A high-speed, large-field-of-view microscopic imaging device and its working method

Abstract

This invention discloses a high-speed, large-field-of-view microscopic imaging device and its operating method. The microscopic imaging device includes a light source system, a microscopic imaging system, a beam deflection system, a photoelectric sensor system, and a control system. After the light from the light source system illuminates the sample, the sample outputs a light signal to be measured. The microscopic imaging system collects the light signal, which is then deflected by the beam deflection system and focused onto the photoelectric sensor system. The control system synchronously controls the light source system, the beam deflection system, and the photoelectric sensor system. The photoelectric sensor system alternately integrates and converts the light signal, thereby achieving higher shooting speed and a larger image size in global exposure mode. This invention solves the problems of inconsistent exposure times and limited image size in traditional high-speed imaging technologies.

Description

Technical Field

[0001] The present invention relates to the field of microscopic imaging technology, and in particular to a high-speed large-viewing-field microscopic imaging device and a working method thereof. Background Art

[0002] One development direction in the field of optical imaging is how to capture high-speed biological processes. By achieving higher imaging speeds, the details of high-speed biological processes can be captured, which can provide a basis for further biological research.

[0003] The acquisition of optical signals is mainly completed by photoelectric conversion devices. Commonly used photoelectric conversion devices include photomultiplier tubes (PMT), charge-coupled devices (CCD), and metal complementary oxide semiconductor (CMOS) technologies. The sensors mainly involved in high-speed imaging are CCD and CMOS devices. With the development of optoelectronic devices in recent years, especially the emergence of scientific research-grade high-speed low-noise cameras (sCMOS), photoelectric sensors can achieve higher frame rates, and the full-frame image acquisition speed can reach 100fps. The frame rate can be further increased by adjusting the image size (ROI), thus meeting the needs of most biological research.

[0004] However, for some ultrafast biological processes, such as the study of the dynamic process of membrane potential, a time resolution of close to 1ms is often required. At this time, even using high-speed sCMOS, the ROI can only be limited to a very small range, usually only 100-200 pixels in height, which limits the acquisition and analysis of large-scale signals.

[0005] At the same time, when using sCMOS directly for high-speed imaging, due to the limitations of the sCMOS structure, a rolling shutter method is usually used. This method can achieve a higher imaging speed, but the exposure time of each row in the image is different, which will cause errors in situations where time analysis is required, making analysis difficult and increasing errors. Summary of the invention

[0006] The present invention provides a high-speed, large-field-of-view microscopic imaging device and a working method thereof, so as to solve one or more technical problems existing in the prior art, and at least provide a beneficial choice or create conditions.

[0007] In a first aspect, an embodiment of the present invention provides a high-speed and large-field-of-view microscopic imaging device, comprising:

[0008] A light source system, used to provide the light source required for microscopic imaging;

[0009] A microscopic imaging system, used to receive and focus light signals emitted by the sample;

[0010] A beam deflection system, used for receiving the optical signal output by the microscopic imaging system, and controlling the position where the optical signal converges to switch between different photoelectric sensors in the photoelectric sensor system and different sub-areas on the sensor photosensitive surface;

[0011] A photoelectric sensor system, comprising at least two photoelectric sensors, wherein a photosensitive surface of each photoelectric sensor comprises N sub-regions, a switching time between two positions is less than a time resolution required by the system, each sensor is used to alternately perform integration of light signals and signal conversion, and after the signal conversion, digital data of images of the N sub-regions on each photoelectric sensor is obtained, and the digital data is transmitted to a control system, wherein N ≥ 2;

[0012] The control system is used to synchronously and orderly control the light source system, the beam deflection system, and the photoelectric sensor system, and to obtain a complete time series of images within the entire field of view based on the digitized data of the images of N sub-areas of each photoelectric sensor.

[0013] Further, each sensor is used to alternately perform integration of light signals and signal conversion, including:

[0014] When the light signal converges to the i-th photoelectric sensor, the i-th photoelectric sensor integrates the light signal, where 1≤i≤M, M is the number of photoelectric sensors, and M≥2;

[0015] During the process of the i-th photoelectric sensor integrating the light signal, the j-th photoelectric sensor that has not yet integrated the light signal converts the light signal it has integrated to obtain digital data of the image of N sub-areas on the j-th photoelectric sensor, where 1≤j≤M and j≠i.

[0016] Furthermore, the light source system includes a light source and a lens, and the light emitted by the light source is converged by the lens and then output.

[0017] Furthermore, the microscopic imaging system includes a microscope objective, a dichroic mirror, a first focusing lens, a reflector, an aperture and a second focusing lens. The light output from the light source system is reflected by the dichroic mirror to the microscope objective and then irradiated onto the sample. The sample emits a light signal, which passes through the microscope objective and the dichroic mirror in sequence. The light signal transmitted from the dichroic mirror passes through the first focusing lens and the reflector in sequence. The light signal reflected from the transmitting mirror passes through the aperture and the second focusing lens in sequence.

