A highly integrated multi-modal microscopic imaging device and method

Abstract

The present invention discloses a highly integrated multimodal microscopic imaging device and method. The device comprises a light source module and an MIID imaging module. The light source module comprises a first light source and a second light source respectively disposed at two ends of a glass slide, wherein the first light source is used for dark field illumination, and the second light source is used for fluorescent illumination. The light source module further comprises a third light source disposed below the glass slide for bright field illumination. A composite medium filter film for compressing the illumination light bandwidth is disposed above the glass slide. The present invention places a light source at the end of the glass slide so that the glass slide acts as a planar waveguide, and does not increase additional light source space, thereby realizing dark field imaging in a meta-lens microscopic imaging system. Bright field, dark field and fluorescent imaging can be quickly realized by switching the power supply of the first light source, the second light source and the third light source, without replacing a microscope or adding or removing other components, and more complete sample information can be observed on a microscopic imaging device.

Description

Technical Field

[0001] The present invention relates to a microscopic imaging device and method, in particular to a highly integrated multi-modal microscopic imaging device and method. Background Art

[0002] Microscopic imaging technology plays an irreplaceable and important role in biological and medical research. At present, a series of microscopic imaging technologies such as bright field, dark field, and fluorescence have been developed. In order to observe different types of samples, different imaging methods are often required. Bright field imaging usually shows sample patterns on a bright background, which can obtain low-frequency information of the sample and is suitable for the observation of strongly absorbing samples. Dark field imaging can observe bright samples against a dark background, and can obtain high-frequency information of the sample, which is suitable for weakly absorbing and clearly outlined samples. Fluorescence imaging has the advantages of high sensitivity, low cost, and selectivity, and is widely used in medical imaging and other fields. By irradiating the sample with excitation light of a specific wavelength to make it emit fluorescence, the internal structure of the sample can be observed. By combining bright field, dark field, and fluorescence imaging, more complete sample information can be observed. However, traditional optical microscopes are large in size. On the one hand, traditional optical microscopes require high numerical aperture objectives to improve resolution. However, high numerical aperture and low aberration lenses not only increase manufacturing costs and assembly complexity, but also increase lens length; on the other hand, a long distance needs to be maintained between the objective lens and the image sensor so that the resolution of the microscope system is not limited by the image sensor. Moreover, the three imaging modes have different optical path structures. Replacing different microscopes not only increases the cost of the microscope, but also makes the operation of the microscope more complicated and increases the difficulty of use. Summary of the invention

[0003] Purpose of the invention: The purpose of the present invention is to provide a highly integrated multimodal microscopic imaging device that can simultaneously realize bright field, dark field and fluorescence imaging. The second purpose of the present invention is to provide a microscopic imaging method that can realize bright field, dark field and fluorescence imaging.

[0004] Technical solution: The highly integrated multimodal microscopic imaging device described in the present invention includes a light source module and a MIID imaging module. The light source module includes a first light source and a second light source respectively placed at both ends of a glass slide, the first light source is used for dark field illumination, and the second light source is used for fluorescent illumination. The light source module also includes a third light source placed under the glass slide for bright field illumination; a composite medium filter film for compressing the light bandwidth is provided above the glass slide.

[0005] Furthermore, it also includes a bottom plate and a power supply device on the bottom plate, wherein the power supply device is connected to the light source module to supply power to the first light source, the second light source and the third light source respectively.

[0006] Furthermore, the glass slide is placed between a loading plate and a clamping plate, four side plates are fixed around the loading plate, and screws pass through the side plates and the clamping plates to adjust the clamping plates to press or loosen the glass slide.

[0007] Furthermore, the sample rack is connected to the guide rail via a slider and slides vertically along the guide rail for focusing; the sample rack is used to place the object plate.

[0008] Furthermore, a groove is provided on the bottom surface of the clamping plate to reserve space for placing a linear polarization film and a quarter wave plate film, the quarter wave plate film includes a left quarter wave plate film and a right quarter wave plate film, the fast axis of the left quarter wave plate film is +45° to the linear polarization film, and the fast axis of the right quarter wave plate film is -45° to the linear polarization film, which are respectively used to modulate the left-handed circular polarization state and the right-handed circular polarization state.

