CN111982865A - Full-slide fluorescence hyperspectral rapid acquisition method and device - Google Patents
Full-slide fluorescence hyperspectral rapid acquisition method and device Download PDFInfo
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Abstract
A full-glass fluorescence hyperspectral rapid acquisition method and a device thereof are disclosed, wherein excitation light emitted by an excitation light source is modulated into slit excitation light by a slit excitation light modulation device, the slit excitation light is projected to the surface of a sample on a glass slide to excite fluorescence, the fluorescence is dispersed by a prism, and a camera acquires a dispersed fluorescence spectrum image of a first camera pixel position; translating the slit exciting light by one pixel position, and collecting a fluorescence spectrum image of the next pixel position until the images of all the pixel positions in the current view field are collected; moving the objective table to enable the microscope objective to be aligned to the next view field position of the glass slide, and repeatedly acquiring fluorescence spectrum images until all the view field images of the glass slide are acquired; and combining the fluorescence spectrum images of all pixel positions in each field to obtain the fluorescence spectrum image of each field, and splicing the spectrum images of all fields to obtain the fluorescence spectrum image of the whole glass slide. The invention can rapidly acquire the fluorescence hyperspectral image of the whole glass slide with high quality.
Description
Technical Field
The invention relates to the field of spectrum acquisition, in particular to a method and a device for rapidly acquiring full-glass fluorescence hyperspectral.
Background
The hyperspectral or hyperspectral image has two spatial dimensions and one spectral dimension, can provide related tissue physiology, morphology and composition information, and is widely applied to disease diagnosis and image-guided surgery. The existing methods for acquiring the spectral image include whiskbroom, pushbroom, staring, snapshot and the like.
The traditional fluorescence detection method is mainly based on the design of a filter, the switching of the filter requires time, and the quality of a measured image is influenced along with mechanical vibration in the process of switching the filter by an electric control platform.
Disclosure of Invention
The invention mainly aims to overcome the defects of the prior art and provides a method and a device for acquiring a full-glass fluorescence spectrum image quickly and with high quality.
In order to achieve the purpose, the invention adopts the following technical scheme:
a full-glass-slide fluorescence spectrum rapid acquisition method comprises the following steps:
s1, moving the slide-carrying stage to align the microscope objective with the first field of view of the slide and to focus the lens of the camera with respect to the first field of view of the slide;
S2, projecting the slit excitation light to the surface of the sample on the glass slide to excite fluorescence, dispersing the excited fluorescence through a prism, and collecting a dispersed fluorescence spectrum image of a first camera pixel position through a camera;
s3, translating the slit excitation light by one pixel position, and collecting the fluorescence spectrum image of the next pixel position until the fluorescence spectrum images of all the pixel positions in the current field of view are collected;
s4, moving the object stage to align the microscope objective lens with the next field position of the slide, and repeating the corresponding processes in the steps S1-S3 until the images of all the fields of the slide are acquired;
s5, combining the fluorescence spectrum images of all pixel positions in each field of view to obtain a fluorescence spectrum image of each field of view;
and S6, obtaining a fluorescence spectrum image of the whole glass slide by image splicing of the spectrum images of all the fields of view.
Further:
in step S1, two light sources of different colors disposed at different spatial positions are used, and the two light sources are turned on when focusing the lens, so that light of the two colors emitted by the two light sources enters the camera through the microscope objective, and an actual focal position is calculated according to the images of the two light sources acquired by the camera and the difference between the two light sources in the spatial positions, thereby controlling the focusing of the lens of the camera.
In step S1, the two light sources are red and green LED lamps, respectively.
In step S2, light emitted from an excitation light source is modulated into parallel light by a collimating lens, the parallel light is projected onto a slit excitation light modulation device, the parallel light is modulated into parallel slit excitation light by the slit excitation light modulation device, the slit excitation light is deflected by 90 degrees by a beam splitter, the slit excitation light in a fluorescence wavelength range is only retained after passing through an optical filter and a tube lens (tube lens), the slit excitation light is projected onto the surface of a sample on a glass slide by the microscope objective lens to excite slit-shaped fluorescence, the fluorescence passes through the microscope objective lens, the tube lens, the optical filter and the beam splitter in sequence, then is dispersed in a direction perpendicular to the slit by the prism, and the dispersed fluorescence enters a camera for imaging.
