An ultra-structured microscopic imaging device and method for dark-field imaging

Abstract

The application discloses a kind of dark-field imaging super-structure microscopic imaging device and dark-field imaging method, the device includes illumination module and MIID imaging module, illumination module includes mounting sheet and LED light bar (1) placed in mounting sheet one end, sample in mounting sheet will incident light scatter;From below to above, linear polarization film for modulating light polarization state and quarter-wave plate film are respectively arranged on mounting sheet, and composite dielectric filter film for compressing light bandwidth, if sample is dyed by fluorescence, composite dielectric filter film also plays the role of cutting off excitation light, imaging module receives left-handed polarization state and right-handed polarization state sample dark-field image synthesis complete dark-field image;The application makes mounting sheet act as plane waveguide, does not increase additional light source space, realizes dark-field imaging and dark-field fluorescence imaging in the microscopic imaging system based on super-structure lens, while reducing imaging chromatic aberration, improves resolution.

Description

Technical Field

[0001] This invention relates to a metamicroscope for dark-field imaging and a dark-field imaging method, belonging to the field of metamicroscopic imaging. Background Technology

[0002] Traditional optical dark-field microscopy uses magnified imaging. The optical image is magnified by a very large factor compared to the observed object. Typically, the microscope objective performs a primary magnification, followed by a series of imaging systems that further magnify the image before it is finally received by a CCD. The distance in the light propagation direction caused by these optical elements constitutes the large image distance of a traditional microscopy system. Based on this large image distance, traditional dark-field microscopes have a sufficiently large image space to accommodate the illumination source. The most classic dark-field illumination method involves precisely emitting a ring of illumination light from around the microscope objective and focusing it onto the sample area. According to the principle of light reflection, the angle of reflection equals the angle of incidence. Therefore, the portion of this focused illumination light directly reflected by the sample slide will redirect to the area where the illumination source is located, instead of being received by the microscope objective located in the center of the ring light source. The sample backscatters the illumination source, changing the direction of reflection, allowing this portion of light to enter the microscope objective and be observed. Since only the light modulated by the sample is observed, and the illumination source itself does not enter the microscope objective, it does not form a bright background; hence, it is called dark-field imaging.

[0003] However, the aforementioned dark-field illumination scheme is not suitable for the MIID imaging system (DOI:10.1117 / 1.AP.2.6.066004) that integrates a metalens with a CMOS image sensor. This is because the system utilizes the ultra-light and ultra-thin advantages of the metalens to directly bond with the CMOS image sensor through same-size imaging (rather than the magnified imaging described above). While this results in high integration, it also reduces the image distance to the millimeter or even hundreds of micrometers level, eliminating the possibility of placing the light source in the large image space provided by the magnified imaging of traditional dark-field microscopes. Therefore, in order to develop dark-field microscopy based on MIID, it is necessary to find another suitable illumination scheme. In addition to reflective ring illumination, existing dark-field imaging methods based on traditional microscopes also include waveguide illumination. Waveguide illumination directly utilizes the total internal reflection characteristics of the sample slide as a medium for light transmission. By incident illumination light from the end face of the slide, the illumination light is prevented from being directly received by the microscope objective, thus achieving a "dark field". While this method effectively avoids the problem of the illumination source utilizing the image space in dark-field imaging, it can only serve traditional microscopic imaging systems and cannot be directly applied to dark-field imaging in MIID, let alone dark-field fluorescence imaging. This is because the working principle of MIID requires the illumination light to have tunable polarization characteristics, and in dark-field fluorescence imaging, since the fluorescence intensity emitted by the sample itself is much smaller than that of the excitation light, a certain method is needed to completely isolate the excitation light. However, existing waveguide illumination methods can neither generate polarized light nor isolate the excitation light. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to provide a meta-microscopic imaging device for dark-field imaging that eliminates chromatic aberration and improves resolution. The second purpose of this invention is to provide a method for dark-field imaging.

