CN116661118A - Imaging method, imaging system, imaging apparatus, computer device, and storage medium - Google Patents
Imaging method, imaging system, imaging apparatus, computer device, and storage medium Download PDFInfo
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/0032—Optical details of illumination, e.g. light-sources, pinholes, beam splitters, slits, fibers
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/0052—Optical details of the image generation
- G02B21/006—Optical details of the image generation focusing arrangements; selection of the plane to be imaged
Abstract
The present application relates to an imaging method, system, apparatus, computer device, and storage medium. The method comprises the following steps: performing linear confocal imaging in the horizontal direction, linear confocal imaging in the vertical direction and wide-field imaging on a target object through a plurality of micromirrors on the micromirror chip to obtain a first imaging result, a second imaging result and a wide-field imaging result of the target object; and calculating a line scanning confocal imaging result of the target object according to the imaging result. By adopting the imaging method provided by the embodiment of the application, confocal line scanning laser irradiation imaging can be respectively carried out in the horizontal direction and the vertical direction, signals in the same visual field are sampled for multiple times in different directions, and the final imaging result is calculated according to the sampling result, so that the accuracy of image signal acquisition can be improved, the imaging speed, the time resolution and the spatial resolution can be improved, the information loss can be reduced, the signal to noise ratio can be improved, and the imaging quality of the final imaging result can be improved.
Description
Technical Field
The present application relates to the field of microscopic imaging, and in particular, to an imaging method, system, apparatus, computer device, and storage medium.
Background
With the continuous development of optical microscopy imaging technology, confocal microscopy imaging technology has emerged. Because of the high stability of confocal microscopy imaging techniques and the high adaptability to different multiple samples, confocal microscopy imaging techniques are important in the field of life science microscopy imaging.
The imaging quality of the existing confocal imaging technology is severely dependent on the size and position of the copolymer Jiao Xiao aperture: the function of removing background to improve the signal to noise ratio is reduced when the small hole is large, and the resolution is not improved at all; the small holes have low passing fluorescent signals, and the signal to noise ratio is correspondingly reduced; if the small holes are deviated, the detected fluorescence signal is greatly reduced. Therefore, in the related art, the confocal image is generally processed through various algorithms (such as a deconvolution wiener filtering algorithm, a maximum entropy algorithm and a Gerchberg-Saxton algorithm), but the quality of the confocal image calculated by the above algorithm has a larger relation with the imaging quality of the original image, and is greatly affected by noise of the original image, so that the quality of the confocal image is poor.
Disclosure of Invention
In view of the foregoing, it is desirable to provide an imaging method, apparatus, computer device, computer-readable storage medium, and computer program product that are capable of improving imaging quality.
In a first aspect, the present application provides an imaging method. The method is applied to an imaging system comprising a micromirror chip comprising a plurality of micromirrors thereon; the method comprises the following steps:
performing linear confocal imaging on a target object in the horizontal direction through a plurality of micromirrors on the micromirror chip to obtain a first imaging result of the target object;
performing linear confocal imaging on the target object in the vertical direction through a plurality of micromirrors on the micromirror chip to obtain a second imaging result of the target object;
performing wide-field imaging on the target object through a plurality of micromirrors on the micromirror chip to obtain a wide-field imaging result of the target object;
and calculating a line scanning confocal imaging result of the target object according to the first imaging result, the second imaging result and the wide-field imaging result.
In one embodiment, the imaging the target object by using the plurality of micromirrors on the micromirror chip to perform linear confocal imaging in a horizontal direction to obtain a first imaging result of the target object includes:
Sequentially determining a plurality of target rows along the horizontal direction of the micromirrors on the micromirror chip according to a preset horizontal order;
starting a micromirror of the target row for each target row on the micromirror chip, and closing the micromirror of the previous row of the target row to enable the micromirror of the target row to irradiate the target object, and performing linear confocal imaging on the target object to obtain an imaging result of the target row;
and obtaining a first imaging result of the target object according to the imaging results of the target rows.
In one embodiment, the performing linear confocal imaging of the target object in a vertical direction through a plurality of micromirrors on the micromirror chip to obtain a second imaging result of the target object includes:
sequentially determining a plurality of target columns along the vertical direction of the micromirrors on the micromirror chip according to a preset vertical sequence;
starting a micromirror of the target column for each target column on the micromirror chip, and closing a micromirror of a previous column of the target column to enable the micromirror of the target column to irradiate the target object, and performing linear confocal imaging on the target object to obtain an imaging result of the target column;
And obtaining a second imaging result of the target object according to the imaging results of the target columns.
In one embodiment, the calculating the line scan confocal imaging result of the target object according to the first imaging result, the second imaging result and the wide-field imaging result includes:
summing the first imaging result and the second imaging result to obtain a target value;
and taking the difference between the target value and the wide-field imaging result as a line scanning confocal imaging result of the target object.
In one embodiment, the method further comprises:
according to the first imaging result, carrying out alignment treatment on the second imaging result to obtain a third imaging result corresponding to the second imaging result;
performing fusion processing on the first imaging result and the third imaging result to obtain a primary fusion result;
and carrying out joint deconvolution calculation according to the primary fusion result to obtain a line scanning confocal imaging result of the target object.
In a second aspect, the present application also provides an imaging system, the system comprising: the device comprises a micro-mirror chip, a laser light source module, an imaging module and a data processing module; wherein the micromirror chip comprises a plurality of micromirrors,
The laser light source module is used for emitting a laser light source;
the micro mirror is used for irradiating the laser light source to a target object so that the imaging module can perform linear confocal imaging on the target object in the horizontal direction to obtain a first imaging result of the target object; performing linear confocal imaging on the target object in the vertical direction to obtain a second imaging result of the target object; performing wide-field imaging on the target object to obtain a wide-field imaging result of the target object;
the data processing module is used for calculating a line scanning confocal imaging result of the target object according to the first imaging result, the second imaging result and the wide-field imaging result.
