Light processing device, fluorescence wide-field microscope, imaging method, medium and apparatus

Abstract

This disclosure relates to the field of microscopy technology, and more particularly to light processing devices, fluorescence wide-field microscopes, imaging methods, media, and equipment. The light processing device includes: a first polarizing beam splitter for splitting incident light into first polarized light and second polarized light with mutually perpendicular polarization directions, wherein the first polarized light enters a first optical path, and the second polarized light enters a second optical path different from the first optical path; a grating located in the first optical path to modulate the first polarized light into structured light after passing through the grating; a second polarizing beam splitter for combining the first polarized light modulated by the grating and the second polarized light not modulated by the grating into an outgoing optical path; and a polarization light selection component located in the outgoing optical path for selectively allowing either the first polarized light or the second polarized light to pass through.

Description

Technical Field

[0001] This disclosure relates to the field of microscopy technology, and more particularly to light processing devices, fluorescence wide-field microscopes, imaging methods, media, and equipment. Background Technology

[0002] Fluorescence wide-field microscopy is known to illuminate the entire sample in a single pass. Therefore, fluorescence is emitted not only at the focal plane but also at the defocus plane (i.e., the regions above and below the focal plane). Furthermore, the fluorescence from the focal plane and the defocus plane overlap, resulting in reduced image contrast at the focal plane and consequently lowering the actual resolution achievable by the microscope.

[0003] To suppress fluorescence at the aforementioned defocused plane, various technical solutions have emerged, such as scanning confocal microscopy and multiphoton excitation microscopy. While these laser point scanning-based imaging methods overcome the limitations of fluorescence wide-field microscopy in terms of resolution and contrast, they also sacrifice the advantages of fast imaging speed and system simplicity.

[0004] Jerome Mertz's research group proposed a hybrid illumination wide-field fluorescence tomography (HiLo) technique that retains the advantages of wide-field fluorescence microscopy while overcoming its poor contrast. The group has published several papers elucidating the relevant principles, such as "Optical sectioning microscopy with planar or structured illumination" (Nature Methods–8:811-819, 2011), "Optically sectioned in vivo imaging with speckle illumination HiLo microscopy" (Journal of Biomedical Optics–16:016014, 2011), and "Wide-field fluorescence sectioning with hybrid speckle and uniform-illumination microscopy" (Optics Letters–33:1819-1821, 2008).

[0005] HiLo technology requires acquiring both a uniformly illuminated image and a structured light (e.g., fringe, speckle) illuminated image. The uniformly illuminated image contains high-frequency information, low-frequency information, and low-frequency information off-focus. Optical tomography requires obtaining both the high-frequency and low-frequency information from the uniformly illuminated image. The low-frequency information can be obtained using the structured light illuminated image, while the high-frequency information can be obtained from the uniformly illuminated image after high-pass filtering. Fusing the high-frequency and low-frequency information yields the full-resolution image, i.e., the optical tomographic image. HiLo technology can achieve results close to those of confocal microscopy, and is simpler, faster, and less expensive than confocal microscopy systems.

[0006] Evident has incorporated HiLo technology into its VS200-SILA scanning slide system (see https: / / www.olympus-lifescience.com / en / solutions-based-systems / vs200 / sila / ). According to the company, SILA (Speckle Illumination Acquisition) uses speckle illumination to obtain high-contrast images. The speckle is generated by inter-mode interference of a coherent light source (laser) passing through a multimode fiber. When uniform illumination is required, a controllable speckle eliminator, typically a high-speed vibrating or rotating device, is used to scramble modes and eliminate speckle, thus achieving uniform illumination. Although the SILA module can be easily mounted onto the VS200, upgrading a standard VS200 scanner to a HiLo-enabled scanner, the requirement to use a laser as the light source to generate the speckle limits the use of HiLo technology in ordinary scanners. The standard VS200 comes with an LED light source, which is sufficient for its normal use. However, if you want to upgrade to the VS200-SILA, you need to replace the LED light source with a laser light source, which will increase the cost of the equipment by hundreds of thousands or even millions of dollars.

[0007] Besides the SILA module, Digital Micromirror Devices (DMDs), Spatial Light Modulators (SLMs), and Liquid Crystal on Silicon (LCoS) microdisplays can also be used to generate structured light. DMDs, SLMs, or LCoS can actively generate any desired pattern, so structured light and uniform light can share the same illumination path; switching between structured light and uniform light is achieved simply by changing the image displayed on the DMD or other devices. However, DMDs, SLMs, and LCoS are generally expensive, and because they are diffraction devices, some high-order diffraction energy is lost during use. Furthermore, SLMs and LCoS require linearly polarized incident light, while the light sources of typical wide-field microscopes are mercury lamps, metal halide lamps, or light-emitting diodes (LEDs), all of which are unpolarized light, resulting in a loss of half the light energy when converted to linearly polarized light.

