Deep ultraviolet fluorescence microscope device capable of obtaining contrast image and distinguishing crystal properties at same time

Abstract

The invention discloses a deep ultraviolet fluorescence microscope device capable of obtaining a contrast image and distinguishing crystal properties at the same time. Wherein the deep ultraviolet light source directly faces the light uniform adjusting lens group mechanism, the optical filter and the dichroic lens, light emitted into the dichroic lens from the optical filter is emitted to the lower path, the crystal plate is sequentially arranged below the dichroic lens, the condenser and the white light LED light source are sequentially arranged below the crystal plate, and a sample is placed in the crystal plate; light emitted by the white light LED light source penetrates through the dichroic lens, a monochromatic light filter, a lens group and a CMOS system without a glass protection lens are sequentially arranged above the dichroic lens, and a high-resolution black and white CMOS detector is arranged in the CMOS system without the glass protection lens. According to the general characteristics of the protein, the deep purple external excitation crystal is used, and the protein crystal can emit fluorescence, so that the vacancy of an upright fluorescence microscope system in the prior art is made up.

Description

Technical Field

[0001] The present invention belongs to the technical field of materials testing using optical means for ultraviolet illumination and microscopes, and specifically relates to a deep ultraviolet fluorescence microscope device that can distinguish crystal properties while obtaining contrast images. Background Art

[0002] In the prior art, X-ray diffraction analysis is used to distinguish salt crystals and protein crystals. The technical source is mainly based on the basic principle of X-ray diffraction and the differences in crystal structures. X-ray diffraction technology uses the diffraction phenomenon of X-rays in crystals to analyze the crystal structure, and its basis is Bragg's equation: 2dhklsinθ = nλ, where dhkl is the interplanar spacing, θ is the incident angle, and λ is the X-ray wavelength. Then the Laue equation laid the foundation for X-ray diffraction technology.

[0003] This technology utilizes the structural differences between salt crystals and protein crystals, where:

[0004] Salt crystals: The structure of salt crystals is relatively simple, usually composed of ions or small molecules, with a small unit cell and a highly ordered arrangement. Therefore, the diffraction pattern of salt crystals usually has strong diffraction peaks with large peak spacings.

[0005] Protein crystals: The structure of protein crystals is complex, composed of macromolecules, with a large unit cell and a relatively disordered arrangement. Their diffraction patterns are usually weak, and obvious diffraction peaks only appear at high resolutions.

[0006] The operating steps of the prior art are to take out the crystals from the protein crystallization well plate, freeze them, and then use an X-ray diffraction instrument to obtain the diffraction pattern. The properties are distinguished based on the structural differences between salt crystals and protein crystals described above.

[0007] However, the existing X-ray diffraction method for distinguishing crystal properties is very cumbersome, time-consuming, and laborious. The cost of using an X-ray diffraction instrument is also very high. If it is determined that it is not a protein crystal, all previous efforts will be in vain. Summary of the Invention

[0008] In view of the problems existing in the background art, the present invention provides a deep ultraviolet fluorescence microscope device that can distinguish the crystal properties while obtaining a comparison image. By optimizing the light source, optical system, and detector, the imaging performance in the deep ultraviolet band is significantly improved, and the deficiencies in the prior art are solved. The technical solution includes: a deep ultraviolet light source, a light ray uniform adjustment lens group mechanism, a filter, a dichroic lens, a deep ultraviolet compatible objective lens, a crystal plate, a monochromatic filter, a lens group, a high-resolution black and white CMOS detector, a CMOS system without a glass protection lens, a white light LED light source, and a condenser. Among them, the deep ultraviolet light source is directly opposite to the light ray uniform adjustment lens group mechanism, the filter, and the dichroic lens. The light entering the dichroic lens from the filter shoots downward. Below the dichroic lens, there are successively arranged a crystal plate, and below the crystal plate, there are successively arranged a condenser and a white light LED light source. A sample is placed inside the crystal plate. The light emitted by the white light LED light source passes through the dichroic lens. Above the dichroic lens, there are successively arranged a monochromatic filter, a lens group, and a CMOS system without a glass protection lens. A high-resolution black and white CMOS detector is arranged in the CMOS system without a glass protection lens.

[0009] Both the deep ultraviolet compatible objective lens and the dichroic lens are made of quartz.

[0010] The wavelength emitted by the deep ultraviolet light source is in the range of 200 - 355 nanometers.

[0011] The wavelength emitted by the deep ultraviolet light source is in the range of 200 - 300 nanometers.