In one embodiment, the system further comprises an imaging control module;
the imaging control module is used for sequentially determining a plurality of target rows along the horizontal direction of the micro mirrors on the micro mirror chip according to a preset horizontal sequence; starting a micromirror of the target row for each target row on the micromirror chip, and closing the micromirror of the previous row of the target row to enable the micromirror of the target row to irradiate the target object, and performing linear confocal imaging on the target object to obtain an imaging result of the target row; and obtaining a first imaging result of the target object according to the imaging results of the target rows.
The imaging control module is further used for sequentially determining a plurality of target columns along the vertical direction of the micromirrors on the micromirror chip according to a preset vertical sequence; starting a micromirror of the target column for each target column on the micromirror chip, and closing a micromirror of a previous column of the target column to enable the micromirror of the target column to irradiate the target object, and performing linear confocal imaging on the target object to obtain an imaging result of the target column; and obtaining a second imaging result of the target object according to the imaging results of the target columns.
In a third aspect, the present application also provides an image forming apparatus, the apparatus comprising:
the first imaging unit is used for carrying out linear confocal imaging on a target object in the horizontal direction through a plurality of micromirrors on the micromirror chip to obtain a first imaging result of the target object;
the second imaging unit is used for carrying out linear confocal imaging on the target object in the vertical direction through a plurality of micro mirrors on the micro mirror chip to obtain a second imaging result of the target object;
the wide-field imaging unit is used for carrying out wide-field imaging on the target object through a plurality of micro mirrors on the micro mirror chip to obtain a wide-field imaging result of the target object;
And the calculating unit is used for calculating a line scanning confocal imaging result of the target object according to the first imaging result, the second imaging result and the wide-field imaging result.
In a fourth aspect, the present application also provides a computer device. The computer device comprises a memory storing a computer program and a processor which when executing the computer program performs the steps of:
performing linear confocal imaging on a target object in the horizontal direction through a plurality of micromirrors on the micromirror chip to obtain a first imaging result of the target object;
performing linear confocal imaging on the target object in the vertical direction through a plurality of micromirrors on the micromirror chip to obtain a second imaging result of the target object;
performing wide-field imaging on the target object through a plurality of micromirrors on the micromirror chip to obtain a wide-field imaging result of the target object;
and calculating a line scanning confocal imaging result of the target object according to the first imaging result, the second imaging result and the wide-field imaging result.
In a fifth aspect, the present application also provides a computer-readable storage medium. The computer readable storage medium having stored thereon a computer program which when executed by a processor performs the steps of:
Performing linear confocal imaging on a target object in the horizontal direction through a plurality of micromirrors on the micromirror chip to obtain a first imaging result of the target object;
performing linear confocal imaging on the target object in the vertical direction through a plurality of micromirrors on the micromirror chip to obtain a second imaging result of the target object;
performing wide-field imaging on the target object through a plurality of micromirrors on the micromirror chip to obtain a wide-field imaging result of the target object;
and calculating a line scanning confocal imaging result of the target object according to the first imaging result, the second imaging result and the wide-field imaging result.
In a sixth aspect, the application also provides a computer program product. The computer program product comprises a computer program which, when executed by a processor, implements the steps of:
performing linear confocal imaging on a target object in the horizontal direction through a plurality of micromirrors on the micromirror chip to obtain a first imaging result of the target object;
performing linear confocal imaging on the target object in the vertical direction through a plurality of micromirrors on the micromirror chip to obtain a second imaging result of the target object;
Performing wide-field imaging on the target object through a plurality of micromirrors on the micromirror chip to obtain a wide-field imaging result of the target object;
and calculating a line scanning confocal imaging result of the target object according to the first imaging result, the second imaging result and the wide-field imaging result.
The imaging method, the imaging device, the computer equipment, the storage medium and the computer program product are used for carrying out linear confocal imaging in the horizontal direction, linear confocal imaging in the vertical direction and wide-field imaging on the target object through a plurality of micromirrors on the micromirror chip to obtain a first imaging result, a second imaging result and a wide-field imaging result of the target object; and calculating a line scanning confocal imaging result of the target object according to the imaging result. By adopting the imaging method provided by the embodiment of the invention, confocal line scanning laser irradiation imaging can be respectively carried out in the horizontal direction and the vertical direction, signals in the same visual field are sampled for multiple times in different directions, and the final imaging result is calculated according to the sampling result, so that the accuracy of image signal acquisition can be improved, the information loss is reduced, the signal-to-noise ratio is improved, the imaging speed and the spatial resolution are improved, and the imaging quality of the final imaging result is improved.
Drawings
FIG. 1 is a block diagram of an imaging system in one embodiment;
FIG. 2 is a flow diagram of an imaging method in one embodiment;
FIG. 3 is a flowchart illustrating a first imaging result step according to an embodiment;
FIG. 4 is a flowchart illustrating a second imaging result step according to an embodiment;
FIG. 5 is a flowchart illustrating steps of line scan confocal imaging of a target object according to one embodiment;
FIG. 6 is a schematic diagram of an imaging system in another embodiment;
FIG. 7A is a schematic diagram of a module 1 of an imaging system according to another embodiment;
FIG. 7B is a schematic diagram of the structure of a module 2 in an imaging system according to another embodiment;
FIG. 7C is a schematic diagram of the structure of a module 3 in an imaging system according to another embodiment;
FIG. 7D is a schematic diagram of the structure of the module 4 in the imaging system according to another embodiment;
FIG. 8 is a schematic diagram of a micromirror chip in one embodiment;
FIG. 9A is a schematic diagram showing the sequence of micromirror opening when imaging in horizontal direction in the imaging system according to one embodiment;
FIG. 9B is a schematic diagram of the sequence in which a sample imaged in a horizontal direction is illuminated in an imaging system according to one embodiment;
FIG. 10A is a schematic diagram showing the sequence of micromirror opening for vertical imaging in an imaging system according to one embodiment;
FIG. 10B is a schematic diagram of the sequence in which a sample imaged in a vertical direction is illuminated in an imaging system according to one embodiment;
FIG. 11A is a schematic diagram of a micromirror in an open state when performing wide-field imaging in an imaging system according to one embodiment;
FIG. 11B is a schematic diagram of the sequence in which a sample is illuminated when performing wide field imaging in an imaging system in one embodiment;
FIG. 12 is a block diagram of an imaging device in one embodiment;
fig. 13 is an internal structural view of a computer device in one embodiment.
