Inclined illumination relief microscopic imaging system based on phase difference light path

Abstract

The invention relates to the technical field of optical systems, in particular to an oblique illumination embossment microscopic imaging system based on a phase difference light path, which comprises a light source, an adjustable oblique illumination phase difference baffle plate, a collecting lens, a sample platform, a phase difference objective lens and an imaging device, the sample platform is located at the position of a rear focal plane of the collecting lens, the phase difference objective lens is located below the sample platform, the center line of the phase difference objective lens coincides with the center line of the collecting lens, the imaging device is located below the phase difference objective lens, and the adjustable inclined illumination phase difference shielding plate comprises a phase difference ring plate, a phase difference ring and a light shielding plate. Parameters of the phase difference ring are matched with multiples of the phase difference objective lens, and the shading plate is installed on the phase difference ring plate in a sliding mode. The system provided by the invention can realize continuous adjustment, and is good in relief observation effect, high in universality, simple in structure and low in cost.

Description

Technical Field

[0001] The present invention relates to the technical field of optical systems, and in particular to an oblique illumination relief microscopic imaging system based on a phase-contrast optical path. Background Art

[0002] In brightfield imaging, light from a light source is divided into direct and diffracted light after passing through the sample. Direct light is undeflected and passes directly through or around the sample. The diffracted light does not decrease in amplitude as it would with an absorbing object. Instead, its propagation speed is slowed by differences in the sample's refractive index or thickness, resulting in a phase lag of approximately 1 / 4 wavelength. When the diffracted light and the undeflected direct light reach the image plane together, they interfere with each other. However, this interference does not significantly reduce the intensity of the light, resulting in extremely low contrast in the final image, making sample details difficult to discern and virtually invisible.

[0003] Phase contrast imaging is a method that converts phase differences caused by the specimen's refractive index into variations in image intensity. By placing an annular aperture (phase ring) in front of the condenser, direct light passes through the specimen in the form of a hollow cone of light and reaches the objective's rear focal plane in the form of a phase ring. Diffracted light is then distributed across the entire rear focal plane of the objective. As light passes through the objective's rear focal plane, a phase ring plate mounted there delays the direct light by 1 / 4 wavelength, reducing the wavelength difference between the direct and diffracted light incident on the phase specimen to 1 / 2 wavelength. At this point, the direct and diffracted light reaching the image plane interfere with each other, enhancing the contrast at the interface. While phase contrast imaging can enhance image contrast, it still lacks a certain degree of three-dimensionality and detail.

[0004] Differential interference contrast (DIC) is another commonly used label-free microscopy imaging method. Light from a light source is converted to plane-polarized light by a polarizer. This light is then split into two perpendicularly polarized beams by a Wollaston prism. After passing through the specimen, the two beams are combined by another Wollaston prism. Finally, an analyzer converts the two beams into intensity information, which gives the specimen a pseudo-relief appearance. DIC can more fully utilize the numerical aperture of the system and improve image contrast. However, it is not compatible with plastic tissue culture vessels, and its optical path is more complex than other methods.

[0005] Oblique illumination technology has been used as a traditional observation technique to improve the visibility of transparent or translucent specimens, especially for imaging various unstained objects. When oblique light hits the surface of the specimen, it produces refraction or diffraction. After the light passes through the objective lens and forms an image, it produces different shadows, which makes the transparent or translucent specimens have light and dark differences, appearing in obvious relief and 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 label-free observation techniques such as differential interference, phase contrast or Hoffman modulation, the equipment cost of oblique illumination is significantly lower, and only a conventional microscope is required.

[0006] However, there are some problems with traditional oblique illumination imaging systems: 1. The area blocked by the aperture to achieve oblique light is fixed and cannot be continuously adjusted to achieve the best relief effect; 2. Conventional microscopes often need to realize both relief and phase contrast functions at the same time, but it is difficult to achieve simple switching between the two functions while maintaining a good relief effect; 3. Conventional oblique illumination imaging systems have poor relief effects and little contrast improvement without the use of additional components for modulation. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide an oblique illumination relief microscopic imaging system based on phase contrast optical path, which can be continuously adjusted and has good relief observation effect, strong versatility, simple structure and low cost.