[0008] Furthermore, gratings can also be used to generate structured light illumination. For example, CN110141182A discloses a microscopic endoscopic imaging method and system based on structured light illumination. However, it requires two sets of illumination light paths: one for generating structured light and the other for generating uniform light, with the two light sources emitting alternately to achieve switching between the two types of light. This illumination design relies on two modulated illumination sources, making it unusable on microscopes equipped with traditional illumination sources such as mercury lamps or metal halide lamps, thus reducing the applicability of the technology and increasing its cost. In particular, as described in the specification, when using high-power LED light sources, each additional fluorescent light path means adding two monochromatic LEDs and related beam combining devices, reducing its scalability and increasing expansion costs.

[0009] In summary, HiLo technology can retain the advantages of fluorescence wide-field microscopy while overcoming its disadvantage of poor contrast. However, existing related technical solutions suffer from problems such as high cost, low light energy utilization, and poor scalability, making it difficult to achieve compatibility with existing fluorescence wide-field microscopy in a simple way. Summary of the Invention

[0010] The purpose of this disclosure is to provide a light processing device, a fluorescence wide-field microscope, an imaging method, a medium, and an apparatus to easily extend a microscope into a HiLo-enabled microscope, and to solve the problems of high cost, low light energy utilization, and poor expandability.

[0011] In a first aspect, this disclosure provides an optical processing apparatus, comprising:

[0012] The first polarizing beam splitter is used to split the incident light into a first polarized light and a second polarized light with polarization directions perpendicular to each other. The first polarized light enters a first optical path, and the second polarized light enters a second optical path that is different from the first optical path.

[0013] A grating is located in the first optical path so that the first polarized light is modulated into structured light after passing through the grating;

[0014] The second polarizing beam splitter is used to combine the first polarized light modulated by the grating and the second polarized light not modulated by the grating into the outgoing optical path.

[0015] A polarization light selection component, located in the outgoing optical path, is used to selectively allow either the first polarized light or the second polarized light to pass through.

[0016] Secondly, this disclosure provides a fluorescence wide-field microscope, including:

[0017] Non-laser light source;

[0018] An optical fiber, wherein the light incident surface of the optical fiber is connected to the non-laser light source;

[0019] The microscope body has a light source inlet and an image sensor, the image sensor being used to acquire images of the sample;

[0020] In the aforementioned optical processing apparatus, the incident light is provided by the emitting surface of the optical fiber, and the emitted light path passes through the light source inlet.

[0021] Thirdly, this disclosure provides an imaging method applied to the aforementioned fluorescence wide-field microscope, characterized in that it includes:

[0022] The image sensor receives a structured light illumination image acquired at a first moment, wherein at the first moment, the first polarized light passes through the polarization light selection component to illuminate the sample, and the second polarized light is blocked by the polarization light selection component.

[0023] Receive a uniformly illuminated image acquired by the image sensor at a second time different from the first time, wherein, at the second time, the second polarized light passes through the polarization light selection component to illuminate the sample, and the first polarized light is blocked by the polarization light selection component;

[0024] The HiLo algorithm is used to process the structured light illumination image and the uniform light illumination image to obtain a HiLo image.

[0025] Fourthly, this disclosure provides a computer-readable storage medium comprising a stored program, wherein the program, when executed, performs the imaging method described above.

[0026] Fifthly, this disclosure provides an electronic device including a memory and a processor, wherein the memory stores a computer program and the processor is configured to execute the imaging method described above through the computer program.

[0027] Compared with the prior art, this disclosure includes at least the following beneficial effects:

[0028] The first polarizing beam splitter splits the incident light into a first polarized light and a second polarized light. The first polarized light enters the first optical path, and the second polarized light enters the second optical path. A grating is placed in the second optical path to modulate the first polarized light into structured light. No grating is placed in the second optical path, so the second polarized light remains uniform light. The second polarizing beam splitter combines the first polarized light and the second polarized light into the outgoing optical path. In the outgoing optical path, a polarization light selection component is used to select whether the outgoing optical path allows the first polarized light or the second polarized light to pass through, thereby realizing the switching between structured light and uniform light.

[0029] Based on this, this disclosure does not require the use of lasers as a light source, nor does it require the use of diffraction devices such as DMD, SLM, or LcoS, nor does it require the use of multiple light sources. It only requires a single light source such as a mercury lamp, metal halide lamp, or LED, which are equipped with a traditional fluorescence wide-field microscope, to achieve the switching between uniform light and structured light.

[0030] Therefore, compared to solutions using lasers as the light source, this disclosure has a lower cost. Compared to solutions using diffraction devices, this disclosure has a lower cost and improves light energy utilization (in the case of mercury lamp light sources, the light energy utilization is more than 5 times that of the DMD solution). Compared to solutions using two light sources, this disclosure has a simpler structure and greater scalability. Attached Figure Description

[0031] Figure 1 A schematic diagram of a light processing apparatus according to Embodiment 1 of the present disclosure is shown.

[0032] Figure 2 A schematic diagram of a light processing apparatus according to Embodiment 2 of this disclosure is shown.

[0033] Figure 3 A flowchart of an imaging method according to Embodiment 4 of this disclosure is shown.