Detailed Description
The present application will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present application more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the application.
The method provided by the embodiment of the application can be applied to the field of fluorescence microscopy imaging, in particular to the field of confocal microscopy imaging. The imaging method provided by the embodiment of the application can be applied to the imaging system 100 shown in the application environment shown in fig. 1, wherein the imaging system comprises a micro-mirror chip 200, a laser light source module 300, an imaging module 400, an imaging control module 500 and a data processing module 600. Wherein the imaging module 400 communicates with the data processing module 600 via a network. The data processing module 600 may be, but is not limited to, various personal computers, notebook computers, smart phones, tablet computers, internet of things devices, and portable wearable devices, where the internet of things devices may be smart speakers, smart televisions, smart air conditioners, smart vehicle devices, etc. The portable wearable device may be a smart watch, smart bracelet, headset, or the like. The data processing module 600 may also be a stand-alone server or a server cluster made up of multiple servers.
In this embodiment, as shown in fig. 2, the imaging method includes the following steps:
step 102, performing linear confocal imaging on the target object in the horizontal direction through a plurality of micromirrors on the micromirror chip to obtain a first imaging result of the target object.
The micromirror chip may be a digital micromirror device DMD, and includes a plurality of micromirrors (micromirror pixels) at a pixel level, for example, a micromirror chip formed by arranging n×n micromirrors, that is, there are N rows of micromirrors and N columns of micromirrors on the micromirror chip. The micro-mirror chip can generate line scanning copolymer Jiao Zhaoming light and realize the function of confocal aperture (pinhole). The target object may be a sample containing a fluorescent marker substance.
Specifically, the imaging system may generate line scanning illumination light in a horizontal direction by turning on a plurality of micromirrors on a micromirror chip, and perform confocal imaging on a target object to obtain a first imaging result of the target object.
In one example, the imaging system may further include a sample placement cell, which may be a conventional sample cell, a living cell culture dish configured to maintain cell viability, or the like, and a detector, to which the target object is placed. The laser light source module generates laser, the imaging system irradiates the laser to the corresponding position of the target object according to the horizontal direction through the micro mirror on the micro mirror chip, so that fluorescent marking substances at the corresponding position irradiated by the laser can be excited to generate fluorescence, and the generated fluorescence can be collected and reflected to the micro mirror on the micro mirror chip. Thus, the micro mirror on the micro mirror chip can reflect fluorescence and perform linear confocal imaging on the detector to obtain a first imaging result of the target object.
And 104, performing linear confocal imaging on the target object in the vertical direction through a plurality of micromirrors on the micromirror chip to obtain a second imaging result of the target object.
Specifically, the imaging system may generate line scanning illumination light in a vertical direction by turning on a plurality of micromirrors on a micromirror chip, and perform confocal imaging on the target object to obtain a second imaging result of the target object.
In one example, the laser light source module generates laser light, the imaging system irradiates the laser light to the corresponding position of the target object in the vertical direction through the micro mirror on the micro mirror chip, so that the fluorescent marking substance of the corresponding position irradiated by the laser light is excited to generate fluorescence, and the generated fluorescence is collected and reflected to the micro mirror on the micro mirror chip. Thus, the micro mirror on the micro mirror chip can reflect fluorescence and perform linear confocal imaging on the detector to obtain a second imaging result of the target object.
And 106, performing wide-field imaging on the target object through a plurality of micromirrors on the micromirror chip to obtain a wide-field imaging result of the target object.
Specifically, the specific process of performing wide-field imaging on the target object can be: the imaging system turns on all the micromirrors on the micromirror chip. Therefore, when the laser light source module generates laser, all the micromirrors on the micromirror chip can irradiate the laser to the target object, fluorescent substances on all the positions on the target object can be excited by the laser to generate fluorescence, the generated fluorescence can be collected and reflected to all the micromirrors on the micromirror chip in an open state, and the micromirrors in the open state can reflect the fluorescence to perform wide-field imaging on the detector to obtain a wide-field imaging result of the target object.
Optionally, the steps 102, 104, 108 do not need to be sequenced in the execution process of the actual application scenario.
Step 108, calculating a line scanning confocal imaging result of the target object according to the first imaging result, the second imaging result and the wide-field imaging result.
Specifically, the imaging system calculates a first imaging result, a second imaging result and a wide-field imaging result, and the first imaging result is obtained by imaging by using line scanning illumination light in a horizontal direction, so that the first imaging result contains a noise image in the horizontal direction; similarly, the second imaging result contains a noise image in the vertical direction, and the wide-field imaging result contains a noise image in the omnidirectional direction. Thus, since the sum of the noise image in the horizontal direction and the noise image in the vertical direction is an omnidirectional noise image, the calculation process of the imaging result containing no noise may be: the sum of the first imaging result and the second imaging result is different from the wide-field imaging result, and the difference is taken as a line scanning confocal imaging result of the target object.
In the imaging method, through a plurality of micro mirrors on the micro mirror chip, linear confocal imaging in the horizontal direction, linear confocal imaging in the vertical direction and wide-field imaging are carried out on a target object, so that a first imaging result, a second imaging result and a wide-field imaging result of the target object are obtained; and calculating a line scanning confocal imaging result of the target object according to the imaging result. By adopting the imaging method provided by the embodiment of the invention, confocal line scanning laser irradiation imaging can be respectively carried out in the horizontal direction and the vertical direction, signals in the same visual field are sampled for multiple times in different directions, and the final imaging result is calculated according to the sampling result, so that the accuracy of image signal acquisition can be improved, the information loss is reduced, the signal to noise ratio is improved, and the imaging quality of the final imaging result is improved.