[0005] Technical solution: The meta-microscopic imaging device for dark-field imaging of the present invention includes an illumination module and a MIID imaging module. The illumination module includes a slide and an LED light strip placed at one end of the slide. The sample in the slide scatters the incident light. A linear polarizing film, a quarter-wave plate film and a composite dielectric filter film are respectively arranged on the top of the slide from bottom to top. The linear polarizing film and the quarter-wave plate film are used to modulate the polarization state of the upwardly scattered incident light, and the composite dielectric filter film is used to compress the bandwidth of the incident light.

[0006] Furthermore, 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 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, which are used to modulate the left-hand circular polarization state and the right-hand circular polarization state, respectively. The composite medium filter film is attached to the left quarter-wave plate film and the right quarter-wave plate film, respectively.

[0007] Furthermore, the lighting module also includes a carrier plate and a clamping plate for holding the substrate and placing the LED light strip. The carrier plate and the clamping plate are connected by two side plates. The clamping plate has a groove below it to allow the linear polarizing film (3) to slide.

[0008] Furthermore, the linear polarizing film is provided with a handle on the side near the LED light strip, and two handles extend from the gaps in the side plate for sliding the linear polarizing film.

[0009] Furthermore, the device of the present invention also includes a two-axis displacement stage for moving the MIID imaging module. The clamping plate and the L-shaped plate are connected by a first adjusting screw. Rotating the first adjusting screw drives the L-shaped plate to move horizontally along the clamping plate (9). The L-shaped plate is also provided with a guide rail. The top plate is connected to the guide rail by a slider and moves vertically along the guide rail (23). The bottom of the top plate is connected to the MIID imaging module.

[0010] Furthermore, the MIID imaging module includes a CIS chip and a metalens integrated thereon.

[0011] Furthermore, the sample in the slide contains a fluorescent dye, which scatters the incident light and is excited to emit fluorescence; the linear polarizing film and the quarter-wave plate film are used to modulate the polarization state of the fluorescence and the upwardly scattered incident light, and the composite medium filter film is used to filter out the upwardly scattered incident light and compress the bandwidth of the fluorescence.

[0012] The dark-field imaging method of the meta-microscopic imaging device of the present invention includes the following steps:

[0013] (1) The sample in the slide will scatter the incident light;

[0014] (2) The incident light scattered upward passes through the linear polarization mode and the quarter-wave plate, and is converted into circularly polarized light by polarization modulation;

[0015] (3) The circularly polarized light is converted into narrow-band circularly polarized light through a composite medium filter film;

[0016] (4) The narrowband circularly polarized light is received by MIID for dark field imaging.

[0017] Furthermore, the quarter-wave plate film includes a left quarter-wave plate film and a right quarter-wave plate film, which modulate the upwardly scattered incident light into a left-hand circularly polarized state and a right-hand circularly polarized state, respectively. The MIID receives the narrow-band circularly polarized light of the two polarization states and synthesizes a complete dark field image.

[0018] Further, the sample in step (1) contains a fluorescent dye, and the sample scatters the incident light and is excited by the incident light to emit fluorescence; in step (2), the upwardly scattered incident light and the fluorescence are converted into circularly polarized light after passing through a linear polarization mode and a quarter-wave plate film, and the composite medium filter film cuts off part of the incident light in the circularly polarized light, and then converts it into narrowband circularly polarized fluorescence.

[0019] Beneficial effects: The advantages of this invention compared with the prior art are: (1) By setting a linear polarizing film, a quarter-wave plate film and a composite medium filter film above the slide, and placing a light source at one end of the slide, the slide acts as a planar waveguide, without increasing the space of the light source, and dark-field imaging in a microscope imaging system with equal-size imaging is realized; (2) Compared with the transmission type bright-field illumination scheme, this invention allows smaller sample details to be more easily observed and improves resolution because only the scattered light from the sample enters the imaging module, while the illumination light does not enter the imaging module to form background interference; (3) The composite medium filter film of this invention narrows the bandwidth of the relatively wide illumination light, thereby reducing the chromatic aberration caused by meta-lens imaging; (4) When the sample is stained with fluorescence, the composite medium filter film of this invention also isolates the illumination light scattered by the sample, allowing only the self-emission fluorescence excited by the light source to pass through, thus realizing dark-field fluorescence imaging. Attached Figure Description

[0020] Figure 1 This is a structural diagram of the meta-microscopic imaging device of the present invention.