[0034] Figure label:

[0035] 1. Collimating lens;

[0036] 2. First polarizing beam splitter;

[0037] 3. Reflected light path;

[0038] 4. Transmission optical path;

[0039] 5. First reflecting mirror;

[0040] 6. Grating;

[0041] 7. Second reflecting mirror;

[0042] 8. Second polarizing beam splitter;

[0043] 9. Lens;

[0044] 10. Liquid crystal variable delay device;

[0045] 11. Polarizing filter;

[0046] 12. Outer shell;

[0047] 13. Light entrance;

[0048] 14. Light outlet;

[0049] 15. Optical path compensation plate;

[0050] 16. Third reflecting mirror;

[0051] 17. Outgoing light path. Detailed Implementation

[0052] The technical solutions of this disclosure will be further described below with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are merely for explaining this disclosure and not for limiting it. Furthermore, it should be noted that, for ease of description, only the parts relevant to this disclosure are shown in the accompanying drawings, and not all of them.

[0053] This disclosure defines certain directional terms. Unless otherwise stated, the directional terms used, such as "up," "down," "left," "right," "inner," and "outer," are used for ease of understanding and therefore do not constitute a limitation on the scope of protection of this disclosure.

[0054] In this disclosure, unless otherwise expressly specified and limited, "above" or "below" the second feature can include direct contact between the first and second features, or contact between the first and second features through another feature between them. Furthermore, "above," "over," and "on top" of the second feature includes the first feature directly above or diagonally above the second feature, or simply indicates that the first feature is at a higher horizontal level than the second feature. "Below," "below," and "under" the second feature includes the first feature directly below or diagonally below the second feature, or simply indicates that the first feature is at a lower horizontal level than the second feature.

[0055] In the description of this disclosure, unless otherwise expressly specified and limited, the terms "connected," "linked," and "fixed" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral part; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; they can refer to the internal communication of two components or the interaction between two components. Those skilled in the art can understand the specific meaning of the above terms in this disclosure based on the specific circumstances.

[0056] The existing technology includes three schemes for achieving the switching between uniform light and structured light.

[0057] The first method uses laser light source interference speckle as structured light illumination, and then high-speed modulation to homogenize the speckle to form uniform illumination. This method relies on a laser light source and is relatively expensive.

[0058] The second method involves using devices such as digital micromirror arrays (DMDs), spatial light modulators (SLMs), and liquid crystal on silicon (LCoS) microdisplays to directly generate patterns of structured or uniform light. All three of these devices are diffractive devices, resulting in low light energy utilization.

[0059] The third method uses gratings to generate structured light illumination, but this requires two sets of illumination paths: one for generating structured light and the other for generating uniform light. This presents two main problems: First, this structure requires time-division multiplexing of the two light sources, making it impossible to use traditional mercury lamps or metal halide lamps in microscopes as illumination sources, thus limiting its applicability. Second, while this method uses high-power LEDs as the light source, which are easy to control, they are less flexible when expanding imaging color channels and are more expensive because adding another color channel requires two more monochrome LEDs and related beam combining devices.

[0060] The inventive concept disclosed herein is as follows:

[0061] First, the incident light emitted by the light source is split into p-polarized light and s-polarized light using a first polarizing beam splitter. The p-polarized light and s-polarized light each enter a beam path. A grating is set in one beam path so that the polarized light is modulated into structured light. No grating is set in the other beam path so that the polarized light remains uniform.

[0062] Then, the p-polarized light and s-polarized light are combined into the output light path by the second polarizing beam splitter;

[0063] Finally, a polarization selection component is used to select whether the outgoing light path allows p-polarized light or s-polarized light to pass through, thereby achieving the switching between structured light and uniform light.

[0064] Based on this inventive concept, this disclosure does not require the use of lasers as light sources, nor does it require the use of diffraction devices such as DMD, SLM, or LcoS, nor does it require the use of multiple light sources. It only requires a single light source such as a mercury lamp, metal halide lamp, or LED, which are equipped with a traditional fluorescence wide-field microscope, to achieve the switching between uniform light and structured light.

[0065] Therefore, compared to solutions using lasers as the light source, this disclosure has a lower cost. Compared to solutions using diffraction devices, this disclosure has a lower cost and improves light energy utilization (in the case of mercury lamp light sources, the light energy utilization is more than 5 times that of the DMD solution). Compared to solutions using two light sources, this disclosure has a simpler structure and greater scalability.

[0066] According to the inventive concept disclosed herein, in one example, a first polarizing beam splitter, a second polarizing beam splitter, a grating, and a polarization light selection component can be integrated into a single housing, thereby allowing for easy mounting onto a fluorescence wide-field microscope (hereinafter referred to as the microscope). This transforms an ordinary microscope into a microscope with HiLo technology, enabling it to achieve imaging effects close to those of a confocal microscope, without affecting its original functions and structure.