In one embodiment, as shown in fig. 3, the specific process of step 104 "performing linear confocal imaging of the target object in the horizontal direction through a plurality of micromirrors on the micromirror chip to obtain the first imaging result" of the target object includes:
step 202, sequentially determining a plurality of target rows along the horizontal direction of the micromirrors on the micromirror chip according to a preset horizontal order.
Specifically, the micromirror chip may be a digital micromirror device DMD, and the micromirror chip includes a plurality of micromirrors (micromirror pixels) at a pixel level, for example, a micromirror chip formed by arranging n×n micromirrors, that is, there are N rows of micromirrors and N columns of micromirrors on the micromirror chip. Thus, the plurality of target rows on the micromirror chip may be sequentially determined in the order of the first and second rows of the micromirror chip according to the preset horizontal order.
Step 204, starting the micro-mirrors of the target row for each target row on the micro-mirror chip, and closing the micro-mirrors of the previous row of the target row, so that the micro-mirrors of the target row irradiate the target object, and performing linear confocal imaging on the target object to obtain an imaging result of the target row.
Specifically, for each target row on the micromirror chip, the imaging system turns on the micromirrors of that target row at the current time and turns off the micromirrors of the other rows so that only the micromirrors of the target row are in an on state at the current time. Thus, the micromirror in the on state can receive the laser light emitted from the laser light source module and irradiate the laser light to the target position of the target object, and fluorescent material marked on the target position of the target object generates fluorescence. The generated fluorescence is collected by an imaging system and reflected to a micro mirror on a target row on a micro mirror chip, the micro mirror on the target row can reflect the fluorescence, and linear confocal imaging is carried out on a detector to obtain an imaging result of the target row.
Wherein the abscissa of the target position of the target object corresponds to the abscissa of the position of the target row on the micromirror chip.
Step 206, obtaining a first imaging result of the target object according to the imaging results of the plurality of target rows.
Wherein the plurality of target rows consists of all rows on the micromirror chip. The imaging system combines a plurality of imaging results of a plurality of target rows to obtain a first imaging result of the target object.
In one example, the imaging system places the target object in the sample cell such that the imaging system can activate a first row of micromirrors on the micromirror chip such that the micromirrors included in the first row can illuminate the target object and perform linear confocal imaging on the target object to obtain an imaging result of the first row. Then, the imaging system can start the micro mirrors of the second row on the micro mirror chip, and simultaneously close the micro mirrors of the first row, so that the micro mirrors included in the second row can irradiate the target object, and the target object is subjected to linear confocal imaging to obtain an imaging result of the second row. Similarly, the executing process is repeated until the last row of the micro mirror chip is opened, and the micro mirrors of the previous row of the last row are closed, so that the micro mirrors included in the last row can irradiate the target object, and the target object is subjected to linear confocal imaging, so that an imaging result of the last row is obtained. And respectively carrying out linear confocal imaging on the target object according to the micro-mirror chip to obtain imaging results of a plurality of rows, and combining to obtain a first imaging result.
In another example, the target line may include two lines and three lines, and the specific line number may be determined according to the actual application scenario.
In this embodiment, the micromirrors are sequentially opened in the horizontal direction, and confocal line scanning laser irradiation imaging is performed in the horizontal direction according to the line scanning mode, so that the accuracy of signal acquisition is improved, the information loss is reduced, the signal-to-noise ratio is improved, and the imaging quality is improved to a certain extent.
In one embodiment, as shown in fig. 4, step 106 "a specific process of performing linear confocal imaging of a target object in a vertical direction through a plurality of micromirrors on a micromirror chip to obtain a second imaging result" of the target object includes:
step 302, sequentially determining a plurality of target columns along the vertical direction of the micromirrors on the micromirror chip according to a preset vertical order.
The preset vertical sequence may be that a plurality of target columns on the micro-mirror chip are determined in sequence according to the arrangement sequence of the first column and the second column of the micro-mirror chip.
Specifically, the imaging system may sequentially determine a plurality of target columns in a vertical order along a vertical direction of the micromirror arrangement on the micromirror chip.
Step 304, for each target column on the micromirror chip, starting the micromirror of the target column, and closing the micromirror of the previous column of the target column, so that the micromirror of the target column irradiates the target object, and linear confocal imaging is performed on the target object, thereby obtaining the imaging result of the target column.
Specifically, for each target column on the micromirror chip, the imaging system turns on the micromirrors of that target column at the current time and turns off the micromirrors of the other columns so that only the micromirrors of the target column are in an on state at the current time. Thus, the micromirror in the on state can receive the laser light emitted from the laser light source module and irradiate the laser light to the target position of the target object, and fluorescent material marked on the target position of the target object generates fluorescence. The generated fluorescence is collected by an imaging system and reflected to a micro mirror on a target column on a micro mirror chip, the micro mirror on the target column can reflect the fluorescence, and linear confocal imaging is carried out on a detector to obtain an imaging result of the target column.
Wherein the abscissa of the target position of the target object corresponds to the abscissa of the position of the target column on the micromirror chip.
Step 306, obtaining a second imaging result of the target object according to the imaging results of the plurality of target columns.
Wherein the plurality of target columns consists of all columns on the micromirror chip. The imaging system combines the plurality of imaging results of the plurality of target columns to obtain a second imaging result of the target object.
