A digital confocal imaging system and method based on digital micromirror

Abstract

The present invention discloses a digital confocal imaging system and method based on a digital micro-mirror. The digital confocal imaging system includes a laser module, a digital micro-reflector module, an objective lens, a dichroic mirror, and a camera. The digital micro-reflector module includes a digital micro-reflector and a controller. The digital micro-reflector includes a plurality of micro-reflector units arranged in an array. The controller is connected to and used to control each micro-reflector unit on the digital micro-reflector to be set to an "on" or "off" state. Laser light emitted by the laser module irradiates the digital micro-reflector. Each micro-reflector unit in the "on" state reflects the laser light to focus on the dichroic mirror. After reflection, the laser light is irradiated onto a sample to be tested through the objective lens. The area on the sample to be tested that is illuminated by the laser emits fluorescence that is irradiated onto the dichroic mirror through the objective lens. After passing through the objective lens, the fluorescence is focused and received by the camera. The present invention significantly improves the temporal resolution of the imaging system and greatly enhances the imaging quality.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescence confocal microscopy, and in particular to a digital confocal imaging system and method based on a digital microscope. Background Art

[0002] In the study of biological cell structures and the interactions between tissues, it is difficult to distinguish different cell structures using conventional wide-field microscopes due to the interconnected and overlapping nature of various biological structures. Therefore, fluorescence microscopy has emerged as a necessary step (Jahr W, Schmid B, Schmied C, et al. Hyperspectral light sheet microscopy [J]. Nature Communications, 2015.). Fluorescence microscopy involves labeling different biological structures in a sample with different fluorescent dyes. Illuminating the sample with light of a specific wavelength causes the different fluorescent dyes to emit fluorescence at different wavelengths upon excitation. Using a spectrometer, images of different spectral bands can be obtained, allowing the identification of different cell structures (Cranfill PJ, Sell BR, Baird MA, et al. Quantitative assessment of fluorescent proteins [J]. Nature Methods.).

[0003] When the fluorescent sample is thick, under widefield illumination, the entire sample is illuminated simultaneously, and light from out-of-focus planes is also collected by the camera, resulting in blurred and divergent images of multiple cell structures in the specimen. Ultimately, the resolution of the microscopic image obtained by widefield illumination fluorescence microscopy is low (Wood SR, Kirkham J, Marsh PD, et al. Architecture of Intact Natural Human Plaque Biofilms Studied by Confocal Laser Scanning Microscopy [J]. Journal of Dental Research, 2000, 79 (1): 21-27.). In order to eliminate the interference of out-of-focus background in the sample, confocal microscopy is required.

[0004] Conventional confocal microscopes can typically only capture fluorescence information from a single point in three-dimensional space at a time. To obtain a confocal image of the entire image, the entire image must be scanned point by point. However, since only one point can be scanned at a time, the scanning speed is not fast enough, resulting in insufficient temporal resolution when imaging rapidly changing living cells, making it impossible to capture key dynamic changes.

[0005] The disclosure of the above background technology content is only used to assist in understanding the concept and technical solution of the present invention. It does not necessarily belong to the prior art of this patent application. In the absence of clear evidence that the above content has been disclosed on the filing date of this patent application, the above background technology should not be used to evaluate the novelty and creativity of this application. Summary of the Invention

[0006] To solve the above technical problems, the present invention proposes a digital confocal imaging system and method based on a digital microscope, which not only improves the scanning speed of the confocal microscope to significantly improve the temporal resolution of the imaging system, but also reduces the loss of fluorescence signals to greatly improve the imaging quality.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The present invention discloses a digital confocal imaging system based on a digital micro-reflector, comprising a laser module, a digital micro-reflector module, an objective lens, a dichroic mirror and a camera.

[0009] The digital micro-reflection module includes a digital micro-reflector and a controller. The digital micro-reflector includes a plurality of micro-reflection units arranged in an array. Each of the micro-reflection units can be deflected at a preset angle around a diagonal line. The controller is connected to and used to control the deflection angle of each micro-reflection unit on the digital micro-reflector so that each micro-reflection unit can be placed in an "on" or "off" state respectively.

[0010] The laser emitted by the laser module irradiates the digital micro-mirror. Each micro-reflection unit in the digital micro-mirror that is in the "on" state reflects the laser to focus and irradiate the dichroic mirror. After being reflected by the dichroic mirror, the laser passes through the objective lens and irradiates the sample to be tested. The area on the sample to be tested that is illuminated by the laser emits fluorescence that passes through the objective lens and irradiates the dichroic mirror. The fluorescence passes through the dichroic mirror and is then focused to be received by the camera.