[0067] Example 1

[0068] This embodiment relates to a light processing device. The light processing device is used to process incident light and can selectively output structured light or uniform light. As mentioned above, the light processing device can be applied to fluorescence wide-field microscopes, thereby upgrading ordinary microscopes to microscopes with HiLo technology. However, the light processing device can also be applied to other scenarios, such as endoscopes, scanners, etc.

[0069] Figure 1 A schematic diagram of a light processing apparatus according to Embodiment 1 of the present disclosure is shown. Figure 1As shown, the light processing device includes a housing 12. The housing 12 includes a light inlet 13 and a light outlet 14. Incident light enters the housing 12 through the light inlet 13, and the emitted light, after being processed by the light processing device, exits through the light outlet 14.

[0070] In this embodiment, the light processing device is applied to a microscope (not shown) as an example. The light inlet 13 is used to connect to the light source of the microscope so that the light emitted by the light source can enter the housing 12. The light outlet 14 is used to connect to the light source inlet of the microscope so that the outgoing light processed by the light processing device is introduced into the microscope.

[0071] In known microscopes, the light source is typically a mercury lamp, a metal halide lamp, or an LED. Incident light emitted from the light source can be introduced into the microscope body via an optical fiber. In this case, the emitting surface of the optical fiber can be inserted into the light inlet 13, thereby introducing the incident light emitted from the light source into the housing 12. The light outlet 14 can be connected to the light source inlet, thereby introducing the emitted light, after being processed by the light processing device, into the microscope body. In other words, in this embodiment, the incident light first enters the housing 12 through the light inlet 13, is then processed inside the housing 12, and finally enters the microscope body through the light outlet 14.

[0072] It should be noted that this disclosure does not involve improvements to the internal structure of the microscope, and the internal structure of the microscope and the corresponding imaging principle will not be described in detail.

[0073] like Figure 1 As shown, the light inlet 13 and the light outlet 14 can be located on the same straight line. However, the straight line containing the light inlet 13 and the straight line containing the light outlet 14 do not have to be collinear. For example, the straight line containing the light inlet 13 and the straight line containing the light outlet 14 can be parallel to each other, intersect each other, etc.

[0074] like Figure 1 As shown, a collimating lens 1 is disposed inside the housing 12. The collimating lens 1 can be aligned with the light inlet 13 to collimate the incident light. Specifically, the light emitting surface of the optical fiber can be disposed upstream of the collimating lens 1 and located at the focal point of the collimating lens 1. It should be noted that, in this disclosure, "upstream" is determined according to the direction of light propagation, pointing from upstream to downstream along the direction of light propagation.

[0075] like Figure 1 As shown, a first polarizing beam splitter 2 is disposed inside the housing 12. Exemplarily, the first polarizing beam splitter 2 can be a polarizing flat beam splitter or a polarizing beam splitter cube. When incident light is incident on the first polarizing beam splitter 2, the incident light is split into first polarized light and second polarized light with polarization directions perpendicular to each other. For example, the first polarized light is s-polarized light, and the second polarized light is p-polarized light.

[0076] It should be noted that the first polarized light and the second polarized light propagate along different optical paths. Specifically, the first polarized light propagates along a first optical path, while the second polarized light propagates along a second optical path, which is different from the first optical path.

[0077] exist Figure 1 In the example shown, the first polarized light is reflected by the first polarizing beam splitter 2 and enters the first optical path, while the second polarized light is transmitted through the first polarizing beam splitter 2 and enters the second optical path. Therefore, the first optical path can be considered as the reflected optical path 3, and the second optical path can be considered as the transmitted optical path 4.

[0078] It should be noted that the first optical path and the second optical path are only used to distinguish them as different optical paths, and do not limit the specific propagation directions of the first optical path and the second optical path. In other examples, the first optical path can be the transmission optical path 4, and the second optical path can be the reflection optical path 3.

[0079] exist Figure 1 In the example shown, a first set of reflectors is provided in the reflected light path 3. Exemplarily, the first set of reflectors may include two reflectors, specifically a first reflector 5 and a second reflector 7. However, in other embodiments, the first set of reflectors may include one reflector (see Embodiment 2 below) or three or more reflectors.

[0080] like Figure 1 As shown, the reflecting surface of the first reflecting mirror 5 is parallel to the reflecting surface of the first polarizing beam splitter 2, and the reflecting surface of the second reflecting mirror 7 is perpendicular to each other. The first polarized light in the first optical path first enters the reflecting surface of the first reflecting mirror 5 at an angle of incidence of 45 degrees, and thus the reflection angle is also 45 degrees. Therefore, after the first polarized light passes through the first reflecting mirror, its propagation direction is deflected by 90 degrees. After being reflected by the first reflecting mirror 5, the first polarized light enters the second reflecting mirror 7 at an angle of incidence of 45 degrees, and thus the reflection angle is also 45 degrees. Therefore, after being reflected by the second reflecting mirror 7, its propagation direction is deflected by another 90 degrees. That is, after being reflected twice by the first reflecting mirror 5 and the second reflecting mirror 7, the propagation direction of the first polarized light is deflected by a total of 180 degrees.