[0012] The deep ultraviolet light emitted by the deep ultraviolet light source passes through the light ray uniform adjustment lens group mechanism and the filter to obtain pure single-wavelength deep ultraviolet light, and then is reflected by the dichroic lens to the deep ultraviolet compatible objective lens and irradiated onto the sample on the crystal plate. The light ray uniform adjustment lens group mechanism is used to optimize the deep ultraviolet light field distribution.

[0013] Subsequently, the light emitted by the sample on the crystal plate passes through the deep ultraviolet compatible objective lens, passes through the dichroic lens and the automatically switchable monochromatic filter, and then passes through the lens group and finally obtains the ultraviolet fluorescence image of the crystal on the high-resolution black and white CMOS detector.

[0014] The white light emitted by the white light LED light source passes through the condenser, passes through the crystal plate and is focused on the sample above, and then the light passes through the deep ultraviolet compatible objective lens and passes through the dichroic lens, thereby realizing high-definition imaging of visible light.

[0015] The monochromatic filter is fixed to the moving device. When performing visible light imaging, that is, when the white light LED light source emits light, the monochromatic filter is moved out from the front of the deep ultraviolet lens group by the moving device.

[0016] The beneficial effects of the present invention are as follows: According to the general properties of proteins, deep ultraviolet light is used to excite the crystal, and the protein crystal will emit fluorescence. Based on this principle, a fluorescence microscope is made. The imaging ability of traditional fluorescence microscopes is limited in the ultraviolet spectral region, especially in the deep ultraviolet band (such as 200 - 300 nm). Due to problems such as insufficient light source intensity, poor light transmission of the optical system, and low fluorescence labeling efficiency, it is difficult to achieve high-resolution and high-sensitivity imaging. The present invention fills the gap in the prior art of lacking an upright fluorescence microscope system specifically optimized for the deep ultraviolet band. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 FIG. is a schematic structural diagram of an embodiment of a deep ultraviolet fluorescence microscope device of the present invention for distinguishing crystal properties while obtaining a comparison image;

[0018] Figure 2 FIG. is an interface diagram for determining that this crystal is a protein crystal in an embodiment of the present invention;

[0019] Figure 3 FIG. is an interface diagram for determining that the substance with this particle shape is not a protein crystal in an embodiment of the present invention.

[0020] Among them, 1 - deep ultraviolet light source, 2 - light ray uniform adjustment lens group mechanism, 3 - filter, 4 - dichroic lens, 5 - deep ultraviolet compatible objective lens, 6 - crystal plate, 7 - monochromatic filter, 8 - lens group, 9 - high-resolution black and white CMOS detector, 10 - CMOS system without glass protection lens, 11 - white light LED light source, 12 - condenser. SPECIFIC EMBODIMENTS

[0021] The present invention will be further described in detail below with reference to the accompanying drawings.

[0022] As Figure 1The embodiment of the present invention shown includes: a deep ultraviolet light source 1, a light ray uniform adjustment lens group mechanism 2, a filter 3, a dichroic lens 4, a deep ultraviolet compatible objective lens 5, a crystal plate 6, a monochromatic filter 7, a lens group 8, a high-resolution black and white CMOS detector 9, a CMOS system 10 without a glass protection lens, and a white light LED light source 11 and a condenser 12. Among them, the deep ultraviolet light source 1 faces the light ray uniform adjustment lens group mechanism 2, the filter 3, and the dichroic lens 4. The light entering the dichroic lens 4 from the filter 3 shoots downward. Below the dichroic lens 4, there are successively arranged 5 and the crystal plate 6. Below the crystal plate 6, there are successively arranged the condenser 12 and the white light LED light source 11. A sample is placed inside the crystal plate 6. The light emitted by the white light LED light source 11 passes through the dichroic lens 4. Above the dichroic lens 4, there are successively arranged the monochromatic filter 7, the lens group 8, and the CMOS system 10 without a glass protection lens. A high-resolution black and white CMOS detector 9 is arranged in the CMOS system 10 without a glass protection lens. The deep ultraviolet compatible objective lens 5 is directly above the sample (stage), and the transmission light source (white light LED light source 11) is directly below the sample (stage).