Light emitted by an excitation light source is modulated by a slit array (slit excitation light) to form the slit excitation light, and the translation of the slit excitation light is realized by moving the objective table.
The light emitted by the excitation light source is modulated by a DMD (digital micromirror device) to form the slit excitation light, and the translation of the slit excitation light is realized by controlling the DMD.
The excitation light source is a mercury lamp.
A full-glass fluorescence spectrum rapid acquisition device comprises a carrying table for carrying a glass slide, a microscope, a camera, an excitation light source, a slit excitation light modulation device, a prism and an image processing device, wherein excitation light emitted by the excitation light source is modulated into slit excitation light by the slit excitation light modulation device, fluorescence is excited on the surface of a sample projected on the glass slide, the excited fluorescence is dispersed by the prism, and a dispersed fluorescence spectrum image of a first camera pixel position is acquired by the camera; translating the slit exciting light by one pixel position, and collecting a fluorescence spectrum image of the next pixel position until the fluorescence spectrum images of all the pixel positions in the current view field are collected; moving the objective table to enable the microscope objective to be aligned to the next view field position of the glass slide, and repeatedly acquiring fluorescence spectrum images until all the images of all the view fields of the glass slide are acquired; the image processing device combines the fluorescence spectrum images of all pixel positions in each view field to obtain the fluorescence spectrum image of each view field, and performs image splicing on the spectrum images of all the view fields to obtain the fluorescence spectrum image of the whole glass slide.
Further:
slit excitation light modulating device is the slit array, the translation of slit excitation light is through removing the objective table realizes, perhaps, slit excitation light modulating device is DMD, the translation of slit excitation light is through control DMD's state realizes.
The focusing device comprises two light sources with different colors and a control device, wherein the two light sources are arranged at different spatial positions, the light with the two colors emitted by the two light sources enters the camera through the microscope objective lens when the lens is focused, and the control device calculates the actual focal position according to the images of the two light sources acquired by the camera and the difference of the two light sources in the spatial positions and controls the lens of the camera to focus.
The invention has the following beneficial effects:
the invention provides a rapid construction method of full-glass fluorescence spectrum, which combines the spectrum image acquisition with fluorescence detection, and can realize rapid and high-quality acquisition of fluorescence hyperspectral image of the whole glass slide (slice), thereby being capable of more accurately and rapidly diagnosing diseases which can not be completed by the traditional method, and having great application prospect in the medical field.
The invention combines spectral image acquisition and fluorescence detection, and provides a method for rapidly acquiring full-glass fluorescence ultra-broad spectrum clusters. The method is designed based on a pushbroom method in spectral image acquisition, slit exciting light is projected onto a sample through a DMD, excited fluorescence is dispersed through a prism, and a camera captures dispersed images to splice fluorescence spectral images of all wavelength bands. The design does not need to switch the filter, and has the advantages of high speed and automatic acquisition of the full-dial fluorescence ultra-broad spectrum image.
The invention has the outstanding advantages that the design of using the filter in the traditional scheme is cancelled, so that the time expenditure and the mechanical vibration in the process of switching the filter are avoided, and the slit light can be quickly and accurately translated.
In a further preferred embodiment, for example, a new focusing device is designed, which includes two light sources of different colors located at different spatial positions and a control device, and when lens focusing is performed, two colors of light emitted by the two light sources enter the camera through the microscope objective, and an actual focal position is calculated according to images of the two light sources acquired by the camera and a difference between the two light sources in the spatial positions, and thereby the lens focusing of the camera is controlled. The traditional focusing is carried out in a manual mode, the efficiency of the manual focusing mode is extremely low, and the automatic acquisition of the full-glass fluorescence spectrum image is difficult to realize by combining with a control program of the DMD. In the preferable scheme of the invention, if the camera is in a defocusing position, because the two light sources with different colors are different in spatial position, the images of the two light sources captured by the camera have difference, the actual focus position can be quickly calculated by calculating the difference between the two images through mutual information, so that the lens can be controlled to realize quick focusing, a large amount of time expenditure in the manual focusing process is avoided, and the quick acquisition of the full-slide hyperspectral image is easily realized by matching with a program for controlling a DMD.