[0081] Therefore, the angle between the transmitted light path 4 and the reflected light path 3 located downstream of the second reflecting mirror 7 is 90 degrees. Consequently, the reflected light path 3 and the transmitted light path 4 intersect at a certain position. Taking the intersection area as the end of the reflected light path 3 and the end of the transmitted light path 4, the ends of the reflected light path 3 and the transmitted light path 4 form an intersection area.

[0082] like Figure 1As shown, a second polarizing beam splitter 8 is also disposed within the housing 12. Exemplarily, the second polarizing beam splitter 8 can be a polarizing flat beam splitter or a polarizing beam splitter cube. The second polarizing beam splitter 8 is located in the aforementioned intersection region, and its orientation is opposite to that of the first polarizing beam splitter 2. Therefore, both the first polarized light and the second polarized light will be incident on the second polarizing beam splitter 8, and after incident on the second polarizing beam splitter 8, the first polarized light and the second polarized light will be combined into the outgoing light path 17.

[0083] Specifically, the first polarized light is reflected by the second polarizing beam splitter 8 after being incident on it, and then enters the outgoing light path 17. The second polarized light is transmitted through the second polarizing beam splitter 8 after being incident on it, and then enters the outgoing light path 17.

[0084] exist Figure 1 In the example shown, no reflector is provided in the second optical path, i.e., the transmission optical path 4, so that the second polarized light is directly incident on the second polarizing beam splitter 8 without reflection. However, in other embodiments, a second set of reflectors may be provided in the second optical path, which will be described in Embodiment 2 below.

[0085] like Figure 1 As shown, in order to convert one of the first polarized light and the second polarized light into structured light, a grating 6 is also provided inside the housing 12. The grating 6 can be a glass substrate. The grating 6 only needs to be placed in either the first optical path or the second optical path. As mentioned above, the first optical path and the second optical path are not absolute and can be interchanged. In this embodiment, the optical path where the grating 6 is located is defined as the first optical path.

[0086] exist Figure 1 In the example shown, the grating 6 can be located between the first polarizing beam splitter 2 and the first reflecting mirror 5, or between the first reflecting mirror 5 and the second reflecting mirror 7, or between the second reflecting mirror 7 and the second polarizing beam splitter 8, or between the first polarizing beam splitter 2 and the second polarizing beam splitter 8.

[0087] Therefore, a grating 6 is provided in the first optical path, while a grating 6 is not provided in the second optical path. Based on this, the first polarized light is modulated into structured light after passing through the grating 6, while the second polarized light remains uniform light because it does not pass through the grating 6.

[0088] like Figure 1 As shown, a lens 9 is also provided inside the outer casing 12. The lens 9 is located in the outgoing light path 17 and is used to transmit the image of the grating 6 to the microscope body. The optical path between the grating 6 and the lens 9 is equal to the focal length of the lens 9. The lens 9 and the objective lens in the microscope body act as a relay lens to project the image of the grating 6 onto the sample. If necessary, another relay lens can be added between the lens 9 and the objective lens.

[0089] like Figure 1 As shown, a polarization light selection component is also provided inside the housing 12. The polarization light selection component is located in the outgoing light path 17 and downstream of the lens 9. The polarization light selection component is used to selectively allow either first polarized light or second polarized light to pass through. When the polarization light selection component allows the first polarized light to pass through, the first polarized light (i.e., structured light) is emitted from the light exit port 14 of the housing 12. When the polarization light selection component allows the second polarized light to pass through, the second polarized light (uniform light) is emitted from the light exit port 14 of the housing 12.

[0090] It should be noted that at any given moment, the polarization light selection component only allows one of the first polarized light and the second polarized light to pass through. That is, when the polarization light selection component allows the first polarized light to pass through, the second polarized light is blocked, and when the polarization light selection component allows the second polarized light to pass through, the first polarized light is blocked.

[0091] like Figure 1 As shown, as an example of a polarization light selection component, the polarization light selection component may include a liquid crystal variable retarder 10, a controller (not shown), and a polarizer 11. The liquid crystal variable retarder 10 and the controller cooperate to adjust the polarization states of a first polarized light and a second polarized light. For example, by simultaneously rotating the polarization states of the first and second polarized lights by 90 degrees, s-polarized light can be adjusted to p-polarized light, and p-polarized light can be adjusted to s-polarized light. Alternatively, the polarization states of the first and second polarized lights may remain unchanged; that is, s-polarized light remains s-polarized light, and p-polarized light remains p-polarized light. The polarizer 11 is located downstream of the liquid crystal variable retarder 10, and its polarization axis is aligned with the first or second polarized light that has not passed through the liquid crystal variable retarder 10, thereby allowing the corresponding polarized light to pass through. Specifically, the polarizer 11 can be aligned with s-polarized light, allowing s-polarized light to pass through the polarizer 11, while blocking p-polarized light. Furthermore, the polarizer 11 can also be aligned with p-polarized light, allowing p-polarized light to pass through the polarizer 11.

