CN114577762A - Digital confocal imaging system and method based on digital micro-reflector - Google Patents
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Abstract
The invention discloses a digital confocal imaging system and a digital confocal imaging method based on a digital micro-reflector, wherein the digital confocal imaging system comprises a laser module, a digital micro-reflection module, an objective lens, a dichroic mirror and a camera, the digital micro-reflection module comprises the digital micro-reflector and a controller, the digital micro-reflector comprises a plurality of micro-reflection units which are arranged in an array manner, and the controller is connected with and used for controlling each micro-reflection unit on the digital micro-reflector to be respectively placed in an 'on' state or an 'off' state; laser emitted by the laser module irradiates the digital micro-reflector, each micro-reflection unit arranged in an 'on' state reflects the laser to irradiate the laser to a dichroic mirror in a focusing manner, the reflected laser irradiates a sample to be detected through the objective lens, fluorescence emitted by a laser-illuminated place on the sample to be detected irradiates the dichroic mirror through the objective lens, and the fluorescence is focused to be received by the camera after penetrating. The invention obviously improves the time resolution of the imaging system and also greatly improves the imaging quality.
Description
Technical Field
The invention relates to the technical field of fluorescence confocal microscopic imaging, in particular to a digital confocal imaging system and method based on a digital microscope.
Background
In the course of studying biological cell structures and tissue interactions, fluorescence microscopy techniques have been developed because it is difficult to distinguish between different cell structures under a conventional wide field microscope due to the interconnection and overlap of various biological structures (Jahr W, Schmid B, Schmied C, et al. Hyperspectral light sheet microscopy [ J ]. Nature Communications, 2015.). Fluorescence microscopy refers to labeling different biological structures of a sample with different fluorescent dyes, irradiating the sample with light with a specific wavelength, exciting the different fluorescent dyes to emit fluorescence with different wavelengths, and obtaining images of different spectral bands by a light-splitting element, so as to distinguish different cell structures (Cranfill P J, Sell B R, Baird MA, et al.
When the fluorescent sample is thick, in the case of wide field illumination, the resolution of the microscopic image obtained by the wide field illuminated fluorescence microscope is low (Wood S R, Kirkham J, Marsh P D, et al. architecture of Integrated Natural Human plasma biological filmstudy Confocal Laser Scanning [ J ]. Journal of dental Research,2000,79(1):21-27.) because the entire sample is illuminated simultaneously and the light from the out-of-focus plane is also collected by the camera, thus blurring and diverging the image of the multiple cellular structures in the sample. In order to eliminate the interference of the out-of-focus background in the sample, confocal microscopy is used.
Conventional confocal microscopes can only acquire fluorescence information of one point in three-dimensional space at one time, and scanning the whole image point by point is required to acquire a confocal image of the whole image. However, since only one point can be scanned at a time, the scanning speed is not fast enough, which makes the time resolution not high enough to capture the critical dynamic change when the living cell biology with fast change is imaged.
The above background disclosure is only for the purpose of assisting understanding of the concept and technical solution of the present invention and does not necessarily belong to the prior art of the present patent application, and should not be used for evaluating the novelty and inventive step of the present application in the case that there is no clear evidence that the above content is disclosed at the filing date of the present patent application.
Disclosure of Invention
In order to solve the above technical problems, the present invention provides a digital confocal imaging system and method based on a digital microscope, which not only increases the scanning speed of the confocal microscope to significantly improve the time resolution of the imaging system, but also reduces the loss of fluorescence signals to greatly improve the imaging quality.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention discloses a digital confocal imaging system based on a digital micro-reflector, which comprises a laser module, a digital micro-reflector module, an objective lens, a dichroic mirror and a camera,
the digital micro-reflection module comprises a digital micro-mirror and a controller, the digital micro-mirror comprises a plurality of micro-reflection units arranged in an array, each micro-reflection unit can deflect a preset angle around a diagonal line, and the controller is connected with and used for controlling the deflection angle of each micro-reflection unit on the digital micro-mirror so as to enable each micro-reflection unit to be respectively placed in an 'on' state or an 'off' state;
the laser module emits laser to irradiate the digital micro-reflector, each micro-reflection unit in the digital micro-reflector in an 'on' state reflects the laser to focus the laser to irradiate the dichroic mirror, the laser is irradiated to a sample to be detected through the objective lens after being reflected on the dichroic mirror, fluorescence emitted by a position, illuminated by the laser, of the sample to be detected is irradiated to the dichroic mirror through the objective lens, and the fluorescence penetrates through the dichroic mirror and then is focused to be received by the camera.
