General relief micro-imaging system based on oblique illuminationTechnical Field
The invention relates to a microscopic imaging system, in particular to a general relief microscopic imaging system based on oblique illumination.
Background
During the lengthy development of microscopes, oblique illumination techniques have been used as a traditional technique for improving the visibility of transparent or translucent specimens, particularly for imaging various undyed objects, such as living cells, crystals, algae, etc. When the oblique light irradiates the surface of the sample, refraction or diffraction is generated, and different shadows are generated after the light rays are imaged through the objective lens, so that the transparent or semi-transparent sample has a brightness difference and is in an obvious relief shape, and the contrast ratio is enhanced. The most common method is to offset part of the condenser aperture or add a fan-shaped stop between the condenser and aperture to form an oblique illumination light path. Compared to non-marker observations such as differential interference, phase contrast, or huffman modulation, the cost of obliquely illuminated equipment is significantly lower, requiring only one microscope of conventional configuration. In addition, in some cases, the oblique illumination technique can enable the observer to see more phase details than differential interference, and the contrast can be significantly enhanced while the resolution is not lower than the bright field resolution.
The relief microscopic imaging system needs to add a condenser side relief flashboard and a eye lens barrel side relief flashboard in a bright field microscopic light path. Wherein the side relief flashboard of the collecting lens is positioned at the front focal plane position of the collecting lens, and a fan-shaped baffle plate is arranged on the flashboard to play a part of shading; the eye lens barrel side relief insertion plate is positioned between the objective lens and the ocular light path, and the insertion plate comprises a plurality of diaphragm holes with different sizes for limiting the clear aperture of the back focal plane of the objective lens. The illumination light forms oblique illumination after passing through the side relief picture peg of the condenser and the condenser to irradiate on the sample, and then the image with relief stereoscopic effect is observed through the objective lens and the ocular lens. In the process, the baffle image on the condenser side relief flashboard can be imaged to the diaphragm hole position on the eye tube side relief flashboard, and the objective lenses with different multiplying powers correspond to diaphragm holes with different sizes so as to meet the requirement of realizing the relief effect. However, conventional relief imaging systems have some problems: 1. the shielding area of the sector baffle plate of the relief flashboard at the side of the collecting lens is fixed, and the objective lens with different multiplying power cannot be adjusted to match the numerical aperture of the objective lens, so that the optimal relief effect and the field uniformity cannot be realized; 2. conventional microscopes often need to realize the relief and phase contrast functions simultaneously, when the relief system is observed by using a phase contrast objective, a phase contrast ring in the objective may form a shielding for a light-transmitting area of a sector-shaped annular plate image on the relief plugboard, so that serious uneven brightness of an image surface is caused; 3. when the eye lens barrel side relief flashboard is used, the flashboard needs to be moved to switch the size of the diaphragm hole so as to match objective lenses with different multiplying powers, the adjusting process is not convenient enough, and the repeated back and forth use can cause abrasion to the clamping groove position, so that the centering precision of the diaphragm hole is affected to deteriorate the imaging effect.
Disclosure of Invention
The invention aims to solve the technical problem of providing a general relief microscopic imaging system based on oblique illumination, which can achieve the best relief observation effect aiming at objective lenses with different multiplying powers, and has the advantages of strong universality, simple structure and low cost.
The technical scheme adopted for solving the technical problems is as follows: the utility model provides a general type relief (sculpture) microscopic imaging system based on oblique illumination, includes light source, condensing lens, sample platform, objective and image device, the light source with the condensing lens between be provided with condensing lens side relief (sculpture) picture peg, condensing lens side relief (sculpture) picture peg be located the front focal plane position of condensing lens and be provided with the circular light-passing hole that condensing lens clear aperture size equals, objective with image device between be provided with the lens cone side relief (sculpture) picture peg, the light-passing hole on be provided with the light screen that can continuously adjust the light-passing area, the lens cone side relief (sculpture) picture peg be provided with circular adjustable diaphragm hole.