[0092] In order for the liquid crystal variable retarder 10 to correctly adjust the polarization state, the angle between the optical axis of the liquid crystal variable retarder 10 and the polarization direction of the first polarized light is 45 degrees, or the angle between the optical axis of the liquid crystal variable retarder 10 and the polarization direction of the second polarized light is 45 degrees.

[0093] A controller is coupled to the liquid crystal variable retarder 10 and is used to control the voltage of the liquid crystal variable retarder 10 to selectively adjust the polarization states of the first polarized light and the second polarized light. Specifically, when the controller applies a first voltage to the liquid crystal variable retarder 10, the liquid crystal variable retarder 10 can simultaneously rotate the polarization states of the first polarized light and the second polarized light by 90 degrees; when the controller applies a second voltage different from the first voltage to the liquid crystal variable retarder 10, the liquid crystal variable retarder 10 does not change the polarization states of the first polarized light and the second polarized light.

[0094] Since the delay of light by the liquid crystal variable retarder 10 is related to the wavelength of light, the values ​​of the first voltage and the second voltage will be slightly different when capturing patterns of different color channels. Users can consult the voltage-delay curves for different wavelengths provided by the liquid crystal variable retarder 10 manufacturer, or pre-test and record the values ​​of the first and second voltages for each wavelength, and create a corresponding wavelength-voltage lookup table. This lookup table can be stored in the imaging program for user use.

[0095] Therefore, the polarization light selection component can select either the first polarized light or the second polarized light to pass through, thereby allowing structured light or uniform light to be emitted from the light outlet 14.

[0096] Example 2

[0097] This embodiment relates to a light processing device, and the structure is similar to that in Embodiment 1, so it will not be described again.

[0098] Figure 2 A schematic diagram of the light processing apparatus according to Embodiment 2 is shown. As shown in Figure 2, in this embodiment, the grating 6 is disposed on the transmission light path 4, therefore the transmission light path 4 is the first light path, and the reflection light path 3 is the second light path. Furthermore, the relative positional relationship between the first polarizing beam splitter 2 and the second polarizing beam splitter 8 differs from that in the embodiment. Specifically, the first polarizing beam splitter 2 and the second polarizing beam splitter 8 are no longer disposed on a horizontal straight line, but are disposed at an angle.

[0099] like Figure 2 As shown, in order to ensure that the ends of the first optical path and the second optical path still form an intersection area at the position of the second polarizing beam splitter 8, not only is a first reflecting mirror group provided on the reflecting optical path 3, but a second reflecting mirror group is also provided on the transmitting optical path 4.

[0100] like Figure 2As shown, the first reflecting mirror group includes a first reflecting mirror 5. The reflecting surface of the first reflecting mirror 5 is parallel to the reflecting surface of the first polarizing beam splitter 2 on one hand, and also parallel to the reflecting surface of the second polarizing beam splitter 8 on the other hand. The second polarized light in the second optical path first enters the reflecting surface of the first reflecting mirror 5 at an angle of incidence of 45 degrees, and thus the reflection angle is also 45 degrees. After passing through the first reflecting mirror, the propagation direction of the second polarized light is deflected by 90 degrees. The second polarized light, after being reflected by the first reflecting mirror 5, enters the second polarizing beam splitter 8.

[0101] like Figure 2 As shown, the second reflector group includes a second reflector 7. The reflecting surface of the second reflector 7 is parallel to both the reflecting surface of the first polarizing beam splitter 2 and the reflecting surface of the second polarizing beam splitter 8. The first polarized light in the first optical path first enters the reflecting surface of the second reflector 7 at an angle of incidence of 45 degrees, and thus the reflection angle is also 45 degrees. After passing through the second reflector 7, the propagation direction of the first polarized light is deflected by 90 degrees. The first polarized light, after being reflected by the second reflector 7, enters the second polarizing beam splitter 8.

[0102] The first polarized light is incident on the second polarizing beam splitter 8 and then transmitted through the second polarizing beam splitter 8 to enter the outgoing light path 17. The second polarized light is incident on the second polarizing beam splitter 8 and then reflected by the second polarizing beam splitter 8 to enter the outgoing light path 17.

[0103] In addition, such as Figure 2 As shown, a third reflecting mirror 16 is also provided on the outgoing light path 17. The third reflecting mirror 16 is located between the second polarizing beam splitter 8 and the lens 9. The third reflecting mirror 16 is used to deflect the propagation direction of the polarized light from the second polarizing beam splitter 8 by 90 degrees, so that it can pass through the lens 9, the liquid crystal variable retarder 10, the polarizer 11, and the light exit port 14. It should be noted that the third reflecting mirror 16 is not necessary; it can be omitted by changing the position of the light exit port 14.

[0104] In addition, such as Figure 2 As shown, an optical path compensation plate 15 is also provided on the second optical path, namely the reflected optical path 3. The distance between the grating 6 and the first polarizing beam splitter 2 is equal to the distance between the optical path compensation plate 15 and the first polarizing beam splitter 2. The material of the optical path compensation plate 15 is the same as that of the grating 6, and the thickness of the optical path compensation plate 15 is the same as that of the grating 6. When the grating 6 is a glass substrate, the optical path compensation plate 15 can be a transparent glass sheet.