[0023] The deep ultraviolet light source mechanism composed of the deep ultraviolet light source 1 and the light ray uniform adjustment lens group mechanism 2 can emit deep ultraviolet light rays in a specific direction. The light ray uniform adjustment lens group mechanism 2 is used to optimize the deep ultraviolet light field distribution. When working, the deep ultraviolet light rays emitted by the deep ultraviolet light source 1 pass through the filter 3 to obtain pure single-wavelength deep ultraviolet light, and then are reflected by the dichroic lens 4 to the deep ultraviolet compatible objective lens 5 and irradiated onto the sample on the crystal plate 6. Then, the light emitted by the sample on the crystal plate 6 passes through the deep ultraviolet compatible objective lens 5, passes through the dichroic lens 4 and the automatically switchable monochromatic filter 7, and then passes through the lens group 8 to obtain the ultraviolet fluorescence image of the crystal on the high-resolution black and white CMOS detector 9.

[0024] The white light source mechanism composed of the white light LED light source 11 and the condenser 12 can emit focused white light in a specific direction. When working, the white light emitted by the white light LED light source 11 passes through the condenser 12, passes through the crystal plate 6 and is focused on the sample above, and then the light passes through the deep ultraviolet compatible objective lens 5 and passes through the dichroic lens 4, thereby realizing high-definition imaging of visible light.

[0025] In this embodiment, the monochromatic filter 7 is fixed to the mobile device. When visible light imaging is performed, i.e., when the white light LED light source 11 emits light, the monochromatic filter 7 will be moved out from in front of the deep ultraviolet lens group 8 by the mobile device, allowing visible light of all wavelengths to be received by the high-resolution black-and-white CMOS detector 9 to obtain a visible light image; thus achieving high-definition imaging of visible light. Specifically, when the fluorescent substance on the sample on the crystal plate 6 is excited, it will emit light with a longer wavelength (fluorescence); these fluorescent signals are collected by the deep ultraviolet compatible objective lens 5 and filtered and guided through the dichroic lens (dichroic beam splitter) and the monochromatic filter 7; the function of the monochromatic filter 7 is to remove unwanted excitation light and other stray light, allowing only fluorescent light of a specific wavelength to pass through and finally be received by the detector; the fluorescent signals after being processed by the filter finally reach the eyepiece and the high-resolution black-and-white CMOS detector 9, are amplified and form a clear image.

[0026] In this embodiment, an infinity-corrected optical system, an autofocus system, and an automatic switching between visible light imaging and ultraviolet imaging modes are adopted to ensure independent dual focus correction of the deep ultraviolet compatible objective lens 5 and the high-resolution black-and-white CMOS detector 9 during visible light and ultraviolet imaging, improving the clarity and contrast of the imaging. Specifically, a deep ultraviolet light source with a wavelength between 200 - 355 nm is used to excite the fluorescent substance in the sample, and a wavelength between 200 - 300 nm is preferably selected; such a light source can provide high-energy photons to cause the electrons of the fluorescent substance to transition to higher energy levels. The excitation light is usually selected through the filter 3 to ensure that only light of a specific wavelength can irradiate the sample; at the same time, the light emitted by the light source is irradiated onto the sample through the deep ultraviolet compatible objective lens 5 to excite the fluorescent substance to emit fluorescence. This design enables the objective lens to not only be used for imaging but also act as a condenser.

[0027] In this embodiment, the deep ultraviolet light source 1 adopts a high-power deep ultraviolet light source, such as a deep ultraviolet laser or a high-intensity deep ultraviolet LED, to ensure sufficient excitation light intensity to increase the intensity of the fluorescent signal.

[0028] In this embodiment, a dedicated deep ultraviolet optical system is adopted, including: a monochromatic filter 7 for deep ultraviolet fluorescence, a high-transmittance deep ultraviolet lens group 8 with a transmittance exceeding 98%, and a deep ultraviolet compatible objective lens 5 to reduce light loss and improve imaging quality; specifically, the deep ultraviolet lens group 8, the deep ultraviolet compatible objective lens 5, and the dichroic lens 4 are all made of quartz.

[0029] In this embodiment, a high-sensitivity deep ultraviolet detector, such as a high-resolution black-and-white CMOS detector 9, is used, and the glass protection lens of the CMOS camera is removed, which can effectively detect fluorescent signals in the deep ultraviolet band.

[0030] During operation, the present invention automatically takes pictures of the same sample with a white light LED light source 11 and a deep ultraviolet light source 1 successively. By comparing the obtained visible light image and ultraviolet fluorescence image, if a crystal can be seen in the visible light image but not in the ultraviolet fluorescence image, it can be intuitively judged that this crystal is a salt crystal; if a crystal can be seen in the visible light image and the same crystal can be seen at the same position in the ultraviolet fluorescence image, it can be intuitively judged that this crystal is a protein crystal.