[0011] Preferably, the digital confocal imaging system further includes a first tube lens, a second tube lens and a third tube lens, the first tube lens is arranged between the laser module and the digital micro-reflector, the second tube lens is arranged between the digital micro-reflector and the dichroic mirror, and the third tube lens is arranged between the dichroic mirror and the camera.

[0012] Preferably, the digital micro-reflector is located at one focal length of the first tube lens and one focal length of the second tube lens, and the objective lens and the camera are respectively located at one focal length of the third tube lens.

[0013] Preferably, the distances between the second tube lens and the third tube lens and the dichroic mirror are equal.

[0014] Preferably, the dichroic mirror is located at the midpoint between the third tube lens and the objective lens.

[0015] Preferably, the camera comprises a plurality of detection units arranged in an array, wherein the plurality of detection units correspond to the plurality of micro-reflection units in a one-to-one manner.

[0016] Preferably, the detection unit and the micro-reflection unit have the same size.

[0017] Preferably, the sizes of the detection unit and the micro-reflection unit are both 4-8 μm.

[0018] The present invention also discloses a digital confocal imaging method based on a digital micro-mirror, which uses the above-mentioned digital confocal imaging system to perform digital confocal imaging on a sample to be measured, comprising the following steps:

[0019] A1: Start the laser module;

[0020] A2: controlling, by the controller, the first group of micro-reflective units in the digital micro-mirror to be in an "on" state, wherein n micro-reflective units are spaced between every two adjacent micro-reflective units in the plurality of micro-reflective units included in the first group of micro-reflective units, where n is a natural number greater than 1;

[0021] A3: collecting the first set of fluorescence data on the camera;

[0022] A4: controlling the digital micro-mirror by the controller so that all the micro-reflective units currently in the “on” state are translated by one unit, so that the i-th group of micro-reflective units in the digital micro-mirror is in the “on” state, where i is a natural number greater than 1;

[0023] A5: collecting the i-th set of fluorescence data on the camera;

[0024] A6: Repeat steps A4 and A5 until all micro-reflective units are scanned;

[0025] A7: Splice the fluorescence data of each group together to obtain a fluorescence confocal image of the sample to be tested.

[0026] Preferably, the camera includes a plurality of detection units arranged in an array, wherein the plurality of detection units correspond one-to-one to the plurality of micro-reflection units, wherein in steps A3 and A5, the camera collects fluorescence data on the detection units corresponding to the first group and the i-th group of micro-reflection units, respectively.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows: the digital confocal imaging system and method based on digital micromirrors proposed in the present invention, on the one hand, utilize digital micromirrors as "illumination pinholes", and at the same time, by extracting the signals of specific detection units, realize the role of "detection pinholes" in a digital manner, eliminate the interference of out-of-focus background, and achieve the confocal effect; on the other hand, the fluorescence does not need to pass through the digital micromirror in the light path, thereby avoiding the loss of fluorescence signals caused by the digital micromirror, improving the signal-to-noise ratio of the image, and greatly improving the imaging quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is an optical path diagram of a digital confocal imaging system based on a digital micromirror proposed in a preferred embodiment of the present invention;

[0029] Figure 2 for Figure 1 Schematic diagram of a digital micromirror as an "illumination pinhole" in a digital confocal imaging system;

[0030] Figure 3 for Figure 1 Schematic diagram of the fluorescence optical path of the digital confocal imaging system;

[0031] Figure 4 Schematic diagram of digital micromirror dot matrix scanning. DETAILED DESCRIPTION

[0032] The following is a detailed description of the embodiments of the present invention. It should be emphasized that the following description is only exemplary and is not intended to limit the scope of the present invention and its application.

[0033] It should be noted that when an element is referred to as being "fixed to" or "disposed on" another element, it can be directly on the other element or indirectly on the other element. When an element is referred to as being "connected to" another element, it can be directly connected to the other element or indirectly connected to the other element. In addition, connection can be used for both fixing and circuit / signal communication.

[0034] It should be understood that the terms "length", "width", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", etc., indicating the orientation or position relationship, are based on the orientation or position relationship shown in the accompanying drawings, and are only for the convenience of describing the embodiments of the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operate in a specific orientation, and therefore cannot be understood as limiting the present invention.