Compared with the prior art, the invention has the advantages that the shielding area of the shielding plate of the side inserting plate of the condensing lens can be adjusted, and the size of the diaphragm hole of the side inserting plate of the eye lens cone can be adjusted, so that the system can achieve the best relief observation effect aiming at objective lenses with different multiplying powers. The system can also be matched with a phase contrast objective lens, so that the universality of the system is improved, and meanwhile, the system is simple in structure and low in implementation cost.
Preferably, the light shielding plate is of a drawing structure, and the light passing area of the light passing hole is changed by the transverse movement of the light shielding plate.
Further, the objective lens may be a phase contrast objective lens.
Preferably, the imaging device comprises a tube mirror, a camera and a PC, wherein the camera is positioned at the focal position of the tube mirror and used for collecting images imaged by the microscope system, and the PC is connected with the camera and used for displaying the images imaged by the microscope system and subsequent processing.
Preferably, a reflective mirror is arranged between the eye tube side relief insert plate and the tube mirror, and light rays emitted from the eye tube side relief insert plate enter the tube mirror through reflection of the reflective mirror. This configuration can effectively reduce the volume of the system.
Preferably, the light passing area is S1, the light passing hole area is S, and on the premise of ensuring that the brightness and uniformity of the imaging field of view meet the requirements of the observed sample, the light passing hole area satisfies the following conditions: when the objective lens is 10 times, S1/S is less than or equal to 0.38; when the objective lens is 20 times, S1/S is less than or equal to 0.35; when the objective lens is 40 times, S1/S is less than or equal to 0.31.
Drawings
Fig. 1 is a schematic structural diagram of a general relief microscopic imaging system based on oblique illumination in the embodiment, wherein the serial number 1 is a light source, the serial number 2 is a condenser side relief image plug board, the serial number 3 is a condenser, the serial number 4 is a platform, the serial number 5 is an objective lens, the serial number 6 is a eyepiece barrel side relief image plug board, the serial number 7 is a reflector, the serial number 8 is a tube lens, the serial number 9 is a camera, and the serial number 10 is a PC;
FIG. 2 is a schematic top view of a condenser side relief insert plate, with number 2-1 being the light passing hole and number 2-2 being the mask;
FIG. 3 is a graph of oral epithelial cells taken at different light transmission area ratios under a 20 objective lens, wherein FIG. a is an image with a light transmission area ratio of 0.39, and FIG. b is an image with a light transmission area ratio of 0.35;
FIG. 4 is a schematic top view of a side relief insert plate of a eyepiece barrel, wherein the number 6-1 is an adjustable diaphragm hole, and the number 6-2 is an adjusting deflector rod;
fig. 5 shows an image of oral epithelial cells taken under each magnification objective lens, fig. a is taken with a 10x objective lens, fig. b is taken with a 20x objective lens, and fig. c is taken with a 40x objective lens;
FIG. 6 is a graph showing imaging effects of a condensing lens side relief insert plate in different states from a phase backing ring, and FIG. a is a graph showing imaging effects when the light passing region is not overlapped with the phase backing ring; and (3) an imaging effect diagram when the light transmission area of the diagram b is overlapped with the phase lining ring.
Detailed Description
The invention is described in further detail below with reference to the embodiments of the drawings.
Example 1:
as shown in fig. 1, the general relief microscopic imaging system based on oblique illumination of the invention comprises a light source 1, a condenser side relief plugboard 2, a condenser 3, a platform 4, an objective lens 5, a eyepiece barrel side relief plugboard 6, a reflector 7, a tube mirror 8, a camera 9 and a PC 10 which are sequentially arranged; the light-gathering side relief flashboard 2 is arranged at the front focal plane position of the light gathering lens 3; the reflector 7 is positioned behind the eye tube side relief picture peg 6 and is placed at 45 degrees with the optical axis, and is used for turning light into 90 degrees to enter the tube mirror 8, and the camera 9 is positioned at the focal position of the tube mirror 8 and is used for collecting images imaged by the microscope system; the PC 10 is connected with the camera 9 through a data line and is used for imaging and subsequent processing of the display system. As shown in fig. 2, the optical collecting lens side relief insert plate 2 comprises an optical through hole 2-1, the optical through hole 2-1 is circular in shape, and the radius R of the circular shape is equal to the radius of the optical collecting lens optical through hole, in this embodiment, r=15.5 mm; a light-transmitting hole is internally provided with a light-shielding plate 2-2, and the light-shielding plate 2-2 and the light-transmitting hole 2-1 form a light-transmitting area and a light-blocking area in a light path; the light shielding plate 2-2 is of a drawing structure and can transversely move in the light transmission hole to change the light transmission area; the light passing area S1 occupies the light passing hole area S, and the following conditions are satisfied: S1/S < 0.4.