[0009] Furthermore, the MIID imaging module includes a CIS chip and a meta-lens.

[0010] The highly integrated multimodal microscopic imaging method of the present invention performs dark field imaging, fluorescence imaging and bright field imaging respectively;

[0011] Dark field imaging: The first incident light emitted by the first light source from one end of the glass slide enters the glass slide and is totally reflected on the upper and lower inner surfaces of the glass slide, and then is scattered by the sample. The upward scattered light is modulated into the first narrow-band light by the composite medium filter film, and the first narrow-band light is received by the MIID imaging module for dark field imaging;

[0012] Fluorescence imaging: The second incident light emitted by the second light source from the other end of the glass slide enters the glass slide and is totally reflected on the upper and lower inner surfaces of the glass slide, and then is excited by the sample to produce fluorescence with a wavelength different from the second incident light. The second incident light is cut off by the composite medium filter film, and the fluorescence is modulated into a second narrow-band light through the composite medium filter film. The second narrow-band light is received by the MIID imaging module for fluorescence imaging;

[0013] Bright field imaging: The third incident light emitted by the third light source from below the glass slide enters the glass slide and is then modulated into a third narrow-band light by the composite medium filter film. The third narrow-band light is received by the MIID imaging module for bright field imaging.

[0014] Furthermore, a power supply device is connected to the first light source, the second light source and the third light source to supply power to the first light source, the second light source and the third light source respectively.

[0015] Furthermore, the MIID imaging module includes a CIS chip and a meta-lens. When the meta-lens is a PB phase meta-lens, the first incident light, the second incident light and the third incident light, after passing through the composite medium filter film, also pass through a linear polarization film and a quarter-wave plate film. The quarter-wave plate film includes a left quarter-wave plate film and a right quarter-wave plate film. The fast axis of the left quarter-wave plate film is +45° to the linear polarization film, and the fast axis of the right quarter-wave plate film is -45° to the linear polarization film, which are respectively used to modulate the left circular polarization state and the right circular polarization state.

[0016] The computer-readable storage medium of the present invention stores a computer program, and when the computer program is executed by a processor, the highly integrated multimodal microscopic imaging method is implemented.

[0017] Beneficial effects: Compared with the prior art, the advantages of the present invention are: (1) by placing a composite dielectric filter film above the glass slide, the dark field and fluorescent light sources are integrated into the two ends of the glass slide, so that the glass slide acts as a planar waveguide, without adding additional light source space, and dark field imaging in the meta-lens microscopic imaging system is realized; (2) the composite dielectric filter film narrows the bandwidth of the wider illumination light, thereby reducing the chromatic aberration caused by meta-lens imaging; (3) when the sample is fluorescently stained, the composite dielectric filter film of the present invention also plays a role in isolating the illumination light scattered by the sample, and only allows the self-emitted fluorescence of the sample excited by the light source to pass, thereby realizing dark field fluorescence imaging; (4) three light sources are integrated in the microscopic imaging device, and three imaging modes can be realized by switching the power of the light source, thereby obtaining more complete information about the sample, and compared with traditional microscopes, it is more portable and simpler to operate, which can reduce the cost of the microscope. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a structural diagram of the highly integrated multimodal microscopic imaging device of the present invention.

[0019] Figure 2 This is a structural diagram of dark field illumination and fluorescent illumination of the present invention.

[0020] Figure 3 This is a structural diagram of the MIID imaging module of the present invention.

[0021] Figure 4 This is a structural diagram of the displacement module and bright field illumination of the present invention.

[0022] Figure 5 This is the dark field HeLa cell imaging effect of an embodiment of the present invention.

[0023] Figure 6 This is the fluorescent HeLa cell imaging effect of an embodiment of the present invention.