[0018] Furthermore, the beam deflection system includes an optical deflector and a third focusing lens, and the control system controls the optical deflector to deflect at different angles, so that the optical signal output from the microscopic imaging system is sequentially focused on each photoelectric sensor and different sub-areas of each photoelectric sensor after passing through the third focusing lens.

[0019] Furthermore, the optical deflector is any one of a galvanometer galvanometer mirror, a piezoelectric resonant galvanometer mirror and a polygonal mirror.

[0020] Furthermore, when the light deflector is a galvanometer galvanometer, the light beam deflection system also includes a triangular beam splitter, and the at least two photoelectric sensors include a first photoelectric sensor and a second photoelectric sensor. The light signal output from the microscopic imaging system passes through the galvanometer needle mirror, the third focusing lens and the triangular beam splitter in sequence, and is focused on the first photoelectric sensor and the second photoelectric sensor in sequence after being split by the triangular beam splitter. The first photoelectric sensor and the second photoelectric sensor collect the light signal emitted by the sample.

[0021] In a second aspect, an embodiment of the present invention further provides a working method of a high-speed and large-field-of-view microscopic imaging device, characterized in that the high-speed and large-field-of-view microscopic imaging device includes a light source system, a microscopic imaging system, a beam deflection system, a photoelectric sensor system and a control system, the photoelectric sensor system includes M photoelectric sensors, and the photosensitive surface of each photoelectric sensor includes N sub-areas, wherein M≥2, N≥2; the method includes:

[0022] Turn on the light source system, the light emitted by the light source system shines on the sample, the sample emits a light signal, and the microscopic imaging system collects the light signal;

[0023] The control system controls the beam deflection system to deflect so that the light signal is sequentially converged on each of the M photoelectric sensors, and when the light signal converges on the i-th photoelectric sensor, the control system controls the light signal to converge on the area 1 to the area N of the i-th photoelectric sensor in sequence, and the i-th photoelectric sensor integrates the light signal, where 1≤i≤M;

[0024] In the process of the i-th photoelectric sensor integrating the light signal, the j-th photoelectric sensor that has not integrated the light signal converts the light signal that has been integrated to obtain digital data of the image of N sub-areas on the j-th photoelectric sensor, and transmits the digital data to the control system, where 1≤j≤M, and j≠i;

[0025] The control system obtains a complete time series of images within the entire field of view according to the digitized data of the images of the N sub-areas of each photoelectric sensor.

[0026] Furthermore, the control system controls the deflection of the beam deflection system so that the position where the light signal converges switches between different photoelectric sensors in the photoelectric sensor system and different sub-areas on the sensor's photosensitive surface, and the switching time between the two positions is less than the time resolution required by the system.

[0027] Furthermore, the light source system is turned on when the direction of the beam deflector is stable, and is turned off during switching between different positions.

[0028] A high-speed, large-field-of-view microscopic imaging device and a working method thereof according to an embodiment of the present invention have at least the following beneficial effects:

[0029] 1. The present invention solves the problem that the traditional imaging method needs to reduce the imaging size during high-speed imaging, and can provide a larger imaging size during high-speed imaging.

[0030] 2. The present invention solves the problem of inconsistent exposure time corresponding to each row when using a rolling shutter method for imaging, thereby ensuring synchronization of the exposure time of each row in the image.

[0031] 3. The present invention uses multiple photoelectric sensors to separate the two processes of light signal integration and integrated signal readout of the photoelectric sensors, thereby preventing the readout process from affecting the integration process, ensuring the synchronization of image exposure, and also increasing the image acquisition speed. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The accompanying drawings are used to provide a further understanding of the technical solution of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the technical solution of the present invention and do not constitute a limitation on the technical solution of the present invention.

[0033] Figure 1 A schematic structural diagram of a high-speed, large-field-of-view microscopic imaging device provided by an embodiment of the present invention;

[0034] Figure 2 A flow chart of a working method of a high-speed and large-field-of-view microscopic imaging device provided by an embodiment of the present invention;

[0035] Figure 3 A schematic structural diagram of a high-speed, large-field-of-view microscopic imaging device provided by another embodiment of the present invention;

[0036] Figure 4 A timing diagram of imaging of a high-speed, large-field-of-view microscopic imaging device provided by an embodiment of the present invention;

[0037] Figure 5 A schematic diagram of a CMOS target surface and sub-images of a high-speed, large-field-of-view microscopic imaging device provided by an embodiment of the present invention;