[0031] As Figure 2 shown, Figure 2 the upper left figure in Figure 2 is the visible light image, Figure 3 shown, Figure 3 the upper right figure in Figure 3 is the ultraviolet fluorescence image. The crystal at the center of the visible light image is a bright crystal in the ultraviolet fluorescence image, and it can be judged that this crystal is a protein crystal; As

[0032] Although the present invention has been described herein with reference to specific embodiments, it should be understood that these embodiments are merely examples of the principles and applications of the present invention. Therefore, it should be understood that many modifications can be made to the exemplary embodiments, and other arrangements can be designed, as long as they do not deviate from the spirit and scope of the present invention as defined by the appended claims. It should be understood that different dependent claims and the features described herein can be combined in a manner different from that described in the original claims. It should also be understood that the features described in connection with a single embodiment can be used in other described embodiments.

Claims

1. A deep ultraviolet fluorescence microscope device for differentiating crystal properties while acquiring a comparison image, characterized in that, Comprising: Deep ultraviolet light source (1), light ray uniform adjustment lens group mechanism (2), filter (3), dichroic lens (4), deep ultraviolet compatible objective lens (5), crystal plate (6), monochromatic filter (7), lens group (8), high-resolution black and white CMOS detector (9), CMOS system without glass protection lens (10), white light LED light source (11) and condenser (12). Among them, the deep ultraviolet light source (1) faces the light ray uniform adjustment lens group mechanism (2), filter (3) and dichroic lens (4). The light entering the dichroic lens (4) from the filter (3) shoots downward. Below the dichroic lens (4), (5) and crystal plate (6) are sequentially arranged. Below the crystal plate (6), the condenser (12) and white light LED light source (11) are sequentially arranged. A sample is placed inside the crystal plate (6). The light emitted by the white light LED light source (11) passes through the dichroic lens (4). Above the dichroic lens (4), the monochromatic filter (7), lens group (8) and CMOS system without glass protection lens (10) are sequentially arranged. A high-resolution black and white CMOS detector (9) is arranged in the CMOS system without glass protection lens (10).

2. The deep ultraviolet fluorescence microscope device for differentiating crystal properties while acquiring comparison images according to claim 1, wherein The deep ultraviolet lens group (8), deep ultraviolet compatible objective lens (5) and dichroic lens (4) are all made of quartz material.

3. A deep ultraviolet fluorescence microscope device for differentiating crystal properties while acquiring a comparison image according to claim 1 or 2, characterized in that, The wavelength emitted by the deep ultraviolet light source (1) is in the range of 200 - 355 nanometers.

4. A deep ultraviolet fluorescence microscope device for differentiating crystal properties while acquiring a comparison image according to claim 3, characterized in that, The wavelength emitted by the deep ultraviolet light source (1) is in the range of 200 - 300 nanometers.

5. A deep ultraviolet fluorescence microscope device for differentiating crystal properties while acquiring a comparison image according to claim 4, characterized in that, The deep ultraviolet light rays emitted by the deep ultraviolet light source (1) pass through the light ray uniform adjustment lens group mechanism (2) and filter (3) to obtain pure single-wavelength deep ultraviolet light, and then are reflected by the dichroic lens (4) to the deep ultraviolet compatible objective lens (5) to irradiate the sample on the crystal plate (6). Subsequently, the light emitted by the sample on the crystal plate (6) passes through the deep ultraviolet compatible objective lens (5), passes through the dichroic lens (4) and the automatically switchable monochromatic filter (7), and then passes through the lens group (8) to obtain the ultraviolet fluorescence image of the crystal on the high-resolution black and white CMOS detector (9).

6. The deep ultraviolet fluorescence microscope device for differentiating crystal properties while acquiring a comparison image according to claim 1, wherein The white light emitted by the white light LED light source (11) passes through the condenser (12), passes through the crystal plate (6) and is focused on the sample above, and then the light passes through the deep ultraviolet compatible objective lens (5) and passes through the dichroic lens (4), thereby realizing high-definition imaging of visible light.

7. A deep ultraviolet fluorescence microscope device for differentiating crystal properties while acquiring a comparison image according to claim 6, characterized in that, The monochromatic filter (7) is fixed to the moving device. When visible light imaging is performed, that is, when the white light LED light source (11) emits light, the monochromatic filter (7) is moved out from in front of the deep ultraviolet lens group (8) by the moving device.

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