[0008] The present invention is achieved through the following technical solutions: An oblique illumination relief microscopy imaging system based on a phase contrast optical path comprises a light source, an adjustable oblique illumination phase contrast baffle, a condenser, a sample platform, a phase contrast objective lens and an imaging device, wherein the adjustable oblique illumination phase contrast baffle is located between the light source and the condenser, the sample platform is located at the rear focal plane of the condenser, the phase contrast objective lens is located below the sample platform and the center lines of the phase contrast objective lens and the condenser coincide, the imaging device is located below the phase contrast objective lens, the adjustable oblique illumination phase contrast baffle comprises a phase difference ring plate, a phase difference ring and a light shielding 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 match the magnification of the phase difference objective lens, and the light shielding plate is slidably mounted on the phase difference ring plate.

[0009] Optimally, the imaging device includes a tube lens, a camera, and a computer connected to the camera. The tube lens is located below the phase contrast objective lens, and the camera is located below the tube lens at the focus of the tube lens.

[0010] The optimized magnification of the phase contrast objective lens is 10 times, 20 times or 40 times.

[0011] Optimized, the phase difference ring includes a first phase difference ring and a second phase difference ring. The two phase difference rings are horizontally arranged on the phase difference ring plate with a gap in between. The parameters of the first phase difference ring match the 10x phase difference objective lens, and the parameters of the second phase difference ring match the 20x phase difference objective lens or the 40x phase difference objective lens.

[0012] Optimized parameters of the first phase difference ring are: inner radius 2.75 mm, ring width 0.85 mm, and parameters of the second phase difference ring are: inner radius 4.90 mm, ring width 1.35 mm.

[0013] Optimized, the sunshade is a pull-out structure.

[0014] Optimally, the light shielding plate is located above or below the phase difference ring plate.

[0015] Furthermore, a shielding position mark for optimal imaging effect is provided on the shading plate.

[0016] Optimally, the sample substrate placed on the sample platform is a plastic substrate or a glass substrate.

[0017] Beneficial effects of the invention: The present invention provides an oblique illumination relief microscopy imaging system based on a phase contrast optical path. Compared with the prior art, the present invention constructs an oblique illumination relief microscopy imaging system based on a phase contrast optical path, so that the image contrast is higher and the relief imaging effect is better. At the same time, the light-transmitting area can be continuously adjusted by adjusting and marking the baffle on the adjustable oblique illumination phase contrast baffle to achieve the best relief observation effect. The entire system can also be switched between phase contrast effect and relief effect by pulling out and blocking the baffle. The system also has the advantages of simple structure and low cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a schematic diagram of the system structure of the present invention.

[0019] Figure 2 It is a schematic diagram of the shielding position of the shielding plate of the present invention.

[0020] Figure 3 The images of human osteosarcoma cells (U2OS) and red blood cells are taken under a 20× phase contrast objective lens of the present invention with no obstruction and a light transmission area accounting for 1 / 3.

[0021] Figure 4 These are U2OS cell images taken under a 20× objective lens of the present invention with light transmission area ratios of 1 / 3, 2 / 3, 1 / 3, and 1 / 8, respectively.

[0022] Figure 5 These are U2OS cell images taken when the light transmission area accounts for 1 / 3 under the phase contrast objective lenses of different magnifications of the present invention.

[0023] Figure 6 These are images of U2OS cells on different substrates taken under a 20× phase contrast objective lens of the present invention.

[0024] Figure 7 These are U2OS cell images taken at 20× with different objective lenses.

[0025] Figure 8 It is a side view of the system of the present invention.

[0026] Figure 9 It is the main view of the system of the present invention.