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 disposed between the laser module and the digital micro-mirror, the second tube lens is disposed between the digital micro-mirror and the dichroic mirror, and the third tube lens is disposed between the dichroic mirror and the camera.
Preferably, the digital micro-mirror is located at a focal length of one of the first tube lens and a focal length of one of the second tube lens, and the objective lens and the camera are located at a focal length of one of the third tube lens, respectively.
Preferably, the distances between the second tube lens and the third tube lens to the dichroic mirror are equal.
Preferably, the dichroic mirror is located at a midpoint position of the third tube lens and the objective lens.
Preferably, the camera comprises a plurality of detection units arranged in an array, wherein the plurality of detection units and the plurality of micro reflection units correspond to each other one by one.
Preferably, the detection unit and the micro-reflection unit have the same size.
Preferably, the size of each of the detection unit and the micro reflection unit is 4-8 μm.
The invention also discloses a digital confocal imaging method based on the digital micro-reflector, which adopts the digital confocal imaging system to carry out digital confocal imaging on a sample to be detected and comprises the following steps:
a1: starting the laser module;
a2: controlling 1 st group of micro reflection units in the digital micro-mirror to be in an 'on' state through the controller, wherein n micro reflection units are arranged between every two adjacent micro reflection units in a plurality of micro reflection units contained in the 1 st group of micro reflection units, and n is a natural number greater than 1;
a3: collecting a1 st set of fluorescence data on the camera;
a4: controlling the digital micro-mirrors through the controller to enable all the micro-reflection units in the 'on' state to translate by one unit, so that the ith group of micro-reflection units in the digital micro-mirrors are in the 'on' state, and i is a natural number greater than 1;
a5: collecting an i-th set of fluorescence data on the camera;
a6: repeating the steps A4 and A5 until all the micro-reflection units are scanned once;
a7: and splicing the fluorescence data of each group to obtain a fluorescence confocal image of the sample to be detected.
Preferably, the camera comprises a plurality of detecting units arranged in an array, wherein the plurality of detecting units and the plurality of micro-reflecting units are in one-to-one correspondence, and the fluorescence data collected by the camera in steps A3 and a5 are respectively fluorescence data on the detecting units corresponding to the 1 st group and the i th group of micro-reflecting units.
Compared with the prior art, the invention has the beneficial effects that: according to the digital confocal imaging system and method based on the digital micro-reflector, on one hand, the digital micro-reflector is used as an illumination pinhole, meanwhile, the signal of a specific detection unit is extracted, the effect of the detection pinhole is realized in a digital mode, the interference of an out-of-focus background is eliminated, and a confocal effect is realized; on the other hand, the light path through which the fluorescence passes does not need to pass through the digital micro-reflector, so that the loss of the digital micro-reflector to the fluorescence signal is avoided, the signal-to-noise ratio of the image is improved, and the imaging quality is greatly improved.
Drawings
Fig. 1 is an optical path diagram of a digital confocal imaging system based on digital micro-mirrors according to a preferred embodiment of the present invention;
FIG. 2 is a schematic diagram of the digital micro-mirror as an "illumination pinhole" in the digital confocal imaging system of FIG. 1;
FIG. 3 is a schematic diagram of a fluorescence optical path of the digital confocal imaging system in FIG. 1;
fig. 4 is a schematic diagram of digital micromirror dot matrix scanning.
Detailed Description
The embodiments of the present invention will be described in detail below. It should be emphasized that the following description is merely exemplary in nature and is not intended to limit the scope of the invention or its application.
It will be understood that when an element is referred to as being "secured to" or "disposed on" another element, it can be directly on the other element or be 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 be indirectly connected to the other element. In addition, the connection may be for either a fixed function or a circuit/signal communication function.
It is to be understood that the terms "length," "width," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," and the like are used in an orientation or positional relationship indicated in the drawings for convenience in describing the embodiments of the present invention and to simplify the description, and are not intended to indicate or imply that the referenced device or element must have a particular orientation, be constructed in a particular orientation, and be in any way limiting of the present invention.
Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of the embodiments of the present invention, "a plurality" means two or more unless specifically limited otherwise.