[0035] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of such features. In the description of the embodiments of the present invention, "plurality" means two or more, unless otherwise specifically defined.

[0036] like Figure 1 As shown, a digital confocal imaging system based on a digital microscope disclosed in a preferred embodiment of the present invention includes a sample 1, an objective lens 2, a dichroic mirror 3, a tube lens 4, an sCMOS camera 5, a tube lens 6, a digital micromirror 7, a tube lens 8, and a laser 9. The sample 1 is located in the focal plane of the objective lens 2. The distance between the objective lens 2 and the tube lens 4 is one-fold the focal length of the tube lens 4. The dichroic mirror 3 is located at the midpoint between the tube lens 4 and the objective lens 2. The distance between the tube lens 6 and the tube lens 4 from the dichroic mirror 3 is equal. The distance between the sCMOS camera 5 and the tube lens 4 is one-fold the focal length of the tube lens 4. The distance between the digital micromirror 7 and the tube lens 6 is also one-fold the focal length of the tube lens 6. The distance between the tube lens 8 and the digital micromirror 7 is one-fold the focal length of the tube lens 8. The distance between the tube lens 8 and the laser 9 is one-fold the focal length of the tube lens 8.

[0037] The digital micromirror device (DMD) is a microelectromechanical system with electronic input and optical output. It consists of numerous small aluminum reflective mirrors, each of which is called a pixel. Each mirror can deflect ±17° around the diagonal of each positive mirror (or pixel). This means the DMD's micromirrors have three stable states: +17°, 0°, and -17°. The size of each micromirror is 5.4 μm, and the micromirror array consists of 1920 × 1080 micromirrors. Each micromirror has three stable states: +17° (on), 0° (no signal), and -17° (off). When a signal "1" is applied to a micromirror, it deflects +17°, and the reflected light passes through the projection lens exactly along the optical axis and forms an image on the screen, forming a bright pixel. When the mirror deviates from its equilibrium position by -17 degrees (a signal "0"), the reflected light cannot pass through the projection lens, resulting in a dark pixel. The binary "1" and "0" states of the control signal correspond to the "on" and "off" states of the micromirror respectively; when a given graphic data control signal sequence is written into the CMOS circuit, the incident light is modulated by the DMD, and the graphic can be displayed on the image plane.

[0038] The core of a confocal microscope consists of a pair of "illumination pinholes" and "detection pinholes." The "illumination pinhole" ensures that the laser light passing through it illuminates only the sample in the focal plane, while out-of-focus samples are not illuminated. The "detection pinhole" allows only fluorescence emitted from the focal plane to pass through, while out-of-focus samples are not. In traditional confocal microscopes, the "illumination pinhole" and "detection pinhole" are usually separate, but they work together through a synchronization program. In a multi-point digital confocal system based on a digital micromirror, there is a pair of dual optical paths: the tube lens 4 and sCMOS camera 5 correspond to the tube lens 6 and digital micromirror 7, respectively. Tube lenses 4 and 6 are identical and are located at equal distances from the dichroic mirror 3. The minimum resolution unit of the digital micromirror 7 is equal in size to the minimum resolution unit of the sCMOS camera 5, and each minimum resolution unit has a one-to-one correspondence. This means that fluorescence emitted by a sample illuminated by a specific microreflector unit of the digital micromirror 7 will only be detected by a specific microdetector unit of the corresponding sCMOS camera 5. The digital micromirror 7 is used as an "illumination pinhole" to illuminate the sample 1 in the focal plane. The fluorescence emitted by the sample 1 is detected by the micro-detection array of the sCMOS camera 5. By extracting the signal of the specific detection unit corresponding to the "illumination pinhole" of the digital micromirror 7 (the signal of the focal plane) and discarding the signals of other detection units (the defocused background signal), the defocused background is eliminated through a digital method, thus realizing the role of the "detection pinhole".

[0039] like Figure 2 As shown, the laser light emitted by the laser 9 hits the digital micro-mirror 7. At this time, the control program controls the digital micro-mirror 7 to turn on a micro-reflection unit every n units, as shown in FIG. Figure 4 As shown, the micro-reflection unit in the "on" state reflects the laser light onto the dichroic mirror 3 and reflects it again, and finally illuminates the focal plane of the sample 1. At this time, the role of the micro-reflection unit in the "on" state of the digital micro-reflection mirror 7 is equivalent to that of an "illumination pinhole".