In one example, the imaging system places the target object in the sample cell such that the imaging system can activate a first column of micromirrors on the micromirror chip such that the micromirrors on the first column can illuminate the target object and perform linear confocal imaging of the target object to obtain the imaging result of the first column. Then, the imaging system can start the second row of micromirrors on the micromirror chip and simultaneously close the micromirrors of the first row, so that the micromirrors included in the second row can irradiate the target object, and perform linear confocal imaging on the target object to obtain an imaging result of the second row. Similarly, the above-mentioned execution process is repeated until the last column on the micromirror chip is opened, and the micromirrors of the previous column of the last column are closed, so that the micromirrors included in the last column can irradiate the target object, and linear confocal imaging is performed on the target object, so as to obtain the imaging result of the last column. And respectively carrying out linear confocal imaging on the target object according to the micro-mirror chip to obtain a plurality of rows of imaging results, and combining to obtain a second imaging result.
In another example, the target column may include two columns and three columns, and the specific column number may be determined according to the actual application scenario.
In this embodiment, the micromirrors are sequentially opened in the vertical direction, and confocal line scanning laser irradiation imaging is performed in the vertical direction according to the line scanning manner, so that the accuracy of signal acquisition is improved, the information loss is reduced, the signal-to-noise ratio is improved, and the imaging quality is improved to a certain extent.
In one embodiment, as shown in fig. 5, step 108 "calculate a line scan confocal imaging result of the target object according to the first imaging result, the second imaging result, and the wide-field imaging result" includes:
step 402, summing the first imaging result and the second imaging result to obtain a target value.
Step 404, taking the difference between the target value and the wide-field imaging result as the line scanning confocal imaging result of the target object.
Specifically, the line scan confocal imaging result of the target object is calculated by the following formula:
I 1 ＝I signal +I background-x ，
I 2 ＝I signal +I background-y ，
I 3 ＝I signal +I background ，
I signal ＝I 1 +I 2 -I 3 ，
wherein I is 1 Is the first imaging result, I 2 Is the second imaging result, I 3 Is the result of the wide field imaging, I signal Is the line scanning confocal imaging result of the target object, I background-x Is a noise image in the horizontal direction, I background-y Is a noise image in the vertical direction.
In this embodiment, by adopting the confocal imaging mode of line scanning, the imaging speed is greatly improved compared with the point scanning confocal imaging mode, and the problems of stability of galvanometer control and image deformation caused by the resonance scanning imaging mode can be avoided due to confocal imaging. For example, in the case of processing 512x512 resolution images, the imaging speed may be increased to 30f/s. The effects of removing imaging background light and improving imaging signal to noise ratio can be achieved through line scanning confocal imaging modes in different directions and adding and subtracting calculation of results of three imaging.
In one possible implementation, the method further includes:
according to the first imaging result, performing alignment processing on the second imaging result to obtain a third imaging result corresponding to the second imaging result; fusing the first imaging result and the third imaging result to obtain a primary fusion result; and carrying out joint deconvolution calculation according to the primary fusion result to obtain a line scanning confocal imaging result of the target object.
Specifically, the imaging system may perform joint deconvolution calculation on the first imaging result SideA and the second imaging result SideB, and the specific calculation process may be: according to the first imaging result, aligning the second imaging result, firstly calculating a normalized cross-correlation value between the first imaging result and the second imaging result, and according to the normalized cross-correlation value, aligning the second imaging result to obtain an aligned second imaging result, namely a third imaging result. The matrix data after the alignment process (alignment of the second imaging result to the first imaging result) may be SideA (first imaging result) and SideB' (third imaging result). Thus, the first imaging result and the second imaging result can be fused, and a primary fusion result is obtained. The specific process of fusion may be: and calculating the sum of the first imaging result and the third imaging result, and carrying out weighted calculation on the sum according to a preset weight to obtain a primary fusion result. The preliminary fusion result is calculated, for example, by the following formula:
Result0＝1/2*(SideA+SideB’)。
In this way, the imaging system may perform a joint deconvolution (joint deconvolution) calculation based on the preliminary fusion result to obtain a line scan confocal imaging result of the target object. The specific calculation process can be as follows:
For i＝1,2,...,N
ResultA＝Resulti-1*BlurA(SideA/BlurA(Resulti-1))；
Result i ＝ResultA*BlurB(SideB’/BlurB(ResultA))
wherein Result is i The result of the joint deconvolution calculation is performed.
In this embodiment, the signal-to-noise ratio and the resolution of the target object (sample) in each arrangement direction are synchronously improved by performing joint deconvolution calculation.
It should be understood that, although the steps in the flowcharts related to the embodiments described above are sequentially shown as indicated by arrows, these steps are not necessarily sequentially performed in the order indicated by the arrows. The steps are not strictly limited to the order of execution unless explicitly recited herein, and the steps may be executed in other orders. Moreover, at least some of the steps in the flowcharts described in the above embodiments may include a plurality of steps or a plurality of stages, which are not necessarily performed at the same time, but may be performed at different times, and the order of the steps or stages is not necessarily performed sequentially, but may be performed alternately or alternately with at least some of the other steps or stages.
Based on the same inventive concept, the embodiment of the application also provides an imaging system for realizing the above-mentioned imaging method. The implementation of the solution provided by the system is similar to that described in the above method, so specific limitations in one or more embodiments of the imaging system provided below may be referred to above as limitations of the imaging method, and will not be repeated here.
In one embodiment, as shown in FIG. 1, there is provided an imaging system 100 comprising: a micromirror chip 200, a laser light source module 300, an imaging module 400, and a data processing module 600; wherein the micromirror chip comprises a plurality of micromirrors:
a laser light source module 300 for emitting a laser light source;
the micro-mirror is used for irradiating the laser light source to the target object so that the imaging module 400 can perform linear confocal imaging on the target object in the horizontal direction to obtain a first imaging result of the target object; performing linear confocal imaging on the target object in the vertical direction to obtain a second imaging result of the target object; performing wide-field imaging on the target object to obtain a wide-field imaging result of the target object;
the data processing module 600 is configured to calculate a line scan confocal imaging result of the target object according to the first imaging result, the second imaging result, and the wide-field imaging result.