[0024] Figure 7 This is the bright field HeLa cell imaging effect of an embodiment of the present invention. DETAILED DESCRIPTION

[0025] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0026] like Figures 1 to 4 As shown, the highly integrated multi-modal microscopic imaging device of the present invention includes a light source module, a MIID imaging module and a displacement module.

[0027] The MIID imaging module is composed of a CIS chip 14 and a meta-lens 21. In this embodiment, the design wavelength of the meta-lens 21 is 470nm. In actual use, meta-lenses with other design wavelengths can be selected, and the choice of the light source must match the design wavelength. In this embodiment, the size of the CIS chip 14 is 30mm×30mm, and it is fixed to four screw columns 20 by four M2 countersunk screws through the through holes on the four corners, ensuring the relative parallelism of the meta-lens 21 and the sample 3. The reason for using countersunk screws is to prevent the height difference between the screw and the surface of the meta-lens substrate from being greater than the imaging object distance, thereby limiting the focus of the MIID. An opening 19 is reserved on the top plate 15 for the USB data cable access of the CIS chip 14. The MIID focusing screw 18 can be inserted into the second threaded hole 27 behind the sample holder 25. The MIID focusing screw 18 is fixed to the knob 16 through the bearing 17. The relative position of the MIID focusing screw 18 and the entire device is fixed. By turning the knob 16, the sample holder 25 can be moved up and down to achieve the purpose of focusing.

[0028] The light source module includes a first light source 101, a second light source 102 and a third light source 103. In this embodiment, the first light source 101 and the second light source 102 are light bars formed by connecting a plurality of LEDs in parallel. The size of a single LED lamp bead is 3.2 mm×2.8 mm×0.7 mm. The first light source 101 and the second light source 102 are leaning against the cylindrical sponge 5 and are respectively fixed to the grooves at both ends of the sample carrier plate 6. The elasticity of the cylindrical sponge 5 is used as a buffer and a fastener for the first light source 101 and the second light source 102. The third light source 103 is an LED lamp bead fixed to the T-shaped plate 24 below the sample holder 25.

[0029] The first light source 101 is used for dark field imaging. A 3.5V DC voltage is applied to the two electrodes to emit natural light with a central wavelength of 470nm and a bandwidth of about 20nm. The second light source 102 is used for fluorescence imaging and can emit natural light with a central wavelength of 365nm, which can excite the sample to emit 470nm fluorescence. The slide 2 is inserted between the carrier plate 6 and the clamping plate 7, and the sample 3 is placed on the slide 2. The two end faces of the slide 2 are in close contact with the first light source 101 and the second light source 102 respectively. In dark field imaging and fluorescence imaging, the slide 2 acts as a flat waveguide, which can evenly guide the incident light emitted by the first light source 101 or the second light source 102 to the sample 3. Part of the illumination light is scattered by the sample, and the propagation direction of the light changes. The light containing the sample information passes upward through the composite medium filter film 4, and then is received by the MIID imaging module above. In this embodiment, the composite medium filter film 4 is a thin sheet with a side length of 1cm, which only allows narrow-band light with a central wavelength of 470nm to pass through and is aligned with the meta-lens 21 of the MIID imaging module. In dark field imaging, the function of the composite medium filter film 4 is to narrow the bandwidth of the relatively wide-band illumination light, thereby reducing the chromatic aberration caused by the meta-lens imaging and improving the imaging contrast; in fluorescence imaging, in addition to reducing the chromatic aberration as mentioned above, it also serves to isolate the illumination light scattered by the above-mentioned sample, and only allows the self-emitted fluorescence of the sample excited by the light source to pass through.

[0030] The four side panels 8 are fixed to the loading plate 6 by screws. The screws can pass through the third circular hole 9 on the clamping plate 7 through the first threaded hole 13 above the side panel 8 and be embedded in the groove 11 on the bottom surface of the clamping plate 7. The clamping plate 7 can clamp or loosen the slide 2 by rotating the screws.