Drawings
FIG. 1 is a schematic diagram of a full-glass fluorescence spectrum fast acquisition device according to an embodiment of the present invention;
FIG. 2 is a schematic diagram of the current field of view fluorescence ultra-broad spectrum stitching according to an embodiment of the present invention;
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.
Fluorescence spectroscopy as referred to herein encompasses the concept of fluorescence hyperspectral or fluorescence hyperspectral.
The invention provides a method and a device for rapidly acquiring a full-glass slide fluorescence spectrum, which combine spectral image acquisition and fluorescence detection to acquire a fluorescence hyperspectral image of a slice, and realize rapid and high-quality acquisition of the fluorescence spectrum image of the whole slide. The method and the device of the invention are beneficial to realizing more accurate disease diagnosis or disease diagnosis which can not be finished by the traditional method.
Referring to fig. 1 to 2, the method for rapidly acquiring a full-glass fluorescence spectrum according to an embodiment of the present invention includes the following steps:
s1, moving the stage 7 carrying the slide 6 to align the microscope objective 8 with the first field of view of the slide 6 and to bring the lens 13 of the camera 14 into focus with respect to the first field of view of the slide 6;
S2, projecting the slit excitation light to the surface of the sample on the glass slide 6 to excite fluorescence, dispersing the excited fluorescence through the prism 12, and collecting a fluorescence spectrum image of the dispersed first camera pixel position through the camera 14;
s3, translating the slit excitation light by one pixel position, and collecting the fluorescence spectrum image of the next pixel position until the fluorescence spectrum images of all the pixel positions in the current field of view are collected;
s4, moving the stage 7 to align the microscope objective 8 with the next field position of the slide 6, and repeating the corresponding processes in steps S1 to S3 until the images of all the fields of the slide 6 are acquired;
s5, combining the fluorescence spectrum images of all pixel positions in each field of view to obtain a fluorescence spectrum image of each field of view;
and S6, obtaining a fluorescence spectrum image of the whole glass slide 6 by carrying out image splicing on the spectrum images of all the fields of view.
In a preferred embodiment, in step S1, two light sources 4 and 5 of different colors disposed at different spatial positions are used, the two light sources 4 and 5 are turned on when the lens 13 is focused, the two colors of light emitted by the two light sources 4 and 5 enter the camera 14 through the microscope objective 8, and an actual focal position is calculated according to the images of the two light sources 4 and 5 acquired by the camera 14 and the difference between the spatial positions of the two light sources 4 and 5, so as to control the lens 13 of the camera 14 to focus. In a preferred embodiment, the two light sources 4, 5 are red and green LED lamps, respectively.
Taking two light sources 4 and 5 adopting red and green LED lamps as an example, the focal distance is determined and the focus is fixed based on the images of the light emitted by the red and green LED lamps. If the camera 14 is in a defocusing position, due to the difference of the two red and green LEDs and the like in the space position, the pictures of the two red and green light channels captured by the camera 14 have difference, the actual focus position can be quickly calculated by calculating the difference between the two images through mutual information, the ultrasonic motor of the lens 13 is moved through the electric control platform, the quick focusing can be realized, and the quick acquisition of the full-glass hyperspectral image can be easily realized by matching with a program for controlling the DMD 3.
In a preferred embodiment, in step S2, light emitted from the excitation light source 1 passes through the collimating lens 2 and is modulated into parallel light, the parallel light is projected onto the slit excitation light modulation device and is modulated into parallel slit excitation light by the slit excitation light modulation device, the slit excitation light is deflected by 90 degrees by the beam splitter 11, only the slit excitation light capable of exciting a fluorescence wavelength range is retained after passing through the optical filter 10 and the tube lens 9(tube lens), the slit fluorescence is excited by the sample surface projected onto the slide glass 6 after passing through the microscope objective 8, the tube lens 9, the optical filter 10, and the beam splitter 11, the fluorescence is dispersed in a direction perpendicular to the slit by the prism 12 after passing through the microscope objective 8, the tube lens 9, the optical filter 10, and the beam splitter 11, and the dispersed fluorescence enters the camera 14 for imaging.