In one example, as shown in fig. 6, the imaging system may include a module 1, a module 2, a module 3, a module 4, a module 5, wherein the module 1 is a microscope body portion; module 2 is a laser light source module 300 and module 3 is a copolymer Jiao Zhaoming module; the module 4 is a confocal detection module; the module 5 is a control module. The module 1 comprises an objective lens (obj), and the module 2 comprises a laser controller (laser driver), laser1 (laser 1), laser2 (laser 2), laser3 (laser 3), an acousto-optic filter (aotf) and a coupling lens (coupling lens); the module 3 comprises a digital micromirror array (DMD), a switchable spatial filter (switchable mask); the module 5 includes a workstation (workstation), an image detector control (to camera), and a stage driver (stage driver). The module 2 and the module 3 in the imaging system are connected by a single mode fiber (SM fiber).
Specifically, as shown in fig. 7A, a detailed illustration of the various components contained in module 1, where 1-1 is a microscope objective; 1-2 is a microscope tube lens; 1-3 and 1-4 are reflectors; 1-5 are microscope platforms; 1-6 are sample cells, can be common sample cells, can also be living cell culture dishes and other devices for keeping cell activity. The microscope stage may be any of manual, electric, piezoelectric.
As shown in fig. 7B, a detailed illustration of each component included in a module 2 (Laser driver, laser source module), where 2-1 is a Laser, and the wavelength and number in the Laser are determined according to the actual application scenario, and the present invention is not limited herein; 2-2 is a laser control system, which controls the laser switch, the output intensity and other parts to control synchronously; 2-3 is a laser Coupling device (Coupling Lens), which can couple the outputs of several lasers into a single mode fiber through the design of an optical path; 2-4 are single mode optical fibers.
As shown in fig. 7C, a detailed illustration of each component included in the module 3, where 3-1 is a fiber collimating lens, which can collimate the laser output from the single-mode fiber; 3-2 is a dichroic mirror capable of reflecting illumination light and transmitting fluorescence emitted by the sample; 3-3 is a digital micromirror device DMD (which is a micromirror chip 200) for generating line scanning copolymerization Jiao Zhaoming light and realizing a function of confocal aperture (pin hole), 3-3 is formed by arranging NxN micro mirror pixels, the arrangement diagram may be as shown in fig. 8, the arrangement diagram is only illustrative, the number N of pixels is different from the actual parts, and the invention is not limited herein, and can be determined according to the actual application scenario; 3-4 and 3-5 are relay lenses; 3-6 and 3-7 are plane mirrors; 3-8 are pattern selectable spatial filters, which can further select the light structure of the line scanning copolymer Jiao Zhaoming to improve the illumination quality.
As shown in fig. 7D, the details of each component included in the module 4 are shown, where 4-1 and 4-2 are relay lenses, 4-2 is an optional part, and optional light splitting parts may be selected, so that fluorescence is separated according to the properties of intensity, optical wavelength, and the like, thereby improving the imaging speed, or expanding the imaging function, and the like; 4-3 is a fluorescent filter plate which can filter laser and transmit fluorescence; 4-5 are imaging sensors, optionally scmos, EMCCD, etc. The module 5 is composed of an image workstation, a controller of a part, and the like, and is not described herein.
In one example, in combination with the above imaging system, the specific implementation procedure of the imaging method provided by the embodiment of the present invention may include:
step 1, as shown in fig. 9A, the micromirrors are turned on in a row, the micromirrors in the turned on state have triangular marks, the micromirrors in other positions are turned off, the micromirrors in the turned on state can irradiate laser onto the corresponding positions of the sample, fluorescent marking substances in the corresponding positions of the sample irradiated by the laser are excited to generate fluorescence, the fluorescence is collected by the objective lens 1-1, passes through the tube mirrors 1-2,3-5,3-7,3-8,3-6,3-4, irradiates back onto the 3-3 digital micromirrors, reflects through the micromirrors in the turned on positions on the digital micromirror chip, passes through the 3-2, finally passes through the module 4, and is imaged on the detector. Fluorescence on the focal plane of the sample irradiates the position of the micromirror in the opened state of the digital micromirror chip and is reflected to the imaging module 4, and fluorescence information on the off focal plane irradiates the position of the micromirror in the closed state of the digital micromirror chip and is deviated from the imaging module to be absorbed, so that the micromirror in the closed state of the digital micromirror chip can play a role in modulating illumination laser and can play a role in confocal small holes, and compared with a traditional confocal system, the complex process of collimating small holes is omitted, and the problem of imaging quality reduction caused by inaccurate collimation of small holes is avoided.
The imaging control module 500 sequentially turns on in a horizontal direction (a direction illustrating an on order of micromirrors), and turns off the micromirrors of the previous row in a preset horizontal order. Thus, as shown in fig. 9B, the sample is also irradiated with the confocal line scanning laser (the corresponding position of the irradiated sample is marked with a triangle), and imaging is sequentially performed, and when the confocal line scanning imaging process is completed, the first imaging result is recorded.
Step 2, as shown in fig. 10A, the micromirrors are turned on in a row, the micromirrors in the turned-on state with triangular marks irradiate the corresponding positions on the sample, and confocal imaging is performed; the imaging control module 500 sequentially opens and closes the previous column in a vertical direction (in the direction of the micromirror on order in the drawing) in a pre-vertical order, and synchronously, as shown in fig. 10B, the sample is also irradiated with the confocal line scanning laser (the corresponding position of the irradiated sample is marked with a triangle), and sequentially performs imaging, and when the confocal line scanning imaging process is completed, a second imaging result is recorded.
Step 3, as shown in fig. 11A, all the micromirrors on the micromirror chip are in an open state, the micromirrors in the open state are marked with triangles, and as shown in fig. 11B, the sample is subjected to wide-field imaging (the corresponding positions of the irradiated sample are marked with triangles), and the result of the wide-field imaging is recorded.
