomes chromatic aberration. Achromatic aberration is to correct the axial chromatic aberration to the two line spectra (C line, F line); apochromatic aberration is to correct the three line spectra (C line, D line, F line).
The objective is designed according to the achromaticity and the flatness of the field of view. It can be divided into the following categories.
Achromatic objective: achromatic objective has corrected the chromatic aberration, spherical aberration, and comatic aberration. The chromatic portion of the achromatic objective has corrected only red and green, so when using achromatic objective, yellow-green filters are often used to reduce aberrations. The aberration of the achromatic objective in the center of the field of view is basically corrected, and as its structure is simple, the cost is low, it is commonly used in a microscope.
Semi-plan achromatic objective: in addition to meeting the requirements of achromatic objective, the curvature of field and astigmatism of the objective should also be properly corrected.
Plan achromatic objective: in addition to meeting the requirements of achromatic objectives, the curvature of field and astigmatism of the objective should also be well corrected. The plan objective provides a very good correction of the image plane curvature in the field of view of the objective, making the entire field of view smooth and easy to observe, especially in measurement it has achieved a more accurate effect.
Plan semi-apochromatic objective: in addition to meeting the requirements of plan achromatic objective, it is necessary to well correct the secondary spectrum of the objective (the axial chromatic aberration of the C line and the F line).
Plan apochromatic objective: in addition to meeting the requirements of plan achromatic objective, it is necessary to very well correct the tertiary spectrum of the objective (the axial chromatic aberration of the C line, the D line and the F line) and spherochromatic aberration. The apochromatic aberration has corrected the chromatic aberration in the range of red, green and purple (basically the entire visible light), and there is basically no limitation on the imaging effect of the light source. Generally, the apochromatic aberration is used in a high magnification objective.
|Objective parfocal distance refers to the imaging distance between the objective shoulder and the uncovered object surface (referred to as the “object distance). It conforms to the microscope design, usually 45mm.
The objective of different magnifications of the compound microscope has different lengths; when the distance between the objective shoulder and the object distance is the same, the focal length may not be adjusted when converting to objectives of different magnifications.
|Objective for focal length is a design parameter of the tube focal length of the microscope that the objective is suitable for.
|The objective working distance is the vertical distance from the foremost surface end of the objective of the microscope to the object surface to be observed.
Generally, the greater the magnification, the higher the resolution of the objective, and the smaller the working distance, the smaller the field of view. Conversely, the smaller the magnification, the lower the resolution of the objective, and the greater the working distance, and greater the field of view.
High-magnification objectives (such as 80X and 100X objectives) have a very short working distance. Be very careful when focusing for observation. Generally, it is after the objective is in position, the axial limit protection is locked, then the objective is moved away from the direction of the observed object.
The relatively greater working distance leaves a relatively large space between the objective and the object to be observed. It is suitable for under microscope operation, and it is also easier to use more illumination methods. The defect is that it may reduce the numerical aperture of the objective, thereby reducing the resolution.
|Numerical aperture, N.A. for short, is the product of the sinusoidal function value of the opening or solid angle of the beam reflected or refracted from the object into the mouth of the objective and the refractive index of the medium between the front lens of the objective and the object.
Simply speaking, it is the magnitude of the luminous flux that can be brought in to the mouth of the objective adapter, the closer the objective to the specimen for observation, the greater the solid angle of the beam entering the mouth of the objective adapter, the greater the N.A. value, and the higher the resolution of the objective.
When the mouth of the objective adapter is unchanged and the working distance between the objective and the specimen is constant, the refractive index of the medium will be of certain meaning. For example, the refractive index of air is 1, water is 1.33, and cedar oil is 1.515, therefore, when using an aqueous medium or cedar oil, a greater N.A. value can be obtained, thereby improving the resolution of the objective.
