Inclined illumination relief microscopic imaging system based on phase difference light path
By designing an oblique illumination relief microscopic imaging system based on phase difference optical path, and utilizing an adjustable oblique illumination phase difference shield and phase difference objective lens, high image contrast and good relief effect microscopic imaging are achieved, solving the problems of difficult adjustment and high cost in traditional systems.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Utility models(China)
- Current Assignee / Owner
- NANKAI UNIV
- Filing Date
- 2025-06-19
- Publication Date
- 2026-04-10
AI Technical Summary
Traditional oblique illumination imaging systems cannot achieve continuous adjustment to achieve the best relief effect, and it is difficult to switch between relief and phase contrast functions at the same time, while also being costly.
Design a slanted illumination relief microscopic imaging system based on phase difference optical path, including an adjustable slanted illumination phase difference shield, a phase difference objective lens and an imaging device. The light transmission area can be continuously adjusted by adjusting the shield, and the system supports switching between relief and phase difference effects.
This invention achieves a microscopic imaging system with high image contrast, good relief effect, simple structure and low cost, and can produce high-quality relief images at different magnifications.
Smart Images
Figure CN224109728U_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The utility model relates to optical system technical field especially relates to a slant illumination embossment microscopic imaging system based on phase difference light path. BACKGROUND
[0002] In bright field imaging, the light emitted by the light source is divided into direct light and diffraction light after passing through the sample. The direct light does not deviate, directly passes through or bypasses the sample, and the diffraction light amplitude does not decrease like the light-absorbing object, but the propagation speed slows down due to the difference in refractive index or thickness of the sample, and the phase lags about 1 / 4 wavelength. When the diffraction light and the non-deflected direct light reach the image plane at the same time, interference occurs between the two, but the light intensity after interference does not decrease significantly, resulting in extremely low contrast of the final image, making it difficult to distinguish the details of the sample and almost invisible.
[0003] Phase contrast imaging is a method of converting the phase difference caused by the refractive index of the sample into imaging intensity change. By placing a ring diaphragm (phase contrast ring) in front of the condenser, the direct light passes through the specimen in the shape of a hollow light cone and reaches the back focal plane of the objective lens in the shape of a phase contrast ring. The diffraction light is distributed on the entire back focal plane of the objective lens. When the light passes through the back focal plane of the objective lens, the phase contrast ring plate installed on the back focal plane of the objective lens will delay the direct light by 1 / 4 wavelength, so that the wavelength difference between the direct light and the diffraction light of the phase sample becomes 1 / 2 wavelength. At this time, the direct light and the diffraction light reaching the image plane will be able to produce interference, thereby improving the contrast of the boundary surface. Although phase contrast imaging can enhance the contrast of the image, it still lacks a certain stereoscopic effect and details.
[0004] Differential interference contrast (DIC) is another commonly used label-free microscope imaging method. The light from the light source is converted into plane polarized light by a polarizer, then divided into two polarized light beams with perpendicular polarization directions by a Wollaston prism, and then combined together by another Wollaston prism after passing through the sample, and finally converted into intensity information by a polarizer, thereby giving the sample a pseudo-relief appearance. DIC can make more full use of the numerical aperture of the system and improve the contrast of the image, but it is not suitable for plastic tissue culture containers, and its optical path is also more complex compared with other methods.
[0005] As a traditional observation technique, oblique illumination has been used to improve the visibility of transparent or translucent samples, especially for imaging various unstained objects. When oblique light is incident on the sample surface, refraction or diffraction occurs, and different shadows are produced after the light is imaged by the objective lens, thereby producing a clear difference between light and dark on the transparent or translucent specimen, and the specimen appears as a clear relief, enhancing the contrast. The most commonly used method is to offset part of the condenser diaphragm or add a fan-shaped block between the condenser and the diaphragm to form an oblique illumination light path. Compared with marker-free observation techniques such as differential interference, phase contrast or Hoffman modulation, the device cost of oblique illumination is significantly lower, and only a conventional microscope is needed.
[0006] However, the conventional oblique illumination imaging system has some problems:
[0007] 1. The area blocked at the diaphragm to achieve oblique light is fixed and cannot be continuously adjusted to achieve the best relief effect;
[0008] 2. The conventional microscope often needs to realize both relief and phase difference functions, but it is difficult to achieve simple switching between the two functions while maintaining good relief effect;
[0009] 3. The conventional oblique illumination imaging system has poor relief effect and little contrast improvement without using additional elements for modulation. Utility model content
[0010] The technical problem to be solved by the utility model is to provide an oblique illumination relief microscopic imaging system based on a phase difference light path, which can realize continuous adjustment and has good relief observation effect, is highly versatile, simple in structure and low in cost.