In a preferred embodiment, the light emitted from the excitation light source 1 is modulated by a DMD 3 (digital micromirror device) to form the slit excitation light, and the translation of the slit excitation light is realized by controlling the state of the DMD 3.
In other embodiments, the light emitted from the excitation light source 1 may also be modulated by a slit array (slit excitation light), and the slit excitation light is translated by moving the stage 7.
Both of the above-described ways can be implemented to project the excitation light of the slit onto the sample. The first method requires the slit position to be changed by moving the stage 7, but requires a high degree of control accuracy for the stage 7 because each movement requires one camera pixel to be moved. The second method can translate the slit by controlling the state of the DMD 3 by a program, and is higher than the first method in speed and accuracy because the DMD 3 needs only about 15 microseconds to flip from one state to another and the control accuracy is extremely high.
In some embodiments, the excitation light source 1 may be a mercury lamp.
Referring to fig. 1 to 2, the full-glass fluorescence spectrum fast acquisition apparatus according to the embodiment of the present invention includes: the device comprises a carrying table 7 for carrying a glass slide 6, a microscope, a camera 14, an excitation light source 1, a slit excitation light modulation device, a prism 12 and an image processing device, wherein excitation light emitted by the excitation light source 1 is modulated into slit excitation light by the slit excitation light modulation device, fluorescence is excited on the surface of a sample projected on the glass slide 6, the excited fluorescence is dispersed through the prism 12, and a dispersed fluorescence spectrum image of a first camera pixel position is collected through the camera 14; translating the slit exciting light by one pixel position, and collecting a fluorescence spectrum image of the next pixel position until the fluorescence spectrum images of all the pixel positions in the current view field are collected; moving the object stage 7 to make the microscope objective 8 align with the next field position of the slide 6, and repeatedly acquiring fluorescence spectrum images until all the images of all the fields of the slide 6 are acquired; the image processing device combines the fluorescence spectrum images of all the pixel positions in each view field to obtain the fluorescence spectrum image of each view field, and performs image splicing on the spectrum images of all the view fields to obtain the fluorescence spectrum image of the whole glass slide 6.
In some embodiments, the slit excitation light modulation device is an array of slits, and the translation of the slit excitation light is achieved by moving the stage 7.
In a preferred embodiment, the slit excitation light modulation device is a DMD3, and the translation of the slit excitation light is realized by controlling the state of the DMD 3.
In a preferred embodiment, the whole-slide fluorescence spectrum fast acquisition device further comprises a focusing device, the focusing device comprises two light sources with different colors arranged at different spatial positions and a control device, when the lens 13 is focused, the two colors of light emitted by the two light sources enter the camera 14 through the microscope objective lens 8, and the control device calculates an actual focal position according to the images of the two light sources acquired by the camera 14 and the difference of the two light sources in the spatial positions, and controls the lens 13 of the camera 14 to focus accordingly.
Specific embodiments of the present invention are further described below.
Fig. 1 is a schematic diagram of a full-glass fluorescence spectrum rapid acquisition device according to an embodiment. Light emitted from the mercury lamp is imaged in the camera 14 through the following procedure.
Is modulated into parallel light after passing through a collimating lens 2; the parallel light is projected on the DMD3 and is modulated into exciting light with parallel slits through the DMD 3; the exciting light of the parallel slit is deflected by 90 degrees after passing through the spectroscope 11; only light in a wavelength range capable of exciting fluorescence is reserved after the exciting light passes through the incident light filter 10 and the tube lens 9; exciting light is projected to the surface of the sample to excite fluorescence after passing through the objective lens 8 of 20 times; the excitation light of the slit can only excite slit-shaped fluorescence, and the fluorescence passes through the objective lens 8, the tube lens 9 and the optical filter 10 in sequence; the fluorescence passes through the prism 12 and is dispersed in the direction perpendicular to the slit; the dispersed light is imaged after passing through a lens 13 and a camera 14. After the fluorescence spectrum of the current pixel position is obtained, the slit exciting light is translated by one pixel position through controlling the DMD3, and the fluorescence spectrum image of the next pixel position is obtained.