[0031] For the meta-lens of PB phase (Pancharatnam–Berry phase), since it only has a modulating effect on circularly polarized light, the groove 11 reserved on the bottom surface of the clamp 7 can be used to place a linear polarization film and a quarter-wave plate film. The quarter-wave plate film includes a left quarter-wave plate film and a right quarter-wave plate film. The fast axis of the left quarter-wave plate film is +45° to the linear polarization film, and the fast axis of the right quarter-wave plate film is -45° to the linear polarization film, which are respectively used to modulate the left-handed circular polarization state and the right-handed circular polarization state.

[0032] The third light source 103 can emit natural light with a central wavelength of 470nm, which illuminates the sample 3 through the first circular hole 26 on the sample holder 25 and the second circular hole 10 on the sample plate 6 in turn, and then passes through the composite medium filter film 4, and is finally received by the MIID imaging module for bright field imaging.

[0033] A circular groove 22 is provided on the bottom plate 31, and a power supply device 23 is embedded in the circular groove 22 for supplying power to the light source module. In this embodiment, the power supply device 23 is a button battery with a diameter of 24 mm, a thickness of 5 mm, and a standard voltage of 3 V. The button battery is connected to the first light source 101, the second light source 102, and the third light source 103 through a single-pole triple-throw switch, and the switch is switched to realize microscopic imaging of three modes. A lead port 12 for connecting the light source module to the power supply device 23 is provided on the object carrier 6.

[0034] The displacement module is used to move the sample 3 to achieve focusing, insert the slide 2 into the carrier plate 6, and place the carrier plate 6 on the sample holder 25. The sample holder 25 is fixed to two sliders 29 by screws, and the sliders can move up and down on the guide rails 28, and the two guide rails are fixed to the rear plate 30 by screws. There is a second threaded hole 27 at the rear of the sample holder, and the sample holder 25 can be moved up and down by turning the screws to achieve the purpose of focusing.

[0035] like Figure 5 FIG. 4 is a diagram showing the effect of dark field imaging of HeLa cells using the device of the present invention; Figure 6 FIG. 4 is a diagram showing the effect of fluorescence imaging of HeLa cells using the device of the present invention; Figure 7 Shown is a diagram showing the effect of bright field imaging of HeLa cells using the device of the present invention.

[0036] The method of the present invention performs dark field imaging, fluorescence imaging and bright field imaging respectively.

[0037] Dark field imaging: The first incident light emitted by the first light source 101 from one end of the glass slide 2 enters the glass slide 2 and is totally reflected on the upper and lower inner surfaces of the glass slide 2, and then is scattered by the sample. The upward scattered light is modulated into a first narrow-band light by the composite medium filter film 4, and the first narrow-band light is received by the MIID imaging module for dark field imaging.

[0038] Fluorescence imaging: The second incident light emitted by the second light source 102 from the other end of the glass slide 2 enters the glass slide 2 and is totally reflected on the upper and lower inner surfaces of the glass slide 2, and then is excited by the sample to emit fluorescence having a wavelength different from that of the second incident light. The second incident light is cut off by the composite medium filter film 4, and the fluorescence is modulated into a second narrow-band light through the composite medium filter film 4. The second narrow-band light is received by the MIID imaging module for fluorescence imaging.

[0039] Bright field imaging: The third incident light emitted by the third light source 103 from below the glass slide 2 enters the glass slide 2 and then passes through the composite medium filter film 4 to be modulated into a third narrow-band light, which is received by the MIID imaging module for bright field imaging.

[0040] The first light source 101, the second light source 102 and the third light source 103 are connected to the power supply device 23 via a single-pole triple-throw switch, and the switches are switched to realize microscopic imaging of three modes.

[0041] When the meta-lens in the MIID is a PB phase meta-lens, the first incident light, the second incident light, and the third incident light, after passing through the composite medium filter film, also pass through a linear polarization film and a quarter wave plate film, wherein the quarter wave plate film includes a left quarter wave plate film and a right quarter wave plate film, wherein the fast axis of the left quarter wave plate film is +45° to the linear polarization film, and the fast axis of the right quarter wave plate film is -45° to the linear polarization film, and are respectively used to modulate the left circular polarization state and the right circular polarization state.