Step 4, data processing, which includes two methods:
in one possible implementation, the removal of background noise in one direction may be performed on a sample imaged by a line scan confocal, so it may be calculated by the following formula:
I1＝Isignal+Ibackground-x
I2＝Isignal+Ibackground-y
I3＝Isignal+Ibackground
Isignal＝I1+I2-I3。
in the embodiment, all background light in the sample imaging process can be removed through calculation, and compared with the point scanning confocal imaging technology, the imaging speed is greatly improved while the imaging signal-to-noise ratio is maintained.
In another possible implementation, the imaging quality of a sample imaged by a line scan confocal can be improved.
And (3) performing linear scanning modes in two directions on the first imaging result and the second imaging result, and performing joint-deconvolution calculation on image signals in the x and y directions to improve imaging quality.
Because the method adopts the line scanning imaging mode, under the condition that the image pixels are n×n, the required sampling frequency is 2*n, and the time required by the traditional confocal microscope is n×n, so the time is shortened, and the efficiency is improved. In summary, joint-deconvolution can shorten the sampling time, improve the sampling efficiency, and improve the resolution.
The imaging method provided by the embodiment of the invention can improve the imaging speed, adopts a line scanning imaging mode, greatly improves the imaging speed compared with a point scanning confocal imaging mode, adopts the DMD as confocal illumination and small detection holes, avoids the problem of image quality reduction caused by error hole size selection and position dislocation, has high algorithm efficiency, and improves the resolution of imaging results in multiple directions.
In one embodiment, the imaging system further comprises an imaging control module 500; the imaging control module 500 is configured to sequentially determine a plurality of target rows along a horizontal direction of a micromirror on a micromirror chip according to a preset horizontal order; starting a micromirror of a target row for each target row on a micromirror chip, and closing the micromirror of the previous row of the target row to enable the micromirror of the target row to irradiate a target object, and performing linear confocal imaging on the target object to obtain an imaging result of the target row; and obtaining a first imaging result of the target object according to the imaging results of the target rows.
The imaging control module 500 is further configured to sequentially determine a plurality of target columns along a vertical direction of the micromirrors on the micromirror chip according to a preset vertical order; starting a micromirror of a target column aiming at each target column on a micromirror chip, and closing a micromirror of a previous column of the target column to enable the micromirror of the target column to irradiate a target object, and performing linear confocal imaging on the target object to obtain an imaging result of the target column; and obtaining a second imaging result of the target object according to the imaging results of the target columns.
Based on the same inventive concept, the embodiment of the application also provides an imaging device for realizing the above-mentioned imaging method. The implementation of the solution provided by the device is similar to that described in the above method, so the specific limitations in one or more embodiments of the imaging device provided below may be referred to above for limitations of the imaging method, and will not be repeated here.
In one embodiment, as shown in fig. 12, there is provided an imaging apparatus 601 including: a first imaging unit 602, a second imaging unit 603, a wide field imaging unit 604, and a computing unit 605, wherein:
a first imaging unit 602, configured to perform linear confocal imaging on a target object in a horizontal direction through a plurality of micromirrors on a micromirror chip, so as to obtain a first imaging result of the target object;
a second imaging unit 603, configured to perform linear confocal imaging on the target object in a vertical direction through a plurality of micromirrors on the micromirror chip, so as to obtain a second imaging result of the target object;
a wide-field imaging unit 604, configured to perform wide-field imaging on the target object through a plurality of micromirrors on the micromirror chip, so as to obtain a wide-field imaging result of the target object;
a calculating unit 605 for calculating a line scanning confocal imaging result of the target object based on the first imaging result, the second imaging result, and the wide-field imaging result.
The respective modules in the above-described image forming apparatus may be implemented in whole or in part by software, hardware, and combinations thereof. The above modules may be embedded in hardware or may be independent of a processor in the computer device, or may be stored in software in a memory in the computer device, so that the processor may call and execute operations corresponding to the above modules.
In one embodiment, a computer device is provided, which may be a server, and the internal structure of which may be as shown in fig. 13. The computer device includes a processor, a memory, and a network interface connected by a system bus. Wherein the processor of the computer device is configured to provide computing and control capabilities. The memory of the computer device includes a non-volatile storage medium and an internal memory. The non-volatile storage medium stores an operating system, computer programs, and a database. The internal memory provides an environment for the operation of the operating system and computer programs in the non-volatile storage media. The database of the computer device is used to store the imaging-related data. The network interface of the computer device is used for communicating with an external terminal through a network connection. The computer program is executed by a processor to implement an imaging method.
It will be appreciated by those skilled in the art that the structure shown in FIG. 13 is merely a block diagram of some of the structures associated with the present inventive arrangements and is not limiting of the computer device to which the present inventive arrangements may be applied, and that a particular computer device may include more or fewer components than shown, or may combine some of the components, or have a different arrangement of components.
In an embodiment, there is also provided a computer device comprising a memory and a processor, the memory having stored therein a computer program, the processor implementing the steps of the method embodiments described above when the computer program is executed.
In one embodiment, a computer-readable storage medium is provided, on which a computer program is stored which, when executed by a processor, carries out the steps of the method embodiments described above.
In an embodiment, a computer program product is provided, comprising a computer program which, when executed by a processor, implements the steps of the method embodiments described above.
The user information (including but not limited to user equipment information, user personal information, etc.) and the data (including but not limited to data for analysis, stored data, presented data, etc.) related to the present application are information and data authorized by the user or sufficiently authorized by each party.