[0040] like Figure 3 As shown, the focal plane of sample 1 illuminated by the laser emits fluorescence. Due to the principle of reversible optical paths, the emitted fluorescence passes through objective lens 2 and reaches the same location illuminated by the laser on dichroic mirror 3. It then converges through tube lens 4 onto sCMOS camera 5. Digital processing then extracts only the signals from the corresponding detection units on the detection array of sCMOS camera 5, achieving digital confocal imaging. Fluorescence emitted from the focal plane of sample 1 only needs to pass through objective lens 2 to reach dichroic mirror 3 and then through tube lens 4 to converge onto sCMOS camera 5. Therefore, the optical path of the fluorescence does not include digital micromirror 7, thus preventing loss of fluorescence signal by digital micromirror 7.

[0041] This technology uses a digital micromirror as an "illumination pinhole" and, by extracting signals from specific detection units, digitally implements the "detection pinhole" function, eliminating interference from out-of-focus backgrounds and achieving a confocal effect. Furthermore, the digital micromirror can scan multiple points at once, significantly improving scanning speed compared to traditional single-point scanning methods.

[0042] A preferred embodiment of the present invention discloses a digital confocal imaging method based on a digital microscope, using the above-mentioned digital confocal imaging system based on a digital microscope, and the specific steps are as follows:

[0043] Step 1: If Figure 2 As shown, turn on the laser 9;

[0044] Step 2: If Figure 4 As shown, the digital micro-mirror 4 is controlled by a control program so that a micro-reflection unit is in an "on" state every n units;

[0045] Step 3: Collect the fluorescence data on the detection unit corresponding to the first group of dot arrays on the sCMOS camera 5, and set the signals of other detection units to 0;

[0046] Step 4: Control the digital micro-mirror 7 through the control program so that all units in the "on" state are translated by one unit;

[0047] Step 5: Collect the fluorescence data on the detection unit corresponding to the second set of dot arrays on the sCMOS camera 5, and set the signals of other detection units to 0;

[0048] Step 6: Repeat steps 4 and 5, sequentially shifting the micro-reflection units in the "on" state, and recording and storing the corresponding fluorescence data until all micro-reflection units have been scanned;

[0049] Step 7: Stitch together the fluorescence data of all the dots to obtain a fluorescence confocal image of the entire field of view.

[0050] In a specific embodiment, the objective lens 2 is a 40x magnification objective lens, and the sample 1 is in the focal plane of the 40x magnification objective lens; the dichroic mirror 3 is located at the midpoint between the tube lens 4 and the objective lens 2, reflecting light with a wavelength of 488 nm and transmitting light with a wavelength other than 488 nm; the tube lens 4 and the tube lens 6 are identical, with a focal length of 200 mm; the distance between the objective lens 2 and the tube lens 4 is 200 mm, which is one times the focal length of the tube lens 4; the number of pixels of the digital micromirror 7 is 1920×1080, and the minimum resolution unit is 1000 nm. The size is 5.4 μm, and the distance between the digital micro-mirror 7 and the tube lens 6 is 200 mm, which is one times the focal length of the tube lens 6; the number of pixels of the sCMOS camera 5 is 1920×1080, the minimum resolution unit size is 5.4 μm, and the distance between the sCMOS camera 5 and the tube lens 4 is 200 mm, which is one times the focal length of the tube lens 4; the distance between the tube lens 8 and the digital micro-mirror 7 is 200 mm, which is one times the focal length of the tube lens 8; and the distance between the tube lens 8 and the laser 9 is 200 mm, which is one times the focal length of the tube lens 8.

[0051] The digital confocal imaging system and method based on a digital microscope provided by the preferred embodiment of the present invention can scan multiple points at a time, thereby improving the scanning speed and achieving rapid scanning. Among them, the digital confocal imaging system uses a digital micro-mirror as an "illumination pinhole" and, by extracting the signal of a specific detection unit, realizes the function of the "detection pinhole" in a digital manner, eliminates the interference of the out-of-focus background, and achieves a confocal effect; compared with the traditional single-point scanning method, the digital micro-mirror can scan multiple points at a time, thereby improving the scanning speed. In addition, the fluorescence signal in this embodiment will not be reflected by the digital micro-mirror, which avoids the loss of the fluorescence signal and improves the signal-to-noise ratio of the image.

[0052] The background section of the present invention may contain background information about the problem or environment of the present invention rather than describing prior art by others. Therefore, the inclusion of content in the background section is not an admission by the applicant that the prior art is available.