Claims

1. A highly integrated multimodal microscopic imaging method, It is characterized in that A highly integrated multimodal microscopic imaging device is used to perform dark field imaging, fluorescence imaging and bright field imaging respectively; the highly integrated multimodal microscopic imaging device comprises a light source module and a MIID imaging module, the light source module comprises a first light source (101) and a second light source (102) respectively disposed at two ends of a glass slide (2), the first light source (101) being used for dark field illumination, the second light source (102) being used for fluorescence illumination, the light source module further comprising a third light source (103) disposed below the glass slide (2) and being used for bright field illumination; a composite medium filter film (4) for compressing the illumination light bandwidth is disposed above the glass slide (2); Dark field imaging: first incident light emitted by a first light source (101) from one end of the glass slide (2) enters the glass slide (2) and is totally reflected on the upper and lower inner surfaces of the glass slide (2), and then is scattered by the sample. The upwardly scattered light passes through the composite medium filter film (4) and is modulated into first narrow-band light. The first narrow-band light is received by the MIID imaging module for dark field imaging. Fluorescence imaging: a second incident light emitted by a second light source (102) from the other end of the glass slide (2) enters the glass slide (2) and is totally reflected on the upper and lower inner surfaces of the glass slide (2), and is then excited by the sample to produce fluorescence having a wavelength different from that of the second incident light. The second incident light is cut off by the composite medium filter film (4), and the fluorescence is modulated into a second narrow-band light by the composite medium filter film (4). The second narrow-band light is received by the MIID imaging module for fluorescence imaging. Bright field imaging: the third incident light emitted by the third light source (103) from below the glass slide (2) enters the glass slide (2) and is then modulated into third narrow-band light by the composite medium filter film (4). The third narrow-band light is received by the MIID imaging module for bright field imaging.

2. The highly integrated multimodal microscopic imaging method according to claim 1, It is characterized in that The MIID imaging module comprises a CIS chip (14) and a meta-lens (21); when the meta-lens (21) is a PB phase meta-lens, the first incident light, the second incident light and the third incident light, after passing through the composite medium filter film (4), further pass through a linear polarization film and a quarter wave plate film; the quarter wave plate film comprises a left quarter wave plate film and a right quarter wave plate film; the fast axis of the left quarter wave plate film is at +45° to the linear polarization film, and the fast axis of the right quarter wave plate film is at -45° to the linear polarization film, and are used to modulate a left circular polarization state and a right circular polarization state, respectively.

3. The highly integrated multimodal microscopic imaging method according to claim 1, It is characterized in that The highly integrated multimodal microscopic imaging device further comprises a base plate (31) and a power supply device (23) on the base plate, wherein the power supply device (23) is connected to the light source module to respectively supply power to the first light source (101), the second light source (102), and the third light source (103).

4. The highly integrated multimodal microscopic imaging method according to claim 1, It is characterized in that The glass slide (2) is placed between a loading plate (6) and a clamping plate (7). Four side plates (8) are fixed around the loading plate (6). Screws pass through the side plates (8) and the clamping plates (7) and are used to adjust the clamping plates (7) to press or loosen the glass slide (2).

5. The highly integrated multimodal microscopic imaging method according to claim 4, It is characterized in that The highly integrated multimodal microscopic imaging device further comprises a sample rack (25), a guide rail (28) and a slider (29); the sample rack (25) is connected to the guide rail (28) via the slider (29) and slides vertically along the guide rail (28) for focusing; the sample rack (25) is used to place the carrier plate (6).

6. The highly integrated multimodal microscopic imaging method according to claim 4, It is characterized in that The bottom surface of the clamping plate (7) is provided with a groove (11) for reserving space for placing a linear polarization film and a quarter wave plate film.

7. A computer-readable storage medium storing a computer program, It is characterized in that When the computer program is executed by a processor, the highly integrated multimodal microscopic imaging method according to any one of claims 1 to 6 is implemented.

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