[0011] The utility model is implemented through the following technical solutions:
[0012] An oblique illumination relief microscopic imaging system based on a phase difference light path, comprising a light source, an adjustable oblique illumination phase difference blocking plate, a condenser, a sample platform, a phase difference objective lens and an imaging device, wherein the adjustable oblique illumination phase difference blocking plate is located between the light source and the condenser, the sample platform is located at the back focal plane of the condenser, the phase difference objective lens is located below the sample platform and the center line of the phase difference objective lens coincides with that of the condenser, and the imaging device is located below the phase difference objective lens, wherein the adjustable oblique illumination phase difference blocking plate comprises a phase difference ring plate, a phase difference ring and a light blocking plate, the phase difference ring is an annular light hole provided on the phase difference ring plate, and the parameters of the phase difference ring are matched with the magnification of the phase difference objective lens, and the light blocking plate is slidably installed on the phase difference ring plate.
[0013] The imaging device comprises a tube lens, a camera and a computer connected with the camera, the tube lens is located below the phase difference objective lens, and the camera is located at the position of the tube lens focal point below the tube lens.
[0014] Optimally, the magnification of the phase difference objective lens is 10 times, 20 times or 40 times.
[0015] Optimally, the phase difference ring comprises a first phase difference ring and a second phase difference ring, both of which are horizontally arranged on the phase difference ring plate with a spacing in between, wherein the parameters of the first phase difference ring are matched with the 10 times phase difference objective lens, and the parameters of the second phase difference ring are matched with the 20 times phase difference objective lens or the 40 times phase difference objective lens.
[0016] Optimally, the parameters of the first phase difference ring are: inner ring radius 2.75mm, ring width 0.85mm, and the parameters of the second phase difference ring are: inner ring radius 4.90mm, ring width 1.35mm.
[0017] Optimally, the light shielding plate is of a pull-out structure.
[0018] Optimally, the light shielding plate is located above or below the phase difference ring plate.
[0019] Further, the light shielding plate is provided with a shielding position mark of optimal imaging effect.
[0020] Optimally, the sample substrate placed on the sample platform is a plastic substrate or a glass substrate.
[0021] The beneficial effects of the utility model are as follows:
[0022] Compared with the prior art, the oblique illumination relief microscopic imaging system based on the phase difference light path of the utility model is constructed based on the phase difference light path, so that the image contrast is higher, the relief imaging effect is better, and the continuous adjustment of the light transmission area can be realized by adjusting and marking the shielding plate on the adjustable oblique illumination phase difference shielding plate, the best relief observation effect is achieved, the phase difference effect and the relief effect of the whole system can be switched by pulling out and shielding the shielding plate, and the system also has the advantages of simple structure and low implementation cost. BRIEF DESCRIPTION OF DRAWINGS
[0023] Figure 1 is the system structure schematic diagram of the utility model.
[0024] Figure 2 is the shielding position schematic diagram of the shielding plate of the utility model.
[0025] Figure 3 is the image of human osteosarcoma cells (U2OS) and red blood cells photographed under the 20x phase difference objective lens of the utility model without shielding and with the light transmission area ratio of 1 / 3.
[0026] Fig. 4(a) is the ordinary phase difference imaging effect diagram of U2OS cells under the 20x phase difference objective lens of the utility model.
[0027] Figure 4(b) is the U2OS cell relief imaging effect diagram when the light passing area ratio is 2 / 3 under the 20x phase difference objective lens of the utility model.
[0028] Figure 4(c) is the U2OS cell relief imaging effect diagram when the light passing area ratio is 1 / 3 under the 20x phase difference objective lens of the utility model.
[0029] Figure 4(d) is the U2OS cell relief imaging effect diagram when the light passing area ratio is 1 / 8 under the 20x phase difference objective lens of the utility model.
[0030] Figure 5 It is the U2OS cell image photographed when the light passing area ratio is 1 / 3 under the different magnification phase difference objective lens of the utility model.
[0031] Figure 6 It is the U2OS cell image photographed on different substrates under the 20x phase difference objective lens of the utility model.
[0032] Figure 7 It is the U2OS cell image photographed under the 20x different objective lens.
[0033] Figure 8 It is the system side view of the utility model.
[0034] Figure 9 It is the system front view of the utility model.