[0053] The above description further details the present invention in conjunction with specific / preferred embodiments, and the specific implementation of the present invention should not be construed as being limited to these descriptions. Persons skilled in the art will appreciate that, without departing from the spirit of the present invention, they may make various substitutions or modifications to the described embodiments, and these substitutions or modifications should be considered to fall within the scope of protection of the present invention. Throughout this specification, reference to terms such as "one embodiment," "some embodiments," "preferred embodiments," "examples," "specific examples," or "some examples" indicates that the specific features, structures, materials, or characteristics described in conjunction with such embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of these terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, those skilled in the art may combine and assemble the different embodiments or examples described in this specification, as well as features from different embodiments or examples, without conflicting opinions. Although the embodiments of the present invention and their advantages have been described in detail, it should be understood that various changes, substitutions, and modifications may be made herein without departing from the scope of the appended claims.

Claims

1. A digital confocal imaging system based on a digital micromirror, characterized in that: Including laser module, digital micro-reflection module, objective lens, dichroic mirror and camera, second tube lens, third tube lens, The digital micro-reflection module includes a digital micro-reflector and a controller. The digital micro-reflector includes a plurality of micro-reflection units arranged in an array. Each of the micro-reflection units can be deflected at a preset angle around a diagonal line. The controller is connected to and used to control the deflection angle of each micro-reflection unit on the digital micro-reflector so that each micro-reflection unit can be placed in an "on" or "off" state. The laser light emitted by the laser module is irradiated onto the digital micro-mirror. Each micro-reflection unit in the digital micro-mirror that is in an "on" state reflects the laser light to focus and irradiate the dichroic mirror. After being reflected by the dichroic mirror, the laser light passes through the objective lens and irradiates the sample to be tested. The portion of the sample to be tested that is illuminated by the laser light emits fluorescence that passes through the objective lens and irradiates the dichroic mirror. The fluorescence passes through the dichroic mirror and is then focused to be received by the camera. The second tube lens is arranged between the digital micro-reflector and the dichroic mirror, and the third tube lens is arranged between the dichroic mirror and the camera. The digital confocal imaging system has a pair of dual optical paths, and the third tube lens and the camera in the optical path correspond to the second tube lens and the digital micro-reflector respectively. The distances between the second tube lens and the third tube lens and the dichroic mirror are equal, and the camera includes a plurality of detection units arranged in an array, and the plurality of detection units correspond one-to-one to the plurality of micro-reflection units.

2. The digital confocal imaging system according to claim 1, wherein: The system further includes a first tube lens, which is arranged between the laser module and the digital micro-reflector.

3. The digital confocal imaging system according to claim 2, characterized in that: The digital micro-reflector is located at one focal length of the first tube lens and one focal length of the second tube lens, and the objective lens and the camera are respectively located at one focal length of the third tube lens.

4. The digital confocal imaging system according to claim 1, wherein: The dichroic mirror is located at a midpoint between the third tube lens and the objective lens.

5. The digital confocal imaging system according to claim 1, wherein: The detection unit and the micro-reflection unit have the same size.

6. The digital confocal imaging system according to claim 5, characterized in that: The sizes of the detection unit and the micro-reflection unit are both 4-8 μm.

7. A digital confocal imaging method based on a digital micromirror, characterized in that: The digital confocal imaging system according to any one of claims 1 to 6 is used to perform digital confocal imaging on a sample to be tested, comprising the following steps: A1: Start the laser module; A2: controlling, by the controller, the first group of micro-reflective units in the digital micro-mirror to be in an "on" state, wherein n micro-reflective units are spaced between every two adjacent micro-reflective units in the first group of micro-reflective units, where n is a natural number greater than 1; A3: collecting the first set of fluorescence data on the camera; A4: controlling the digital micro-mirror by the controller so that all the micro-reflective units currently in the "on" state are translated by one unit, so that the i-th group of micro-reflective units in the digital micro-mirror is in the "on" state, where i is a natural number greater than 1; A5: collecting the i-th set of fluorescence data on the camera; A6: Repeat steps A4 and A5 until all micro-reflective units are scanned; A7: Splice the fluorescence data of each group together to obtain a fluorescence confocal image of the sample to be tested.

8. The digital confocal imaging method according to claim 7, characterized in that: The camera includes a plurality of detection units arranged in an array, wherein the plurality of detection units correspond one to one with the plurality of micro-reflection units, wherein in steps A3 and A5, the camera collects fluorescence data on the detection units corresponding to the first group and the i-th group of micro-reflection units, respectively.

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