[0021] Figure 2 This is a structural diagram of the illumination module of the metamicroscopic imaging device of the present invention.

[0022] Figure 3 This is a structural diagram of the biaxial displacement stage of the metamicroscopic imaging device of the present invention.

[0023] Figure 4 This is a structural diagram of the MIID imaging module of the metamicroscopic imaging device of the present invention.

[0024] Figure 5 This is a flowchart of the dark-field imaging method of the metamicroscopic imaging device of the present invention. Detailed Implementation

[0025] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0026] like Figure 1 As shown, the meta-microscopic imaging device including dark-field imaging includes an illumination module, a biaxial displacement stage, and a MIID imaging module.

[0027] like Figure 2As shown, the lighting module includes an LED strip 1, a slide 2, a linear polarizing film 3, a left quarter-wave plate 4, a right quarter-wave plate 5, a composite dielectric filter 6, a sponge strip 7, a carrier plate 8, a clamping plate 9, and two side plates 11. In this embodiment, the light source is the LED strip 1. Each LED bead measures 3.2mm × 2.8mm × 0.7mm. Applying a 3.5V DC voltage to its two electrodes allows it to emit natural light with a center wavelength of 470nm and a bandwidth of approximately 20nm. Several LEDs are arranged in parallel to form the LED strip 1. The LED light source meets the requirements for brightness, wavelength, and uniformity, while also being relatively small in size. Traditional microscope light sources are much larger and cannot meet the miniaturization requirements of MIID (Multi-Instrumentation Device). The LED strip 1 rests diagonally against the cylindrical sponge strip 7 and is fixed together in the space reserved between the carrier plate 8 and the clamping plate 9. The elasticity of the sponge strip serves as both a buffer and a clamping element for the LED strip. Lead wires are used to power the LED strip from its two electrodes.

[0028] The slide 2 is inserted between the carrier plate 8 and the clamping plate 9. The linear polarizing film 3 is located above the slide 2. A quarter-wave plate film and a composite medium filter film 6 are attached to the linear polarizing film 3. The quarter-wave plate film includes a left quarter-wave plate film 4 and a right quarter-wave plate film 5, on which the composite medium filter film 6 is attached respectively. In this embodiment, the left quarter-wave plate film 4, the right quarter-wave plate film 5, and the composite medium filter film 6 are all thin sheets with a side length of 1 cm. The cover glass of the slide 2 is 170 μm thick, the linear polarizing film 3 is 170 μm thick, and the left quarter-wave plate film 4 and the right quarter-wave plate film 5 are... The thickness of the composite medium filter film 5 is 50 μm, the thickness of the composite medium filter film 6 is 4.73 μm, and the imaging object distance is 800 μm. The fast axis of the left quarter-wave plate film 4 is at +45° to the orientation of the linear polarizing film 3, and the fast axis of the right quarter-wave plate film 5 is at -45° to the orientation of the linear polarizing film 3. The linear polarizing film 3 is also provided with a handle 17 at one end near the LED light strip 1. The two side plates 11 are respectively provided with slits 18 to accommodate the handle 17. When the handle 17 is moved, the left quarter-wave plate film 4 and the right quarter-wave plate film 5 can be adjusted to be aligned with the meta-lens 32 of the MIID imaging module.

[0029] Two side plates 11 are fixed to the two sides of the carrier plate 8 perpendicular to the plate 2 by side plate fixing screws 12. The side plate set screws 13 pass through the side plate screw holes 14 on the side plate 11 and are embedded in the grooves 15 on the clamping plate 9. By rotating the side plate set screws 13, the clamping plate 9 can clamp the plate 2 downward. Since the clamping plate 9 has a groove 16 reserved on the side facing the plate 2, the linear polarizing film 3 between the two can still move when the clamping plate 9 clamps the plate 2.