[0027] In the figure: 1. Light source; 2. Adjustable oblique illumination phase contrast shield; 3. Condenser; 4. Sample platform; 5. Phase contrast objective lens; 6. Tube lens; 7. Camera; 8. Computer; 9. Phase contrast ring plate; 10. Phase contrast ring; 11. Shield; 12. Shield position mark. DETAILED DESCRIPTION

[0028] An oblique illumination relief microscopic imaging system based on phase contrast optical path, the system structure diagram is as follows Figure 1 As shown, the system side view is as follows Figure 8 As shown, the main view of the system is as follows Figure 9 As shown, it includes a light source 1, an adjustable oblique illumination phase difference baffle 2, a condenser 3, a sample platform 4, a phase difference objective lens 5 and an imaging device. The adjustable oblique illumination phase difference baffle is located between the light source and the condenser, the sample platform is located at the rear focal plane of the condenser, the phase difference objective lens is located below the sample platform and the center lines of the phase difference objective lens and the condenser coincide with each other, the imaging device is located below the phase difference objective lens, the adjustable oblique illumination phase difference baffle includes a phase difference ring plate 9, a phase difference ring 10 and a light shielding plate 11, the phase difference ring is an annular light hole provided on the phase difference ring plate, and the parameters of the phase difference ring match the magnification of the phase difference objective lens, and the light shielding plate is slidably mounted on the phase difference ring plate.

[0029] Due to the provision of an adjustable oblique illumination phase contrast baffle and a phase contrast objective lens, the phase difference ring is an annular light hole provided on the phase difference ring plate, and the parameters of the phase difference ring match the magnification of the phase difference objective lens. In addition, the baffle is slidably mounted on the phase difference ring plate. This can make the image contrast constructed based on the phase difference optical path higher and the relief imaging effect better. At the same time, the light transmission area can be continuously adjusted by adjusting and marking the baffle, achieving the best relief observation effect. Moreover, it can be achieved with only a conventionally configured microscope, with a simple structure and low implementation cost.

[0030] Optimized, the imaging device includes a tube lens 6, a camera 7 and a computer 8 connected to the camera. The tube lens is located below the phase contrast objective lens, and the camera is located below the tube lens at the focus of the tube lens, which is convenient for collecting images imaged by the microscope system and transmitting the images to the computer display system for imaging and subsequent processing.

[0031] Optimized, the magnification of the phase difference objective is 10x, 20x or 40x, the phase difference ring includes a first phase difference ring and a second phase difference ring, the two phase difference rings are horizontally arranged on the phase difference ring plate with a gap in the middle, wherein the parameters of the first phase difference ring match the 10x phase difference objective, and the parameters of the second phase difference ring match the 20x phase difference objective or the 40x phase difference objective.

[0032] Two phase difference rings are set up to be suitable for phase difference objectives of different magnifications. When replacing phase difference objectives of different magnifications, there is no need to replace the adjustable oblique illumination phase difference baffle. A better relief imaging effect can be achieved by simply moving the position of the baffle. The system structure is relatively simple, the implementation cost is lower, and the efficiency is higher.

[0033] The optimized parameters of the first phase difference ring are: inner radius of the ring 2.75 mm, ring width 0.85 mm. This parameter matches the 10x phase difference objective lens and can achieve better relief imaging effect.

[0034] The parameters of the second phase difference ring are: inner radius 4.90 mm, ring width 1.35 mm. This parameter matches the 20x phase difference objective lens or the 40x phase difference objective lens, and can achieve better relief imaging effects.

[0035] Optimized, the shading plate is a pull-out structure, and the shading plate can be located above or below the phase difference ring plate, which makes it more convenient to slide the shading plate so as to achieve continuous adjustment of the light-transmitting area and achieve the best relief observation effect.

[0036] Furthermore, a shielding position mark 12 for optimal imaging effect is provided on the shading plate, which facilitates and quickly finds the shielding position for optimal imaging effect, so as to quickly adjust different light transmission area ratios, thereby greatly improving the imaging efficiency.

[0037] Optimally, the sample substrate placed on the sample platform is a plastic substrate or a glass substrate.

[0038] The schematic diagram of the shielding position of the shielding plate is as follows Figure 2 As shown, (a) is a schematic diagram showing a completely unobstructed situation, (b) is a schematic diagram showing a 1 / 3 obstructed situation, and (c) is a schematic diagram showing a 2 / 3 obstructed situation. In the figure, ring #2 represents the second phase difference ring, and ring #1 represents the first phase difference ring.

[0039] In this embodiment, the phase difference ring R = 4.90 mm, the ring width L = 1.35 mm, the magnification of the phase difference objective lens is 20×, and the baffle and the phase difference ring form a light-transmitting area and a light-blocking area in the optical path. The light-transmitting area can be changed by moving the position of the baffle.