As shown in fig. 1, the digital confocal imaging system based on a digital microscope disclosed in the 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, where the sample 1 is located at a focal plane of the objective lens 2, a distance between the objective lens 2 and the tube lens 4 is a focal length of the tube lens 4, the dichroic mirror 3 is located at a midpoint between the tube lens 4 and the objective lens 2, distances between the tube lens 6 and the dichroic mirror 3 are equal to each other, a distance between the sCMOS camera 5 and the tube lens 4 is a focal length of the tube lens 4, and a distance between the digital micromirror 7 and the tube lens 6 is a focal length of the tube lens 6. The distance between the tube lens 8 and the digital micro-reflector 7 is one focal length of the tube lens 8. The tube lens 8 is at a distance from the laser 9 that is one focal length of the tube lens 8.
Among them, the Digital Micromirror Device (DMD) is an electronic input, optical output micro electro mechanical system, which is composed of many small aluminum reflective mirrors, each of which is called a pixel. Each mirror surface can deflect +/-17 degrees around the diagonal of each small mirror (or one pixel) in the positive direction, namely the micro-mirror of the DMD has three states of +17 degrees, 0 degrees and-17 degrees, the size of a micro-reflection unit (micro-mirror) is 5.4 mu m, and the number of micro-mirror arrays is 1920 multiplied by 1080. Each micro-reflection unit has three stable states: +17 ° (on), 0 ° (no signal), -17 ° (off). When a signal '1' is given to the micro-reflection unit, the micro-reflection unit deflects by +17 degrees, and reflected light is imaged on a screen through a projection objective just along the direction of an optical axis to form a bright pixel; when the mirror is-17 degrees off equilibrium (signal "0"), the reflected beam will not pass through the projection lens and thus appear as a dark pixel. The binary states of '1' and '0' of the control signal respectively correspond to the two states of 'on' and 'off' of the micro-mirror; when a given pattern data control signal sequence is written in the CMOS circuit, the incident light is modulated by the DMD, and a pattern can be displayed on the image plane.
The focus microscope is characterized by a pair of ' illumination pinhole ' and ' detection pinhole ', wherein the illumination pinhole ' only illuminates a sample at a focal plane through laser light passing through the illumination pinhole, and the sample at an out-of-focus plane is not illuminated. The "detection pinhole" functions to allow only the fluorescence emitted from the focal plane to pass through, while the fluorescence emitted from the defocused plane does not. In conventional confocal microscopes, the "illumination pinhole" and the "detection pinhole" are usually separate and are coordinated by a synchronization procedure. A pair of dual light paths are arranged in a multipoint digital confocal system based on a digital micro-reflector, and a tube lens 4 and an sCMOS camera 5 in the light paths respectively correspond to a tube lens 6 and a digital micro-reflector 7. The tube lens 4 and the tube lens 6 are identical, and the distances from the dichroic mirror 3 are also equal. The minimum resolution cells of the digital micromirror 7 and the minimum resolution cells of the sscmos camera 5 are equal in size, and each minimum resolution cell corresponds to each other one by one, that is, the fluorescence emitted by the sample illuminated by a specific micro-reflection cell of the digital micro-reflection 7 is only detected by a specific micro-detection cell of the corresponding sscmos camera 5. The digital micro-mirror 7 is used as an illumination pinhole to illuminate the sample 1 of the focal plane, the fluorescence emitted by the sample 1 is detected by the micro-detection array of the sCMOS camera 5, and the signals (signals of the focal plane) of the specific detection unit corresponding to the illumination pinhole of the digital micro-mirror 7 are extracted, and the signals (signals of the defocusing background) of other detection units are abandoned, so that the defocusing background is eliminated through a digitization method, and the effect of the detection pinhole is realized.
As shown in fig. 2, the laser emitted from the laser 9 is applied to the digital micromirror 7, and at this time, the digital micromirror 7 is controlled by the control program every n units so that one micro-reflective unit is in an "on" state, as shown in fig. 4, the micro-reflective unit in the "on" state reflects the laser onto the dichroic mirror 3 for re-reflection, and finally illuminates the focal plane of the sample 1, and the micro-mirror in the "on" state of the digital micromirror 7 plays a role as a "pinhole illumination".