[0105] By adding an optical path compensation plate 15, the optical path lengths of the first and second optical paths can be made completely consistent, thereby improving the lighting quality.

[0106] Example 3

[0107] This embodiment relates to a fluorescence wide-field microscope. The fluorescence wide-field microscope includes a light source (especially a non-laser light source), an optical fiber, a microscope body, and a light processing device. The light-incident surface of the optical fiber is connected to the non-laser light source, and the light-exit surface of the optical fiber is connected to the light processing device. The outgoing light path 17 of the light processing device passes through the light source inlet of the microscope body, so that the light emitted from the light source can enter the microscope body through the optical fiber and the light processing device. Furthermore, the microscope body has an image sensor for acquiring images of the sample.

[0108] As mentioned above, structured light or uniform light can be incident into the light source inlet through the light processing device, so that the image sensor can obtain structured light illumination images or uniform light illumination images.

[0109] For example, the light source can be a mercury lamp, a metal halide lamp, or an LED.

[0110] Example 4

[0111] This embodiment relates to an imaging method that can be applied to the fluorescence wide-field microscope of Example 3.

[0112] Figure 3 A flowchart of the imaging method according to Embodiment 4 is shown. Figure 3 As shown, the imaging methods include:

[0113] Step 310: Receive the structured light illumination image acquired by the image sensor at a first moment, wherein, at the first moment, first polarized light passes through the polarization light selection component to illuminate the sample, and second polarized light is blocked by the polarization light selection component;

[0114] Step 320: Receive a uniform illumination image acquired by the image sensor at a second time, which is different from the first time, wherein, at the second time, second polarized light passes through the polarization light selection component to illuminate the sample, and first polarized light is blocked by the polarization light selection component;

[0115] Step 330: Process the structured light illumination image and the uniform light illumination image using the HiLo algorithm to obtain the HiLo image.

[0116] It should be noted that the order of steps 310 and 320 can be arbitrarily interchanged. That is, as long as the structured light illumination image and the uniform light illumination image are obtained before step 330, the order in which the structured light illumination image and the uniform light illumination image are obtained will not affect the execution of step 330.

[0117] Furthermore, the HiLo algorithm is a publicly available technology in this field and will not be described in detail here.

[0118] Specifically, in one example, to obtain the green fluorescent protein (GFP) channel of a sample, then:

[0119] For step 310, the first voltage of the GFP channel is determined to be 2.6V according to the wavelength-voltage lookup table. The controller applies a voltage of 2.6V to the liquid crystal variable delay unit 10, so that the structured light can be incident from the light outlet 14 into the interior of the microscope body, and the image sensor acquires the structured light illumination image.

[0120] For step 320, the second voltage of the GFP channel is determined to be 10V according to the wavelength-voltage lookup table. The controller applies a voltage of 10V to the liquid crystal variable delay unit 10, so that uniform light can enter the microscope body from the light outlet 14 and the image sensor acquires the uniform light illumination image.

[0121] For step 330, the HiLo algorithm is used to process the structured light illumination image and the uniform light illumination image to obtain the HiLo image.

[0122] If it is necessary to obtain a three-dimensional image of the sample in this field of view, steps 310 to 330 can be repeated after selecting different depths when adjusting the focal plane. This will obtain a series of HiLo images of different layers of the sample. Stacking these images together will yield a high-contrast three-dimensional image of the sample.

[0123] If you need to capture a high-contrast image of the Red Fluorescent Protein (RFP) channel, simply find the first and second voltages of the RFP channel using the wavelength-voltage lookup table, and then repeat steps 310 to 330.

[0124] Example 5

[0125] This embodiment relates to a computer-readable storage medium, which includes a stored program, wherein the program executes the imaging method of embodiment 4 when it runs.

[0126] Example 6

[0127] This embodiment relates to an electronic device, including a memory and a processor. The memory stores a computer program, and the processor is configured to execute the imaging method of Embodiment 4 through the computer program.

[0128] Based on the above description of the implementation methods, those skilled in the art will clearly understand that this disclosure can be implemented using software and necessary general-purpose hardware, and of course, it can also be implemented using hardware, but in many cases the former is a better implementation method. Based on this understanding, the technical solution of this disclosure, in essence, or the part that contributes to the prior art, can be embodied in the form of a software product. This computer software product can be stored in a computer-readable storage medium, such as a computer floppy disk, read-only memory (ROM), random access memory (RAM), flash memory, hard disk, or optical disk, etc., including several instructions to cause a computer device (which may be a personal computer, server, or network device, etc.) to execute the methods described in this disclosure.

[0129] Although this disclosure has been described in detail above with general descriptions, specific embodiments, and experiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, such modifications or improvements made without departing from the spirit of this disclosure are all within the scope of protection claimed by this disclosure.