[0040] The light from the light source passes through the phase difference ring on the adjustable oblique illumination phase difference baffle that is not blocked by the baffle, and then part of the hollow light column passes through the condenser to form a partial hollow light cone that illuminates the sample on the sample platform from one side. The light after passing through the sample then passes through the phase difference objective lens and tube lens to focus the image on the camera. The contrast-enhanced image with a relief effect is observed through a computer connected to the camera.

[0041] The light-passing area S1 of the phase difference ring can be adjusted according to the objective lenses of different magnifications and numerical apertures to optimize the relief effect under different objective lenses. The objective lens used in this embodiment is a 20× 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 and between.

[0042] By setting a shielding position mark for optimal imaging effect at a corresponding position on the shielding plate, the shielding position mark for optimal imaging effect is aligned with the rear end of the phase difference ring plate, thereby quickly achieving adjustment of different light transmission area ratios.

[0043] Figure 3 Images of human osteosarcoma cells U2OS and red blood cells taken under a 20× phase contrast objective lens with no obstruction and with a light transmission area of ​​1 / 3. (a) shows the normal phase contrast effect image of adherent cells under a 20× phase contrast objective lens with no obstruction, (b) shows the relief effect image of adherent cells under a 20× phase contrast objective lens with a light transmission area of ​​1 / 3, (c) shows the normal phase contrast effect image of red blood cells under a 20× phase contrast objective lens with no obstruction, and (d) shows the relief effect image of red blood cells under a 20× phase contrast objective lens with a light transmission area of ​​1 / 3.

[0044] from Figure 3 It can be seen that the image with 2 / 3 of the phase difference ring blocked has an obvious relief effect and better contrast compared with the ordinary phase difference image, which shows that the present invention can achieve a good relief imaging effect compared with the ordinary phase difference imaging.

[0045] Figure 4 Figure 3: Images of U2OS cells taken under a 20× phase contrast objective with light transmission area ratios of 1 / 3, 2 / 3, 1 / 3, and 1 / 8, respectively. (a) represents the ordinary phase contrast imaging effect of U2OS cells under a 20× phase contrast objective, (b) represents the relief imaging effect of U2OS cells under a 20× phase contrast objective with a light transmission area ratio of 2 / 3, (c) represents the relief imaging effect of U2OS cells under a 20× phase contrast objective with a light transmission area ratio of 1 / 3, and (d) represents the relief imaging effect of U2OS cells under a 20× phase contrast objective with a light transmission area ratio of 1 / 8.

[0046] from Figure 4As can be seen in the figure, when the light-transmitting area ratio is between 2 / 3 and 1 / 3, the image relief effect is good, indicating that adjusting the light-transmitting area ratio within a certain range can effectively achieve a good relief effect. When the light-transmitting area ratio is 1 / 8, the field of view brightness and uniformity are poor, indicating that excessive occlusion will lead to poor relief effect.

[0047] Figure 5 Figure 3 shows the images of U2OS cells taken under phase contrast objectives of different magnifications, including ordinary phase contrast imaging and images of U2OS cells taken when the light transmission area accounts for 1 / 3. (a) shows the effect of ordinary phase contrast imaging of U2OS cells under a 10× phase contrast objective when the U2OS cells are fully blocked, (b) shows the effect of relief imaging of U2OS cells under a 10× phase contrast objective when the light transmission area accounts for 1 / 3, (c) shows the effect of ordinary phase contrast imaging of U2OS cells under a 20× phase contrast objective when the U2OS cells are fully blocked, (d) shows the effect of relief imaging of U2OS cells under a 20× phase contrast objective when the light transmission area accounts for 1 / 3, (e) shows the effect of ordinary phase contrast imaging of U2OS cells under a 40× phase contrast objective when the U2OS cells are fully blocked, and (f) shows the effect of relief imaging of U2OS cells under a 40× phase contrast objective when the light transmission area accounts for 1 / 3.

[0048] from Figure 5 It can be seen that this system can produce good relief imaging effects for phase contrast objectives of different magnifications.