Light emitted by the light source 1 passes through a light-passing hole on the light collecting lens side relief picture peg 2, which is not blocked by the light shielding plate 2-2, then passes through the light collecting lens 3 from the off-axis direction to form oblique light, irradiates onto a sample of the platform 4 from one side, passes through the objective lens 5 and the eye lens barrel side relief picture peg 6, finally focuses an image on the camera 9 through the reflector 7 and the tube lens 8, and observes a contrast enhancement image with relief effect through the PC 10 connected with the camera 9.
The light passing area S1 can be adjusted according to objective lenses with different multiplying powers and numerical apertures, and the relief effect under different objective lenses is optimized. The objective lens used in this embodiment is three types of objective lenses of 10x (na=0.25), 20x (na=0.4) and 40x (na=0.6), and the area of the light-transmitting area is experimentally adjusted to find the ratio of the light-transmitting area under the optimal relief effect of each objective lens as follows: when a 10-fold objective lens is used, the ratio of the light-passing area S1 to the light-passing hole area S is: s1/s=0.37; when a 20-fold objective lens is used, the ratio of the light-passing area S1 to the light-passing hole area S is: s1/s=0.32; when a 40-fold objective lens is used, the ratio of the light-passing area S1 to the light-passing hole area S is as follows: s1/s=0.3.
Fig. 3 is an image of oral epithelial cells taken under a 20x objective lens with a light transmission area ratio of 0.39 and 0.35, respectively, and an image with a light transmission area ratio of 0.35 can be seen to have a relief effect and a contrast ratio superior to those of 0.39, which indicates that the relief effect can be effectively optimized by adjusting the light transmission area ratio.
As shown in fig. 4, the eye tube side relief insert plate 6 comprises an adjustable aperture hole 6-1, the adjustable aperture hole 6-1 is a conventional adjustable aperture formed by overlapping a plurality of blades, and the radius r of the aperture hole is adjusted by pulling the adjusting deflector rod 6-2 to drive and change the overlapping part of the blades; the diaphragm aperture radius r should satisfy:wherein R' is the spot radius of the back focal plane of the objective lens 5, R is the radius of a light passing hole 2-1 in the condenser side relief insert plate 2, f1 is the combined focal length of all lenses in front of the back focal plane of the objective lens 5, and f2 is the focal length of the condenser 3; in this embodiment, the focal length f2=47 mm, the 10x objective f1=19.2 mm, r' =4.8 mm, and the corresponding aperture diameter is 9.6mm;20x objective f1=12.1 mm, r' =5mm, corresponding diaphragm aperture diameter 8mm;40x objective f1=9.2 mm, r' =5.7 mm, corresponding toThe diaphragm aperture diameter is 6.1mm.
Fig. 5 shows that in this embodiment, by adjusting the size of the light-passing area on the condenser side relief plug board 2 and the size of the diaphragm aperture 6-1 on the eye tube side relief plug board 6, the cell nucleus and the cell shape of the transparent cell can be seen to have obvious relief stereoscopic effect, and the detection capability of the transparent cell is remarkably improved.
In this embodiment, the objective lens 5 may be replaced with a phase-contrast objective lens, but it should be noted that the size of the light passing area on the condenser side relief insert plate 2 should be adjusted so that the image on the back focal plane of the objective lens 5 does not overlap with the inner phase collar in the objective lens. As shown in fig. 6, when the light passing area overlaps the phase collar, the brightness uniformity of the imaging surface is affected to cause uneven field of view, which affects the final imaging effect.