Claims

1. A light processing apparatus, characterized in that, include: The first polarizing beam splitter (2) is used to split the incident light into a first polarized light and a second polarized light with polarization directions perpendicular to each other. The first polarized light enters the first optical path, and the second polarized light enters the second optical path, which is different from the first optical path. A grating (6) is located in the first optical path so that the first polarized light is modulated into structured light after passing through the grating (6); The second polarizing beam splitter (8) is used to combine the first polarized light modulated by the grating (6) and the second polarized light not modulated by the grating (6) into the outgoing optical path. A polarization light selection component, located in the outgoing optical path, is used to selectively allow either the first polarized light or the second polarized light to pass through.

2. The light processing apparatus according to claim 1, characterized in that, The optical processing device further includes: A collimating lens (1) is located upstream of the first polarizing beam splitter (2).

3. The light processing apparatus according to claim 1, characterized in that, One of the first optical path and the second optical path is a reflective optical path (3), and the other of the first optical path and the second optical path is a transmittance optical path (4); A first set of reflectors is provided in the reflected light path (3), and the polarized light in the reflected light path (3) is guided to the second polarizing beam splitter (8) after being reflected by the first set of reflectors. A second set of reflectors is provided in the transmission optical path (4). The polarized light in the transmission optical path (4) is guided to the second polarizing beam splitter (8) after being reflected by the second set of reflectors; or the polarized light in the transmission optical path (4) is guided directly to the second polarizing beam splitter (8) without being reflected.

4. The light processing apparatus according to claim 1, characterized in that, The optical processing device further includes: An optical path compensation plate (15) is located in the second optical path. The distance between the grating (6) and the first polarizing beam splitter (2) is equal to the distance between the optical path compensation plate (15) and the first polarizing beam splitter (2). The material of the optical path compensation plate (15) is the same as that of the grating (6), and the thickness of the optical path compensation plate (15) is the same as that of the grating (6).

5. The light processing apparatus according to claim 1, characterized in that, The optical processing device further includes: The lens (9) is located in the outgoing light path, and the optical path between the grating (6) and the lens (9) is equal to the focal length of the lens (9).

6. The light processing apparatus according to claim 1, characterized in that, The optical processing device further includes: The outer casing (12) has a light inlet (13) and a light outlet (14), wherein the light inlet (13) is used to introduce the incident light, and the outgoing light path passes through the light outlet (14). Among them, at least the first polarizing beam splitter (2), the grating (6), and the second polarizing beam splitter (8) are located inside the housing (12).

7. The light processing apparatus according to any one of claims 1 to 6, characterized in that, The polarization selection component includes: A liquid crystal variable delay device (10) is provided, wherein the angle between the optical axis of the liquid crystal variable delay device (10) and the polarization direction of the first polarized light is 45 degrees, or the angle between the optical axis of the liquid crystal variable delay device (10) and the polarization direction of the second polarized light is 45 degrees. A controller, coupled to the liquid crystal variable delay device (10), is used to control the voltage of the liquid crystal variable delay device (10) to selectively adjust the polarization state of the first polarized light and the second polarized light; A polarizer (11) is located downstream of the liquid crystal variable delay unit (10), and the polarization axis of the polarizer (11) is aligned with the first polarized light or the second polarized light that has not passed through the liquid crystal variable delay unit (10).

8. The light processing apparatus according to claim 7, characterized in that, When the controller applies a first voltage to the liquid crystal variable delay unit (10), the liquid crystal variable delay unit (10) can simultaneously rotate the polarization states of the first polarized light and the second polarized light by 90 degrees. When the controller applies a second voltage different from the first voltage to the liquid crystal variable delay device (10), the liquid crystal variable delay device (10) does not change the polarization state of the first polarized light and the second polarized light.

9. A fluorescence wide-field microscope, characterized in that, include: Non-laser light source; An optical fiber, wherein the light incident surface of the optical fiber is connected to the non-laser light source; The microscope body has a light source inlet and an image sensor, the image sensor being used to acquire images of the sample; The optical processing apparatus according to any one of claims 1 to 8, wherein the incident light is provided by the emitting surface of the optical fiber, and the emitted light path passes through the light source inlet.

10. An imaging method, applied to the fluorescence wide-field microscope according to claim 9, characterized in that, include: The image sensor receives a structured light illumination image acquired at a first moment, wherein at the first moment, the first polarized light passes through the polarization light selection component to illuminate the sample, and the second polarized light is blocked by the polarization light selection component. Receive a uniformly illuminated image acquired by the image sensor at a second time different from the first time, wherein, at the second time, the second polarized light passes through the polarization light selection component to illuminate the sample, and the first polarized light is blocked by the polarization light selection component; The HiLo algorithm is used to process the structured light illumination image and the uniform light illumination image to obtain a HiLo image.

11. A computer-readable storage medium, characterized in that, The computer-readable storage medium includes a stored program, wherein the program, when executed, performs the imaging method of claim 10.

12. An electronic device, characterized in that, It includes a memory and a processor, wherein the memory stores a computer program and the processor is configured to execute the imaging method of claim 10 through the computer program.

Images