[0030] like Figure 3As shown, the two-axis displacement stage includes an L-shaped plate 21, guide rails 23, a slider 25, and a top plate 26. A first adjusting screw 19 passes through a clamping plate through-hole 20 above the clamping plate 9 and is screwed into an L-shaped plate screw hole 22 at the bottom of the L-shaped plate 21. Rotating the first adjusting screw 19 causes the L-shaped plate 21 to slide horizontally on the clamping plate 9, resulting in a horizontal relative movement between the meta-lens 32 and the sample region 10, thus adjusting the region of interest. Two guide rails 23 are fixed to the slider 25 by top plate fixing screws 27. A second adjusting screw 28 passes through an L-shaped plate through-hole 29 at the top of the L-shaped plate 21 and is screwed into a top plate screw hole 30 on the side of the top plate 26 near the guide rails 23. Rotating the second adjusting screw 28 causes the top plate 26 to slide vertically along the guide rails 23, achieving focusing of the MIID imaging module.

[0031] like Figure 4 As shown, the MIID imaging module includes a CIS chip 31 and a meta-lens 32. The CIS chip 31 also has other interfaces that can be developed and utilized, through which a ribbon cable can be led out to power the LED strip 1. The CIS chip 31 measures 30mm × 30mm and is fixed to four screw posts 34 by four M2 countersunk screws 33 through through holes at its four corners, ensuring the relative parallelism between the meta-lens 32 and the substrate 2. Countersunk screws are used to prevent the height difference between the screw and the meta-lens substrate surface from exceeding the object distance for imaging, thus limiting focusing. An opening 35 is provided on the top plate 26 for the USB data cable connection of the CIS chip 31.

[0032] When one end of the slide 2 extends close to the LED strip 1, the slide 2 itself acts as a planar waveguide, guiding the light emitted by the LED strip to the sample area 10. When the light encounters the sample, it is scattered by the sample. This portion of the light carrying sample information changes its propagation direction, passing upwards sequentially through the left quarter-wave plate 4 (or the right quarter-wave plate 5) and the composite dielectric filter 6, and is then received by the MIID imaging module above for dark-field imaging. The function of the composite dielectric filter 6 is to narrow the bandwidth of the relatively wide illumination light, thereby reducing the chromatic aberration caused by the meta-lens imaging. When the sample in the slide 2 is stained with fluorescent dye, the sample not only scatters the light emitted by the LED strip but also emits fluorescence. In addition to reducing chromatic aberration, the composite dielectric filter 6 also isolates the illumination light scattered by the sample, allowing only the self-emitted fluorescence excited by the light source to pass through, thus achieving dark-field fluorescence imaging.

[0033] like Figure 5 As shown, the dark-field imaging method of the present invention includes the following steps:

[0034] (1) Install the substrate 2, turn on the LED light strip 1, the light emitted by the LED light strip 1 enters the substrate 2, and undergoes total internal reflection on its upper and lower inner surfaces to propagate forward continuously. After the light propagates to the sample area 10, it is scattered by the sample and changes its original propagation direction.

[0035] (2) The upward-scattered light is modulated into linearly polarized light after passing through the linear polarizing film 3, and then modulated into left-hand circularly polarized light after passing through the left quarter-wave plate film 4 whose fast axis is at +45° to the linear polarizing film 3.

[0036] (3) After passing through the composite medium filter film 6, it is modulated into narrowband left-hand circularly polarized light;

[0037] (4) Finally, the dark field image of the sample illuminated by narrowband left-handed circularly polarized light is received by MIID;

[0038] (5) Repeat steps (2) to (4) and replace the left quarter-wave plate film 4 with the right quarter-wave plate film 5 whose fast axis is at -45° with the linear polarization film 3 to obtain the dark field image of the sample illuminated by narrow-band right-hand circularly polarized light.

[0039] (6) The dark field image of the sample illuminated by narrowband left-hand circularly polarized light and the dark field image of the sample illuminated by narrowband right-hand circularly polarized light are processed to synthesize a complete dark field image.

[0040] When the sample in slide 2 has been stained with fluorescent dye, the light emitted by LED strip 1 is used for excitation, which is called excitation light. The sample is excited by the excitation light and emits fluorescence. In step (3), the composite medium filter film 6 cuts off the excitation light and only allows the fluorescence to pass through, finally obtaining a dark field fluorescence image with the excitation light cut off.