[0049] Figure 6 Images of U2OS cells on different substrates taken under a 20× phase contrast objective lens, where (a) represents the ordinary phase contrast imaging effect of U2OS cells on a glass substrate taken under a 20× phase contrast objective lens, (b) represents the relief imaging effect of U2OS cells on a glass substrate taken under a 20× phase contrast objective lens when the light transmission area accounts for 2 / 3, (c) represents the ordinary phase contrast imaging effect of U2OS cells on a plastic substrate taken under a 20× phase contrast objective lens, and (d) represents the relief imaging effect of U2OS cells on a plastic substrate taken under a 20× phase contrast objective lens when the light transmission area accounts for 2 / 3.

[0050] from Figure 6 It can be seen that this system can produce good relief imaging effects for samples on different substrates.

[0051] Figure 7 These are images of U2OS cells taken under different 20× objective lenses, among which (a) represents the ordinary phase contrast imaging effect of U2OS cells under a 20× phase contrast objective lens, (b) represents the relief imaging effect of U2OS cells under a 20× phase contrast objective lens when the light transmission area accounts for 2 / 3, (c) represents the bright field imaging effect of U2OS cells under a 20× ordinary objective lens, and (d) represents the bright field imaging effect of U2OS cells under a 20× ordinary objective lens when the light transmission area accounts for 2 / 3.

[0052] from Figure 7 It can be seen that the microscope system can only achieve a better relief effect when using phase contrast objectives.

[0053] The 1 / 3 oblique lighting marked in the above figure means that the light-transmitting area accounts for 1 / 3.

[0054] In summary, the present invention provides an oblique illumination relief microscopy imaging system based on phase difference light path, which has high image contrast and good relief imaging effect. At the same time, by adjusting and marking the baffle on the adjustable oblique illumination phase difference baffle, continuous adjustment of the light-transmitting area is achieved to achieve the best relief observation effect. The entire system can also be switched between phase difference effect and relief effect by pulling out and blocking the baffle. The system structure is simple and the implementation cost is low.

[0055] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. An oblique illumination relief microscopic imaging system based on phase contrast optical path, characterized by: The invention comprises a light source, an adjustable oblique illumination phase difference baffle, a condenser, a sample platform, a phase difference objective lens and an imaging device. The adjustable oblique illumination phase difference baffle is located between the light source and the condenser, the sample platform is located at the rear focal plane of the condenser, the phase difference objective lens is located below the sample platform, and the center lines of the phase difference objective lens and the condenser coincide with each other. The imaging device is located below the phase difference objective lens. The adjustable oblique illumination phase difference baffle comprises a phase difference ring plate, a phase difference ring and a light shielding 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 match the magnification of the phase difference objective lens. The light shielding plate is slidably mounted on the phase difference ring plate.

2. The oblique illumination relief microscopy imaging system based on phase contrast optical path according to claim 1, characterized in that: The imaging device comprises a tube lens, a camera and a computer connected to the camera. The tube lens is located below the phase contrast objective lens, and the camera is located below the tube lens at the focus of the tube lens.

3. The oblique illumination relief microscopy imaging system based on phase contrast optical path according to claim 1, characterized in that: The magnification of the phase contrast objective lens is 10 times, 20 times or 40 times.

4. The oblique illumination relief microscopic imaging system based on phase contrast optical path according to claim 3, characterized in that: The phase difference ring includes a first phase difference ring and a second phase difference ring. The two phase difference rings are horizontally arranged on the phase difference ring plate with a gap in between. The parameters of the first phase difference ring match the 10x phase difference objective lens, and the parameters of the second phase difference ring match the 20x phase difference objective lens or the 40x phase difference objective lens.

5. The oblique illumination relief microscopic imaging system based on phase contrast 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 contrast optical path according to claim 1, characterized in that: The shading plate is a pull-out structure.

7. The oblique illumination relief microscopy imaging system based on phase contrast 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 microscopy imaging system based on phase contrast optical path according to claim 1, characterized in that: The shading plate is provided with a shading position mark for optimal imaging effect.

9. The oblique illumination relief microscopic imaging system based on phase contrast optical path according to claim 1, characterized in that: The sample substrate placed on the sample platform is a plastic substrate or a glass substrate.

Images