[0035] In the figure: 1, light source; 2, adjustable oblique illumination phase difference blocking plate; 3, condenser; 4, sample platform; 5, phase difference objective lens; 6, tube lens; 7, camera; 8, computer; 9, phase difference ring plate; 10, phase difference ring; 11, light shield; 12, blocking position mark. DETAILED DESCRIPTION
[0036] An oblique illumination relief microscopic imaging system based on phase difference light path, the system structure schematic diagram as shown in Figure 1 As shown in the system side view Figure 8 As shown in the system front view Figure 9 It includes light source 1, adjustable oblique illumination phase difference blocking plate 2, condenser 3, sample platform 4, phase difference objective lens 5 and imaging device, adjustable oblique illumination phase difference blocking plate is located between light source and condenser, sample platform is located at the back focal plane position of condenser, phase difference objective lens is located below the sample platform and the center line of phase difference objective lens and condenser coincides, imaging device is located below phase difference objective lens, adjustable oblique illumination phase difference blocking plate includes phase difference ring plate 9, phase difference ring 10 and light shield 11, phase difference ring is annular light passing hole and is arranged on phase difference ring plate, and the parameter of phase difference ring is matched with the multiple of phase difference objective lens, and the light shield is slidably installed on the phase difference ring plate.
[0037] The adjustable oblique illumination phase difference blocking plate and the phase difference objective lens are arranged, the phase difference ring is arranged on the phase difference ring plate, the parameters of the phase difference ring are matched with the magnification of the phase difference objective lens, the light blocking plate is slidably arranged on the phase difference ring plate, the image contrast based on the phase difference light path is higher, the relief imaging effect is better, the continuous adjustment of the light transmission area can be realized by adjusting and marking the blocking plate, and the best relief observation effect is achieved. Only one conventional microscope is needed to realize the structure, and the structure is simple and the implementation cost is low.
[0038] The optimized imaging device comprises a tube lens 6, a camera 7 and a computer 8 connected with the camera, the tube lens is arranged below the phase difference objective lens, the camera is arranged below the tube lens at the position of the tube lens focal point, the image of the microscopic system imaging is collected, and the image is transmitted to the computer display system for imaging and subsequent processing.
[0039] The optimized phase difference objective lens has a magnification of 10 times, 20 times or 40 times, the phase difference ring comprises a first phase difference ring and a second phase difference ring, the two phase difference rings are arranged horizontally on the phase difference ring plate and have a spacing therebetween, the parameters of the first phase difference ring are matched with the 10 times phase difference objective lens, and the parameters of the second phase difference ring are matched with the 20 times phase difference objective lens or the 40 times phase difference objective lens.
[0040] The two phase difference rings can be respectively applied to phase difference objective lenses with different magnifications, when the phase difference objective lens with different magnifications is replaced, the adjustable oblique illumination phase difference blocking plate does not need to be replaced, and only the position of the light blocking plate needs to be moved, so that the better relief imaging effect is achieved, the system structure is relatively simple, the implementation cost is lower, and the efficiency is higher.
[0041] The optimized first phase difference ring has parameters of an inner ring radius of 2.75 mm and an inner ring width of 0.85 mm, the parameters are matched with the 10 times phase difference objective lens, and the better relief imaging effect can be achieved.
[0042] The second phase difference ring has parameters of an inner ring radius of 4.90 mm and an inner ring width of 1.35 mm, the parameters are matched with the 20 times phase difference objective lens or the 40 times phase difference objective lens, and the better relief imaging effect can be achieved.
[0043] The optimized light blocking plate has a pull-out structure, the light blocking plate can be arranged above or below the phase difference ring plate, the sliding of the light blocking plate is more convenient, the continuous adjustment of the light transmission area is realized, and the best relief observation effect is achieved.
[0044] Further, the light blocking plate is provided with a blocking position mark 12 of the optimal imaging effect, the blocking position of the optimal imaging effect is conveniently and quickly found, the adjustment of different light transmission area ratios is quickly realized, and the imaging efficiency is greatly improved.
[0045] The sample substrate placed on the optimized sample platform is a plastic substrate or a glass substrate.
[0046] The schematic diagram of the blocking position of the blocking plate is shown in FIG. 2, wherein (a) represents a schematic diagram in a case of no blocking, (b) represents a schematic diagram in a case of blocking 1 / 3, and (c) represents a schematic diagram in a case of blocking 2 / 3. In the figures, ring #2 represents a second phase difference ring, and ring #1 represents a first phase difference ring. Figure 2
[0047] In the embodiment, the phase difference ring R = 4.90 mm, the ring width L = 1.35 mm, and the phase difference objective lens has a magnification of 20x. The blocking plate and the phase difference ring form a light passing area and a light blocking area in the optical path. By moving the position of the blocking plate, the light passing area can be changed.