Claims

1. A dark-field imaging super-structured microscope imaging device, comprising: The application relates to a lighting module and a MIID imaging module, the lighting module comprising a mounting plate (2) and an LED light bar (1) arranged at one end of the mounting plate (2), wherein a sample in the mounting plate (2) scatters incident light; a linear polarization film (3), a quarter-wave film and a composite medium filter film (6) are arranged above the mounting plate (2) from bottom to top, the linear polarization film (3) and the quarter-wave film are used for modulating the polarization state of upward scattered incident light, and the composite medium filter film (6) is used for compressing the bandwidth of the incident light with the polarization state. The quarter-wave film comprises a left quarter-wave film (4) and a right quarter-wave film (5), the fast axis of the left quarter-wave film (4) is +45 DEG relative to the linear polarization film (3), the fast axis of the right quarter-wave film (5) is -45 DEG relative to the linear polarization film (3), and the left quarter-wave film (4) and the right quarter-wave film (5) are respectively used for modulating left circular polarization and right circular polarization; the composite medium filter film (6) is respectively pasted on the left quarter-wave film (4) and the right quarter-wave film (5). The lighting module further comprises a carrier plate (8) and a clamping plate (9) for clamping the mounting plate (2) and placing the LED light bar (1), the carrier plate (8) and the clamping plate (9) are connected through two side plates (11), and a groove (16) for providing sliding space for the linear polarization film (3) is arranged below the clamping plate (9). A handle (17) is arranged on one side of the linear polarization film (3) close to the LED light bar (1), two handle (17) respectively extend from the slits (18) on the side plates (11), and are used for sliding the linear polarization film (3).

2. The dark-field imaging meta-microscopy device of claim 1, wherein, A two-axis displacement table for moving the MIID imaging module is further provided, the clamping plate (9) and an L-shaped plate (21) are connected through a first adjusting screw (19), the first adjusting screw (19) is rotated to drive the L-shaped plate (21) to move horizontally along the clamping plate (9); a guide rail (23) is further arranged on the L-shaped plate (21), a top plate (26) is connected with the guide rail (23) through a sliding block (25) and moves vertically along the guide rail (23), and the MIID imaging module is connected with the bottom of the top plate (26).

3. The dark-field imaging meta-microscopy device of claim 1, wherein, The MIID imaging module comprises a CIS chip (31) and an integrated super-structure lens (32).

4. The dark-field imaging super-structuring microscopical imaging device according to any one of claims 1 to 3, characterized in that, The sample of the mounting plate (2) contains fluorescent dye, the sample scatters the incident light and excites fluorescence; the linear polarization film (3) and the quarter-wave film are used for modulating the polarization state of the fluorescence and the upward scattered incident light, and the composite medium filter film (6) is used for filtering the upward scattered incident light and compressing the bandwidth of the fluorescence.

5. A dark-field imaging method based on the superlens microscopic imaging device of claim 1, characterized in that, The application further discloses a method for dark-field imaging of a sample, comprising the following steps: (1) the sample in the mounting plate (2) scatters incident light; (2) the upward scattered incident light passes through the linear polarization film (3) and the quarter-wave film, and is converted into circularly polarized light through polarization modulation; (3) the circularly polarized light passes through the composite medium filter film (6) and is converted into narrow-band circularly polarized light; (4) the narrow-band circularly polarized light is received by the MIID and is subjected to dark-field imaging.

6. The dark-field imaging method of a metamaterial microscope device according to claim 5, wherein, The quarter-wave plate film includes a left quarter-wave plate film (4) and a right quarter-wave plate film (5) for modulating the upward scattered incident light into left-handed circularly polarized state and right-handed circularly polarized state respectively, and the MIID receives the two polarized states of narrow-band circularly polarized light to synthesize a complete dark-field image.

7. The dark-field imaging method of the super-constructive microscopic imaging device according to claim 5, characterized in that, The sample in step (1) contains fluorescent dyes, which scatter incident light and are excited to emit fluorescence by the incident light; in step (2), the upward scattered incident light and the fluorescence are converted into circularly polarized light after passing through the linear polarizing film (3) and the quarter-wave plate film, and the composite medium filter film (6) cuts off the incident light part in the circularly polarized light, and then converts into narrow-band circularly polarized fluorescence.

Images