As shown in fig. 3, the sample 1 emits fluorescence where the focal plane is illuminated by the laser, and the emitted fluorescence passes through the objective lens 2 to the same position where the dichroic mirror 3 is illuminated by the laser according to the principle of reversible optical path, and then is converged onto the sCMOS camera 5 through the tube lens 4. Then, only the signals of the corresponding detection units on the detection array of the sCMOS camera 5 are extracted through digital processing, and digital confocal is realized. The fluorescence excited from the focal plane of the sample 1 only needs to pass through the objective lens 2 to reach the dichroic mirror 3, and then is converged onto the sCMOS camera 5 through the tube lens 4, so that the digital micro-mirror 7 is not included in the light path through which the fluorescence passes, and the loss of the digital micro-mirror 7 to the fluorescence signal is avoided.
The technology utilizes the digital micro-reflector as an illumination pinhole, simultaneously realizes the function of the detection pinhole in a digital mode by extracting signals of a specific detection unit, eliminates the interference of an out-of-focus background and realizes a confocal effect. Meanwhile, the digital micro-mirror can scan a plurality of points at one time, and compared with the traditional single-point scanning mode, the scanning speed is greatly improved.
The preferred embodiment of the invention discloses a digital confocal imaging method based on a digital microscope, which adopts the digital confocal imaging system based on the digital microscope and comprises the following specific steps:
step 1: as shown in fig. 2, the laser 9 is turned on;
step 2: as shown in fig. 4, the digital micromirror 4 is controlled by a control program such that every n cells make one micro-reflective cell in an "on" state;
and step 3: collecting fluorescence data on detection units corresponding to the 1 st group of dot matrixes on an sCMOS camera 5, and setting signals of other detection units as 0;
and 4, step 4: controlling the digital micro-mirror 7 by a control program to make all the units in the 'on' state translate by one unit;
and 5: collecting fluorescence data on detection units corresponding to the 2 nd group of dot matrixes on an sCMOS camera 5, and setting signals of other detection units as 0;
step 6: repeating the step 4 and the step 5, sequentially translating the micro reflection units in the 'on' state, and recording and storing the corresponding fluorescence data until all the micro reflection units are scanned once;
and 7: and splicing the fluorescence data of all the dot matrixes to obtain a fluorescence confocal image of the whole field of view.
In a specific embodiment, the objective lens 2 is a 40-times magnification objective lens, and the sample 1 is located in a focal plane of the 40-times magnification objective lens; the dichroic mirror 3 is positioned at the midpoint position of the tube lens 4 and the objective lens 2, reflects light with the wavelength of 488nm, and transmits light except the wavelength of 488 nm; the tube lens 4 and the tube lens 6 are completely the same, and the focal lengths are both 200 mm; the distance between the objective lens 2 and the tube lens 4 is 200mm which is one time of the focal length of the tube lens 4; the number of pixels of the digital micro-reflector 7 is 1920 multiplied by 1080, the size of a minimum resolution unit is 5.4 mu m, and the distance between the digital micro-reflector 7 and the tube lens 6 is 200mm which is one time of the focal length of the tube lens 6; the pixel number of the sCMOS camera 5 is 1920 multiplied by 1080, the size of a minimum resolution unit is 5.4 mu m, and the distance between the sCMOS camera 5 and the tube lens 4 is 200mm which is one time of the focal length of the tube lens 4; the distance between the tube lens 8 and the digital micro-reflector 7 is 200mm which is one time of the focal length of the tube lens 8; the distance between the tube lens 8 and the laser 9 is 200mm which is one focal length of the tube lens 8.
The digital confocal imaging system and method based on the digital microscope provided by the preferred embodiment of the invention can scan a plurality of points at one time, thereby improving the scanning speed and realizing rapid scanning. The digital confocal imaging system utilizes the digital micro-reflector as an illumination pinhole, and simultaneously realizes the function of a detection pinhole in a digital mode by extracting signals of a specific detection unit, eliminates the interference of an out-of-focus background and realizes a confocal effect; compared with the traditional single-point scanning mode, the scanning speed is improved by utilizing the digital micro-mirror to scan a plurality of points at one time. In addition, the fluorescent signal in the embodiment can not be reflected by the digital micro-reflector, so that the loss of the fluorescent signal is avoided, and the signal-to-noise ratio of the image is improved.
The background of the invention may contain background information related to the problem or environment of the present invention rather than the prior art described by others. Accordingly, the inclusion in the background section is not an admission of prior art by the applicant.