[0048] The light emitted by the light source passes through the phase difference blocking plate without being blocked by the blocking plate. Subsequently, part of the hollow light column passes through the condenser lens, forming a part of the hollow light cone irradiating the sample on the sample platform from one side. The light after passing through the sample is focused on the camera through the phase difference objective lens and the tube lens. The contrast-enhanced image with the relief effect is observed through the computer connected with the camera.
[0049] The light passing area S1 of the phase difference ring can be adjusted according to different magnifications and numerical apertures of the objective lens to optimize the relief effect under different objective lenses. In the embodiment, the objective lens used is a 20x phase difference objective lens. By adjusting the light passing area through experiments, it can be found that the ratio of the light passing area S1 to the light passing hole area S under the optimal relief effect of the phase difference objective lens is between 1 / 3 and 2 / 3.
[0050] By setting the optimal imaging effect blocking position mark on the corresponding position of the blocking plate, the optimal imaging effect blocking position mark is aligned with the rear end of the phase difference ring plate, so that the adjustment of different light passing area ratios can be quickly realized.
[0051] Figure 3 FIG. 4 shows the images of human osteosarcoma cells U2OS and red blood cells taken under the conditions of no blocking and the light passing area ratio of 1 / 3 under the 20x phase difference objective lens, wherein (a) represents the ordinary phase difference effect diagram of the adherent cells under the 20x phase difference objective lens without blocking, (b) represents the relief effect diagram of the adherent cells under the 20x phase difference objective lens with the light passing area ratio of 1 / 3, (c) represents the ordinary phase difference effect diagram of the red blood cells under the 20x phase difference objective lens without blocking, and (d) represents the relief effect diagram of the red blood cells under the 20x phase difference objective lens with the light passing area ratio of 1 / 3.
[0052] Figure 3 It can be seen that the image of the 2 / 3 difference ring shielding has obvious relief effect and better contrast compared with the ordinary difference image, which shows that the utility model can realize good relief imaging effect compared with the ordinary difference imaging.
[0053] 20x difference objective lens under the light area ratio of 1, 2 / 3, 1 / 3 and 1 / 8 under the U2OS cell image photographed respectively as shown in figure 4 (a), 4 (b), 4 (c), 4 (d), wherein figure 4 (a) represents the U2OS cell ordinary difference imaging effect diagram under 20x difference objective lens, figure 4 (b) represents the U2OS cell relief imaging effect diagram under 20x difference objective lens when the light area ratio is 2 / 3, figure 4 (c) represents the U2OS cell relief imaging effect diagram under 20x difference objective lens when the light area ratio is 1 / 3, and figure 4 (d) represents the U2OS cell relief imaging effect diagram under 20x difference objective lens when the light area ratio is 1 / 8.
[0054] From figure 4 (a), 4 (b), 4 (c), 4 (d), it can be seen that when the light area ratio is between 2 / 3 and 1 / 3, the relief effect of the image is better, which shows that adjusting the light area ratio within a certain range can effectively realize good relief effect. When the light area ratio is 1 / 8, the field brightness and uniformity are poor, which shows that excessive shielding can lead to poor relief effect.
[0055] Figure 5 For the U2OS cell images photographed under different magnification difference objective lens ordinary difference imaging and light area ratio of 1 / 3, wherein (a) represents the U2OS cell ordinary difference imaging effect diagram under 10x difference objective lens full shielding, (b) represents the U2OS cell relief imaging effect diagram under 10x difference objective lens when the light area ratio is 1 / 3, (c) represents the U2OS cell ordinary difference imaging effect diagram under 20x difference objective lens full shielding, (d) represents the U2OS cell relief imaging effect diagram under 20x difference objective lens when the light area ratio is 1 / 3, (e) represents the U2OS cell ordinary difference imaging effect diagram under 40x difference objective lens full shielding, and (f) represents the U2OS cell relief imaging effect diagram under 40x difference objective lens when the light area ratio is 1 / 3.
[0056] From Figure 5 It can be seen that the system can produce good relief imaging effect for different magnification difference objective lenses.