In this manner, fluorescence spectrum images of all pixel positions of the current field of view are acquired. And when the fluorescent spectrum images of all pixel positions in one field of view are acquired, moving to the next field of view. And combining the fluorescence spectrum images of all the pixel positions in each field to obtain the fluorescence spectrum image of each field, and splicing the spectrum images of all the fields to obtain the fluorescence spectrum image of the whole glass slide 6.
In one embodiment, the method of acquiring a fluorescence spectrum image of the entire slide 6 includes the steps of:
moving the stage 7 to align the objective lens 8 with the position of the first field of view;
turning on the two red and green LED lamps, acquiring images by the camera 14, calculating the position of a focus according to the images of the two red and green light channels, and adjusting the focus by moving the ultrasonic motor of the lens 13;
the DMD 3 projects slit light onto a sample to excite fluorescence, the excited linear fluorescence is dispersed through the prism 12 and captured by the camera 14, the DMD 3 is regulated and controlled to move the slit, dispersion images of different pixel positions are obtained, and the hyperspectral image of the current field of view can be obtained by combining different rows of the dispersion images.
Assuming that there are 80 camera pixel locations between adjacent slits of the set projection, one fluorescence slit is divided into 76 by the dispersed spectral range. The algorithm flow for acquiring the hyperspectral image is as follows:
Regulating and controlling a slit array to be positioned at the current view field position to acquire a picture p1，p1According to the wavelength, the wave length can be divided into 76 different wavelength bands p1si，i＝1，2，3…76。
Secondly, regulating and controlling the position of the projected slit through the DMD to obtain images p of different pixel positionsjJ is 1, 2, 3 …, 80. Each corresponding picture can be divided into 76 different wavelength bands, and an image p with different wavelength bands is obtainedjsi，j＝1，2，3…80，i＝1，2，3…76
Combining the images of the 80 pictures in different wavelength bands to obtain the ultra-broad spectrum image of the current field of view. P is to bejs1Combining the images of 1, 2, 3 … 80 to obtain the current field wavelength s1Similarly, combining other columns can obtain an image s of 76 wavelength bands of the current field of view1～s76。
And after the hyperspectral image of the current field of view is acquired, moving the objective table to the next field of view position, and repeating the process from the adjustment of the focal point until the images of all the fields of view of the slide are acquired.
And after acquiring the fluorescence spectrum images of all the fields of view, carrying out image splicing through imageJ to obtain the fluorescence spectrum image of the whole glass slide.
Fig. 2 is a schematic diagram of a method for obtaining a fluorescence spectrum cube of a current field by stitching 80 images acquired in one field. Assuming that there are 80 pixel positions between adjacent slits, moving the slits in the current field of view will obtain 80 images, dividing each image into 76 columns according to the wavelength difference, and splicing the same columns of different images to obtain fluorescence images corresponding to different wavelengths in the current field of view.
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 full-glass-slide fluorescence spectrum rapid acquisition method is characterized by comprising the following steps:
s1, moving the slide-carrying stage to align the microscope objective with the first field of view of the slide and to focus the lens of the camera with respect to the first field of view of the slide;
s2, projecting the slit excitation light to the surface of the sample on the glass slide to excite fluorescence, dispersing the excited fluorescence through a prism, and collecting a dispersed fluorescence spectrum image of a first camera pixel position through a camera;
s3, translating the slit excitation light by one pixel position, and collecting the fluorescence spectrum image of the next pixel position until the fluorescence spectrum images of all the pixel positions in the current field of view are collected;
s4, moving the object stage to align the microscope objective lens with the next field position of the slide, and repeating the corresponding processes in the steps S1-S3 until the images of all the fields of the slide are acquired;
s5, combining the fluorescence spectrum images of all pixel positions in each field of view to obtain a fluorescence spectrum image of each field of view;
and S6, obtaining a fluorescence spectrum image of the whole glass slide by image splicing of the spectrum images of all the fields of view.