The foregoing is a more detailed description of the invention in connection with specific/preferred embodiments and is not intended to limit the practice of the invention to those descriptions. It will be apparent to those skilled in the art that various substitutions and modifications can be made to the described embodiments without departing from the spirit of the invention, and these substitutions and modifications should be considered to fall within the scope of the invention. In the description herein, references to the description of the term "one embodiment," "some embodiments," "preferred embodiments," "an example," "a specific example," or "some examples" or the like are intended to mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction. Although embodiments of the present invention and their advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the scope of the invention as defined by the appended claims.
Claims (10)
1. A digital confocal imaging system based on a digital micro-reflector is characterized by comprising a laser module, a digital micro-reflector module, an objective lens, a dichroic mirror and a camera,
the digital micro-reflection module comprises a digital micro-mirror and a controller, the digital micro-mirror comprises a plurality of micro-reflection units arranged in an array, each micro-reflection unit can deflect a preset angle around a diagonal line, and the controller is connected with and used for controlling the deflection angle of each micro-reflection unit on the digital micro-mirror so as to enable each micro-reflection unit to be respectively placed in an 'on' state or an 'off' state;
the laser module emits laser to irradiate the digital micro-reflector, each micro-reflection unit in the digital micro-reflector, which is in an 'on' state, reflects the laser to focus the laser to irradiate the dichroic mirror, the laser is irradiated on a sample to be detected through the objective lens after being reflected on the dichroic mirror, fluorescence emitted by a part, illuminated by the laser, of the sample to be detected is irradiated on the dichroic mirror through the objective lens, and the fluorescence penetrates through the dichroic mirror and then is focused to be received by the camera.
2. The digital confocal imaging system of claim 1, further comprising a first tube lens disposed between the laser module and the digital micro-mirror, a second tube lens disposed between the digital micro-mirror and the dichroic mirror, and a third tube lens disposed between the dichroic mirror and the camera.
3. The digital confocal imaging system of claim 2, wherein the digital micro-mirror is located at a focal length of the first tube lens and a focal length of the second tube lens, and the objective lens and the camera are located at a focal length of the third tube lens, respectively.
4. The digital confocal imaging system of claim 2, wherein the second tube lens and the third tube lens are equidistant from the dichroic mirror.
5. The digital confocal imaging system of claim 2, wherein the dichroic mirror is located at a midpoint of the third tube lens and the objective lens.
6. The digital confocal imaging system of any one of claims 1 to 5, wherein the camera comprises a plurality of detection units arranged in an array, wherein the plurality of detection units and the plurality of micro-reflection units correspond one to one.
7. The digital confocal imaging system of claim 6, wherein the detection unit and the micro-reflection unit are the same size.
8. The digital confocal imaging system of claim 7, wherein the detection unit and the micro-reflection unit are each 4-8 μm in size.
9. A digital confocal imaging method based on digital micro-mirrors, characterized in that the digital confocal imaging system of any one of claims 1 to 8 is used for digital confocal imaging of a sample to be measured, comprising the following steps:
a1: starting the laser module;
a2: controlling 1 st group of micro reflection units in the digital micro-mirror to be in an 'on' state through the controller, wherein n micro reflection units are arranged between every two adjacent micro reflection units in a plurality of micro reflection units contained in the 1 st group of micro reflection units, and n is a natural number greater than 1;
a3: collecting a1 st set of fluorescence data on the camera;
a4: controlling the digital micro-mirrors through the controller to enable all the micro-reflection units in the 'on' state to translate by one unit, so that the ith group of micro-reflection units in the digital micro-mirrors are in the 'on' state, and i is a natural number greater than 1;
a5: collecting an i-th set of fluorescence data on the camera;
a6: repeating the steps A4 and A5 until all the micro-reflection units are scanned once;
a7: and splicing the fluorescence data of each group to obtain a fluorescence confocal image of the sample to be detected.
10. The digital confocal imaging method of claim 9, wherein the camera comprises a plurality of detecting units arranged in an array, wherein the plurality of detecting units and the plurality of micro-reflecting units are in one-to-one correspondence, and wherein the fluorescence data collected by the camera in steps A3 and a5 are respectively fluorescence data on the detecting units corresponding to the 1 st group and the i th group of micro-reflecting units.
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