N.A. = refractive index of the medium X sin solid angle of the beam of the object entering the front lens frame of the objective/ 2
Numerical aperture of the objective. Usually, there is a calculation method for the magnification of the microscope. That is, the magnification of the microscope cannot exceed 1000X of the objective. For example, the numerical aperture of a 100X objective is 1.25, when using a 10X eyepiece, the total magnification is 1000X, far below 1.25 X 1000 = 1250X, then the image seen in the eyepiece is relatively clear; if a 20X eyepiece is used, the total magnification will reach 2000X, much higher than 1250X, then eventhoughthe image actually seen by the 20X eyepiece is relatively large, the effect will be relatively poor.
|The thickness of the cover glass affects the parfocal distance of the objective. Usually, in the design of the focal length of the objective,the thickness of the cover glass should be considered, and the standard is 0.17mm.
|The use of different media between the objective and the object to be observed is to change and improve the resolution. For example, the refractive index of air is 1, water is 1.33, and cedar oil is 1.515. Therefore, when using an aqueous medium or cedar oil, a greater N.A. value can be obtained, thereby increasing the resolution of the objective.
Air medium is called dry objective, where oil is used as medium iscalled oil immersion objective, and water medium is called water immersion objective.
However, because of the working distance of the objective, when the working distance of the objective is too long, the use of liquid medium will be relatively more difficult, and it is generally used only on high magnification objective having a shorter working distance, such as objectives of 60X, 80X and 100X.
When using oil immersion objective, first add a drop of cedar oil (objective oil) on the cover glass, then adjust the focus (fine adjustment) knob, and carefully observe it from under the side of the objective of the microscope, until the oil immersion objective is immersed in the cedar oil and close to the cover glass of the specimen, then use the eyepiece to observe, and use the fine focus knob to lift the tube until the clear imageof the specimen is clearly seen.
The cedar oil should be added in an appropriate amount. After the oil immersion objective is used, it is necessary to use a piece of lens wiping tissue to dip xylene to wipe off the cedar oil, and then wipe dry the lens thoroughly with a lens wiping tissue.
|The front end of the objective is equipped with a spring device. When the working distance of the objective is too short, focusing can easily make the objective contact the object to be observed, thereby damaging the object to be observed or the front lens. At this time, the spring acts to recover the front end of the objective lens. It is usually used on high magnification objectives with very short working distances.
|For microscopes of different manufacturers and different models, the thread size of their objectives may also be different.
In general, the objective threads are available in two standard sizes, allowing similar objectives between different manufacturers to be used interchangeably.
One is the British system: RMS type objective thread: 4/5in X 1/36in,
One is metric: M25 X 0.75mm thread.
|Illumination base is a modular light source component, suitable for microscope stand base that has no light source of itself, and it is usually dedicated components supporting some stands.
Illumination base typically includes at least one bottom lighting, and there are also illumination base that includes the circuit portion of the upper light source.
|Focus mechanism, the coarse / fine focus knobs are in a coaxial center position, they are connected together by a gear reduction mechanism, which can be coarse/ fine focus adjusted at any time during the entire stroke.
Generally, the coarse focus diameter is relatively big, which is inside close to the body of the microscope, and the fine focus diameter is relatively small, which is outside of the body of the microscope. Coarse focus adjustment is used to quickly move to find the image, and the fine focus adjustment is used to finely adjust the clarity of the image. Generally, the minimum read value of the fine focus adjustment can be accurate to 1 micron, and single circle can reach a stroke of 0.1 mm. Mechanical fine focus plays a very important role in the accuracy of the microscope resolution. If the fine focus accuracy is not enough, or cannot be stabilized at the sharpest focusing position, the image will be out of focus and become blurred.
The tightness of coarse focus is generally adjustable. Generally, on one side of the knob (usually on the right side), there is a textured knob on the inside of the coarse knob, which is tightened if rotated clockwise; and loosened if rotated counterclockwise.