2. The method for fast acquiring fluorescence spectrum of full-glass slide as claimed in claim 1, wherein in step S1, two light sources with different colors are used and arranged at different spatial positions, the two light sources are turned on when the lens is focused, so that the two colors of light emitted by the two light sources enter the camera through the microscope objective, and the actual focal position is calculated according to the images of the two light sources acquired by the camera and the difference of the two light sources in spatial position, thereby controlling the lens of the camera to focus.
3. The method for fast acquiring full-glass fluorescence spectrum according to claim 2, wherein in step S1, the two light sources are red and green LED lamps, respectively.
4. The method for fast obtaining the fluorescence spectrum of the full-glass slide as claimed in any one of claims 1 to 3, wherein in step S2, the light emitted from the excitation light source is modulated into parallel light after passing through the collimating lens, the parallel light is projected on a slit excitation light modulation device and modulated into parallel slit excitation light by the slit excitation light modulation device, the slit exciting light is deflected by 90 degrees after passing through the spectroscope, only the slit exciting light capable of exciting the fluorescence wavelength range is reserved after passing through the optical filter and the lens barrel lens, the slit exciting light is projected to the surface of the sample on the glass slide after passing through the microscope objective lens to excite slit-shaped fluorescence, the fluorescence passes through the microscope objective lens, the tube lens, the optical filter and the spectroscope in sequence, and then the prism is used for carrying out dispersion in the direction vertical to the slit, and the dispersed fluorescence enters a camera for imaging.
5. The method for rapid acquisition of fluorescence spectrum from full-glass slide according to any of claims 1 to 4, wherein the light emitted from the excitation light source is modulated by the slit array to form the slit excitation light, and the translation of the slit excitation light is realized by moving the stage.
6. The method for rapidly acquiring the fluorescence spectrum of the full-glass slide according to any one of claims 1 to 4, wherein light emitted from an excitation light source is modulated by a DMD to form the slit excitation light, and the translation of the slit excitation light is realized by controlling the state of the DMD.
7. The full-glass fluorescence spectrum rapid acquisition method according to any one of claims 1 to 6, wherein the excitation light source is a mercury lamp.
8. A full-glass fluorescence spectrum rapid acquisition device is characterized by comprising a slide glass bearing object stage, a microscope, a camera, an excitation light source, a slit excitation light modulation device, a prism and an image processing device, wherein excitation light emitted by the excitation light source is modulated into slit excitation light by the slit excitation light modulation device, fluorescence is excited on the surface of a sample projected onto the slide glass, the excited fluorescence is dispersed through the prism, and a dispersed fluorescence spectrum image of a first camera pixel position is acquired through the camera; translating the slit exciting light by one pixel position, and collecting a fluorescence spectrum image of the next pixel position until the fluorescence spectrum images of all the pixel positions in the current view field are collected; moving the objective table to enable the microscope objective to be aligned to the next view field position of the glass slide, and repeatedly acquiring fluorescence spectrum images until all the images of all the view fields of the glass slide are acquired; the image processing device combines the fluorescence spectrum images of all pixel positions in each view field to obtain the fluorescence spectrum image of each view field, and performs image splicing on the spectrum images of all the view fields to obtain the fluorescence spectrum image of the whole glass slide.
9. The full-slide fluorescence spectrum fast acquisition device according to claim 8, wherein the slit excitation light modulation device is a slit array, and the translation of the slit excitation light is realized by moving the stage, or the slit excitation light modulation device is a DMD, and the translation of the slit excitation light is realized by controlling the DMD.
10. The full-glass fluorescence spectrum fast acquisition device according to claim 8 or 9, further comprising a focusing device, wherein the focusing device comprises two light sources with different colors arranged at different spatial positions and a control device, when lens focusing is performed, the two colors of light emitted by the two light sources enter the camera through the microscope objective, and the control device calculates an actual focal position according to the images of the two light sources acquired by the camera and the difference of the two light sources in the spatial positions, and controls the focusing of the lens of the camera accordingly.
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