[0057] Figure 6U2OS cell images taken under 20x different objectives on different substrates, wherein (a) represents the ordinary phase contrast imaging effect of U2OS cells on a glass substrate taken under 20x phase contrast objective, (b) represents the relief imaging effect of U2OS cells on a glass substrate taken under 20x phase contrast objective with a light transmission area ratio of 2 / 3, (c) represents the ordinary phase contrast imaging effect of U2OS cells on a plastic substrate taken under 20x phase contrast objective, (d) represents the relief imaging effect of U2OS cells on a plastic substrate taken under 20x phase contrast objective with a light transmission area ratio of 2 / 3.
[0058] From Figure 6 It can be seen that the system can produce good relief imaging effect on samples on different substrates.
[0059] Figure 7 U2OS cell images taken under 20x different objectives, wherein (a) represents the ordinary phase contrast imaging effect of U2OS cells under 20x phase contrast objective, (b) represents the relief imaging effect of U2OS cells under 20x phase contrast objective with a light transmission area ratio of 2 / 3, (c) represents the bright field imaging effect of U2OS cells under 20x ordinary objective, (d) represents the bright field imaging effect of U2OS cells under 20x ordinary objective with a light transmission area ratio of 2 / 3.
[0060] From Figure 7 It can be seen that only when the phase contrast objective is used, the microscopic system can achieve good relief effect.
[0061] 1 / 3 oblique illumination marked in the above figure is a light transmission area ratio of 1 / 3.
[0062] In summary, the oblique illumination relief microscopic imaging system based on phase contrast light path provided by the present application has high image contrast and good relief imaging effect, and by adjusting and marking the shutter on the adjustable oblique illumination phase contrast shutter, continuous adjustment of the light transmission area is realized to achieve the best relief observation effect, and the system can be switched between phase contrast effect and relief effect by pulling out and shielding the shutter, and the system structure is simple and the implementation cost is low.
[0063] The above only describes preferred embodiments of the present application and is not used to limit the present application, and for those skilled in the art, the present application can have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.
Claims
1. A slanted illumination relief microscopic imaging system based on phase difference optical path, characterized in that: The device includes a light source, an adjustable oblique illumination phase contrast shield, a condenser lens, a sample platform, a phase contrast objective lens, and an imaging device. The adjustable oblique illumination phase contrast shield is located between the light source and the condenser lens. The sample platform is located at the back focal plane of the condenser lens. The phase contrast objective lens is located below the sample platform, and its center line coincides with that of the condenser lens. The imaging device is located below the phase contrast objective lens. The adjustable oblique illumination phase contrast shield includes a phase contrast ring plate, a phase contrast ring, and a light-shielding plate. The phase contrast ring has an annular light-transmitting hole on the phase contrast ring plate, and the parameters of the phase contrast ring match the magnification of the phase contrast objective lens. The light-shielding plate is slidably mounted on the phase contrast ring plate.
2. The oblique illumination relief microscopic imaging system based on phase difference optical path according to claim 1, characterized in that: The imaging device includes a tube mirror, a camera, and a computer connected to the camera. The tube mirror is located below the phase contrast objective lens, and the camera is located below the tube mirror at the focal point of the tube mirror.
3. The oblique illumination relief microscopic imaging system based on phase difference optical path according to claim 1, characterized in that: The phase contrast objective lens has a magnification of 10x, 20x, or 40x.
4. The oblique illumination relief microscopic imaging system based on phase difference optical path according to claim 3, characterized in that: The phase contrast ring includes a first phase contrast ring and a second phase contrast ring. The two phase contrast rings are arranged horizontally on the phase contrast ring plate with a gap in the middle. The parameters of the first phase contrast ring are matched with a 10x phase contrast objective lens, and the parameters of the second phase contrast ring are matched with a 20x or 40x phase contrast objective lens.
5. The oblique illumination relief microscopic imaging system based on phase difference optical path according to claim 4, characterized in that: The parameters of the first phase difference ring are: inner radius 2.75 mm, ring width 0.85 mm, and the parameters of the second phase difference ring are: inner radius 4.90 mm, ring width 1.35 mm.
6. The oblique illumination relief microscopic imaging system based on phase difference optical path according to claim 1, characterized in that: The light-shielding panel has a pull-out structure.
7. The oblique illumination relief microscopic imaging system based on phase difference optical path according to claim 1, characterized in that: The light-shielding plate is located above or below the phase difference ring plate.
8. The oblique illumination relief microscopic imaging system based on phase difference optical path according to claim 1, characterized in that: The light-shielding plate is marked with the occlusion position for optimal imaging effect.
9. The oblique illumination relief microscopic imaging system based on phase difference optical path according to claim 1, characterized in that: The sample substrate placed on the sample platform is either a plastic substrate or a glass substrate.