In the process of focusing, direct focusing should not be on the objective of high magnification; instead, find the object of low magnification first, and gradually adjust to high magnification. Usually, the coarse focus knob is rotated first, and when the objective lens is gradually lowered or the platform is gradually rising, find the object, and then adjust with the fine focus, until the object image in the field of view is clear. Generally, when changing from low magnification to high magnification objective, one only need to slightly adjust the fine focus knob to make the object image clear. During the process, the distance between the objective and the specimen should be observed from the side, to understand the critical value of the object distance between the lens and the specimen.
When using a high magnification objective, since the distance between the objective and the specimen is very close, after the image is found, the coarse focus knob cannot generally be used, and the fine focus knob can only be used to avoid excessive distance of movement, damaging the objective and the slide or specimen.
By using the characteristics of the fine focus, the height or thickness of the observed object can be roughly measured under the microscope, such as measuring the thickness of the cell or tissue, the thickness of the cover glass, and the thickness of small objects that cannot be measured by various conventional measuring instruments.
Method of measurement: place the object to be measured at the center of the field of view of the stage. After the image is clearly focused, try to use the highest magnification objective as much as possible, and align the adapter of the top feature point of the object to be measured. After adjusting clear, record the position of scale of the fine focus knob. Then, move the objective down to the adapter of the lowest feature point of the object to be measured, and record the position of scale of the fine focus knob. Then, according to the above fine focus, record the number of rounds of movement, and based on the parameters of conversion of each round into stroke (see the microscope fine focus knob parameters), the number of rounds is converted into the total stroke, which is the height of the object to be measured. If it is repeated a few times for average, a more accurate measurement can be obtained.
|Different microscope bodies, different human operations, and different requirements for observation and operation, all require adjustment of the pre-tightening force of the stand that support microscope body.
Facing the stand just right, use both hands to reverse the force to adjust the tightness. (face the knob of one side just right, clockwise is to tighten, counterclockwise is to loosen)
In general, after long-time use, the knob will be loose, and adjustment is necessary.
|Stage backlight window size refers to the size of the window through which the transmitted light passes under the stage on the XY table plane of the stage.
This window is usually covered with a piece of glass. For some stages with accuracy requirements in the XY horizontal direction, the horizontal plane of the glass can be adjusted by the height of the screws on the four corners below, and the consistency with the height of the stage plane is guaranteed.
|Abbe condenser is a kind of bright field condenser, a condenser that can only finitely correct the spherical aberration, but not the chromatic aberration. When the numerical aperture of its objectives is higher than 0.6, Abbe condenser will show chromatic aberration and spherical aberration.
|EPI-fluorescence kit, also known as the incidence fluorescence kit or reflection fluorescence kit.
It is an accessory device that excites and emits fluorescence for fluorescent microscope illumination, forming an illumination system that consists an illumination source that can emit ultraviolet light, a configured fluorescent condenser, and filters for excitation and emission of fluorescence etc.
Fluorescence microscope requires a complete set of filters. The fluorescence excitation block is the core component used to excite fluorescent specimens and observe fluorescence in a fluorescence microscope. The excitation and emission filters, after combination, are placed in a filter cube. When the user replaces the filter, it is relatively convenient. The component consisting of several filter blocks in the excitation module includes:
Exciter filter: selects the light that passes through a certain wave band to excite the specimen to produce fluorescence.
Dichroic mirror (commonly known as a mirror): set at a 45-degree angle in the cube, reflect the excitation light downwards and transmit the fluorescent upwards.
Barrier filter: transmit the emitted light, that is, fluorescence, and at the same time block the reflected light in the various stray light and excitation light.
Fluorescence is excited by light of a specific wavelength, and the emitted light that is excited has also a specific wavelength. Therefore, it is necessary to select suitable excitation blocks (mainly selecting the light wavelength parameters of excitation light filter, dichroic mirror and barrier filter).