Those skilled in the art will appreciate that implementing all or part of the above described methods may be accomplished by way of a computer program stored on a non-transitory computer readable storage medium, which when executed, may comprise the steps of the embodiments of the methods described above. Any reference to memory, database, or other medium used in embodiments provided herein may include at least one of non-volatile and volatile memory. The nonvolatile Memory may include Read-Only Memory (ROM), magnetic tape, floppy disk, flash Memory, optical Memory, high density embedded nonvolatile Memory, resistive random access Memory (ReRAM), magnetic random access Memory (Magnetoresistive Random Access Memory, MRAM), ferroelectric Memory (Ferroelectric Random Access Memory, FRAM), phase change Memory (Phase Change Memory, PCM), graphene Memory, and the like. Volatile memory can include random access memory (Random Access Memory, RAM) or external cache memory, and the like. By way of illustration, and not limitation, RAM can be in the form of a variety of forms, such as static random access memory (Static Random Access Memory, SRAM) or dynamic random access memory (Dynamic Random Access Memory, DRAM), and the like. The databases referred to in the embodiments provided herein may include at least one of a relational database and a non-relational database. The non-relational database may include, but is not limited to, a blockchain-based distributed database, and the like. The processor referred to in the embodiments provided in the present application may be a general-purpose processor, a central processing unit, a graphics processor, a digital signal processor, a programmable logic unit, a data processing logic unit based on quantum computing, or the like, but is not limited thereto.
The technical features of the above embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The foregoing examples illustrate only a few embodiments of the application and are described in detail herein without thereby limiting the scope of the application. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the application, which are all within the scope of the application. Accordingly, the scope of the application should be assessed as that of the appended claims.
Claims (10)
1. An imaging method, wherein the method is applied to an imaging system comprising a micromirror chip, the micromirror chip comprising a plurality of micromirrors thereon; the method comprises the following steps:
performing linear confocal imaging on a target object in the horizontal direction through a plurality of micromirrors on the micromirror chip to obtain a first imaging result of the target object;
performing linear confocal imaging on the target object in the vertical direction through a plurality of micromirrors on the micromirror chip to obtain a second imaging result of the target object;
Performing wide-field imaging on the target object through a plurality of micromirrors on the micromirror chip to obtain a wide-field imaging result of the target object;
and calculating a line scanning confocal imaging result of the target object according to the first imaging result, the second imaging result and the wide-field imaging result.
2. The method of claim 1, wherein the performing linear confocal imaging of the target object in the horizontal direction by the plurality of micromirrors on the micromirror chip to obtain the first imaging result of the target object comprises:
sequentially determining a plurality of target rows along the horizontal direction of the micromirrors on the micromirror chip according to a preset horizontal order;
starting a micromirror of the target row for each target row on the micromirror chip, and closing the micromirror of the previous row of the target row to enable the micromirror of the target row to irradiate the target object, and performing linear confocal imaging on the target object to obtain an imaging result of the target row;
and obtaining a first imaging result of the target object according to the imaging results of the target rows.
3. The method of claim 1, wherein said performing linear confocal imaging of said target object in a vertical direction via a plurality of micromirrors on said micromirror chip to obtain a second imaging result of said target object comprises:
Sequentially determining a plurality of target columns along the vertical direction of the micromirrors on the micromirror chip according to a preset vertical sequence;
starting a micromirror of the target column for each target column on the micromirror chip, and closing a micromirror of a previous column of the target column to enable the micromirror of the target column to irradiate the target object, and performing linear confocal imaging on the target object to obtain an imaging result of the target column;
and obtaining a second imaging result of the target object according to the imaging results of the target columns.
4. The method of claim 1, wherein the calculating a line scan confocal imaging result of the target object based on the first imaging result, the second imaging result, and the wide-field imaging result comprises:
summing the first imaging result and the second imaging result to obtain a target value;
and taking the difference between the target value and the wide-field imaging result as a line scanning confocal imaging result of the target object.
5. The method according to claim 1, wherein the method further comprises:
according to the first imaging result, carrying out alignment treatment on the second imaging result to obtain a third imaging result corresponding to the second imaging result;
Performing fusion processing on the first imaging result and the third imaging result to obtain a primary fusion result;
and carrying out joint deconvolution calculation according to the primary fusion result to obtain a line scanning confocal imaging result of the target object.
6. An imaging system, the system comprising: the device comprises a micro-mirror chip, a laser light source module, an imaging module and a data processing module; wherein the micromirror chip comprises a plurality of micromirrors,
the laser light source module is used for emitting a laser light source;
the micro mirror is used for irradiating the laser light source to a target object so that the imaging module can perform linear confocal imaging on the target object in the horizontal direction to obtain a first imaging result of the target object; performing linear confocal imaging on the target object in the vertical direction to obtain a second imaging result of the target object; performing wide-field imaging on the target object to obtain a wide-field imaging result of the target object;
the data processing module is used for calculating a line scanning confocal imaging result of the target object according to the first imaging result, the second imaging result and the wide-field imaging result.
7. The system of claim 6, further comprising an imaging control module;
the imaging control module is used for sequentially determining a plurality of target rows along the horizontal direction of the micro mirrors on the micro mirror chip according to a preset horizontal sequence; starting a micromirror of the target row for each target row on the micromirror chip, and closing the micromirror of the previous row of the target row to enable the micromirror of the target row to irradiate the target object, and performing linear confocal imaging on the target object to obtain an imaging result of the target row; obtaining a first imaging result of the target object according to the imaging results of the target rows;
the imaging control module is further used for sequentially determining a plurality of target columns along the vertical direction of the micromirrors on the micromirror chip according to a preset vertical sequence; starting a micromirror of the target column for each target column on the micromirror chip, and closing a micromirror of a previous column of the target column to enable the micromirror of the target column to irradiate the target object, and performing linear confocal imaging on the target object to obtain an imaging result of the target column; and obtaining a second imaging result of the target object according to the imaging results of the target columns.
8. A computer device comprising a memory and a processor, the memory storing a computer program, characterized in that the processor implements the steps of the method of any one of claims 1 to 5 when the computer program is executed.
9. A computer readable storage medium, on which a computer program is stored, characterized in that the computer program, when being executed by a processor, implements the steps of the method of any of claims 1 to 5.
10. A computer program product comprising a computer program, characterized in that the computer program, when being executed by a processor, implements the steps of the method according to any one of claims 1 to 5.
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