The excitation module is generally named after the basic color tone. The first letter represents the excitation spectrum region of the wavelength, that is, the color tone. For example, UV represents ultraviolet light, V represents violet, B represents blue, and G represents green. The subsequent letter represents the glass, and the numbers represent the model features. For example, BG12 is a kind of blue glass, B is the first letter of blue, and G is the first letter of glass.
|Coupler/C-mount adapter is an adapter commonly used for connection between the C-adapter camera (industrial camera) and a microscope.
|On the coupler/C-mount-adapter, there is an adjustable device to adjust the focal length.
|Different coupler/C-mount-adapters are suitable for different microscopes. For some, some adapter accessories need to be replaced. See the applicable range of each coupler/C-mount-adapter for details.
|Coupler magnification refers to the line field magnification of the coupler/C-mount-adapter. With different magnifications of the adapter lens, images of different magnifications and fields of view can be obtained. The size of the image field of view is related to the sensor size and the coupler/C-mount-adapter magnification.
Camera image field of view (mm) = sensor diagonal / coupler/C-mount-adapter magnification.
For example: 1/2 inch sensor size, 0.5X coupler/C-mount-adapter coupler, field of view FOV (mm) = 8mm / 0.5 = 16mm.
The field of view number of the microscope 10X eyepiece is usually designed to be 18, 20, 22, 23mm, less than 1 inch (25.4mm). Since most commonly used camera sensor sizes are 1/3 and 1/2 inches, this makes the image field of view on the display always smaller than the field of view of the eyepiece for observation, and the visual perception becomes inconsistent when simultaneously viewed on both the eyepiece and the display. If it is changed to a 0.5X coupler/C-mount-adapter, the microscope image magnification is reduced by 1/2 and the field of view is doubled, then the image captured by the camera will be close to the range observed in the eyepiece.
Some adapters are designed without a lens, and their optical magnification is considered 1X.
|At present, the coupler/C-mount adapter generally adopts the C/CS-Mount adapter to match with the industrial camera. For details, please refer to "Camera Lens Mount".
|Sneeze guard is mainly to provide isolation and protection between the operator and the object being observed.
When using ultraviolet light, although the ultraviolet radiation dose is not high, long time observation can have a harmful effect on the human eye, and therefore it is necessary to use sneeze guard to isolate.
The sneeze guard can also isolate harmful, radiating, and volatile substances to ensure the safety and health of the operator. On the other hand, it can also prevent people from exhaling, sneezing, etc. during operation, thereby polluting the observed objects.
|After unpacking, carefully inspect the various random accessories and parts in the package to avoid omissions. In order to save space and ensure safety of components, some components will be placed outside the inner packaging box, so be careful of their inspection.
For special packaging, it is generally after opening the box, all packaging boxes, protective foam, plastic bags should be kept for a period of time. If there is a problem during the return period, you can return or exchange the original. After the return period (usually 10-30 days, according to the manufacturer’s Instruction of Terms of Service), these packaging boxes may be disposed of if there is no problem.
|Microscope Optical Data Sheet
|Objective Working Distance
|BM05102221 (10X Dia. 20mm)
|Field of View(mm)
|1. Magnification=Objective Optical Magnification * Body Magnification * Eyepiece Optical Magnification
|2. Field of View=Eyepiece Field of View /（Objective Optical Magnification*Body Magnification）
|3. The Darker background items are Standard items, the white background items are optional items.
|Video Microscope Optical Data Sheet
|1. Magnification=Objective Optical Magnification * Body Magnification * Coupler Magnification
|M1.5 1pc M2.5 1pc M3 1pc
|Product Instructions/Operation Manual
|Inner Packing Material
|Ancillary Packaging Materials
|Minimum Packaging Quantity
|Transportation Carton Material
|Transportation Carton Dimensions(1)
|Transportation Carton Dimensions(2)
|Total Gross Weight of Transportation(kilogram)
|Total Gross Weight of Transportation(pound)