[0038] Explanation of the accompanying drawings: 1-light source system; 2-sample; 3-microscope imaging system; 4-beam deflection system; 5-photoelectric sensor system; 6-control system; 1-1-laser; 1-2-lens; 3-1-microscope objective; 3-2-dichroic mirror; 3-3-first focusing lens; 3-4-reflector; 3-5-aperture; 3-6-second focusing lens; 4-1-galvanometer galvanometer; 4-2-third focusing lens; 4-3-triangular beam splitter; 5-1-first photoelectric sensor; 5-2-second photoelectric sensor. DETAILED DESCRIPTION

[0039] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0040] It should be noted that, although the functional modules are divided in the system schematic diagram and the logical order is shown in the flowchart, in some cases, the steps shown or described may be performed in a different order than the module division in the system or the order in the flowchart. The terms "first", "second", etc. in the specification, claims and the above drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence.

[0041] Figure 1 Provided is a high-speed, large-field-of-view microscopic imaging device, comprising a light source system 1, a microscopic imaging system 3, a beam deflection system 4, a photoelectric sensor system 5, and a control system 6;

[0042] Light source system 1, used to provide light source required for microscopic imaging;

[0043] A microscopic imaging system 3, used for receiving and focusing light signals emitted by the sample 2;

[0044] The beam deflection system 4 is used to receive the optical signal output by the microscopic imaging system 3 and control the convergence position of the optical signal to switch between different photoelectric sensors in the photoelectric sensor system 5 and different sub-areas on the sensor photosensitive surface;

[0045] The photoelectric sensor system 5 includes at least two photoelectric sensors, the photosensitive surface of each photoelectric sensor includes N sub-areas, the switching time between two positions is less than the time resolution required by the system, each sensor is used to alternately integrate the light signal and convert the signal, and after the signal conversion, the digital data of the image of the N sub-areas on each photoelectric sensor is obtained, and the digital data is transmitted to the control system;

[0046] The control system 6 is used to synchronously and orderly control the light source system 1, the beam deflection system 4, and the photoelectric sensor system 5, and to obtain a complete time series of images within the entire field of view based on the digitized data of the images of N sub-areas of each photoelectric sensor.

[0047] Specifically, the control system 6 is used to synchronously and orderly control the light source system 1, the beam deflection system 4, and the photoelectric sensor system 5 ( Figure 1 The connection relationship of the control system 6 is not shown in the figure), the light source system 1 is used to provide the light source required for microscopic imaging such as exciting fluorescence or bright field illumination, and the sample 2 is placed on the light path where the light source is located, so that the light source shines on the sample 2, and the sample 2 can be excited or illuminated by bright field illumination so that the sample 2 reflects light. The microscopic imaging system 3 is used to receive and converge the light signal emitted by the sample 2 and perform imaging, the beam deflection system 4 controls the light signal emitted by the sample to converge to different photoelectric sensors and different positions of the photoelectric sensors, and the photoelectric sensor system 5 is used to alternately integrate the light signal and convert the signal. The photoelectric sensor at the position where the light signal converges integrates the light signal, and the photoelectric sensor that does not integrate the light signal converts and transmits the signal, and finally obtains a complete time series image within the entire field of view, thereby realizing exposure time synchronization at ultra-fast shooting speeds and larger imaging sizes.

[0048] In one embodiment, the light source system 1 includes a light source and a lens, and the light emitted by the light source is output after being converged by the lens. The light source can be an illumination light source, or a laser light source, or a light source for exciting fluorescence or bright field illumination, for example, an LED lamp, a mercury lamp, a halogen lamp, etc., or a laser, such as a pulsed laser.

[0049] Figure 1 Working method of medium-speed and large-field microscopic imaging device, such as Figure 2 As shown, including:

[0050] S21, turning on the light source system, irradiating the sample with light emitted by the light source system, causing the sample to emit a light signal, and the microscopic imaging system to collect the light signal;

[0051] S22, the control system controls the light beam deflection system to deflect, so that the light signal is sequentially converged on each of the M photoelectric sensors, and when the light signal converges on the i-th photoelectric sensor, the control system controls the light signal to converge on the area 1 to the area N of the i-th photoelectric sensor in sequence, and the i-th photoelectric sensor integrates the light signal, where 1≤i≤M, and M≥2;

[0052] S23, when the i-th photoelectric sensor integrates the light signal, the j-th photoelectric sensor that has not integrated the light signal converts the light signal that has been integrated to obtain digital data of the image of the N sub-areas on the j-th photoelectric sensor, and transmits the digital data to the control system, wherein 1≤j≤M, and j≠i;

[0053] S24. The control system obtains a complete time series of images within the entire field of view according to the digitized data of the images of the N sub-areas of each photoelectric sensor.

[0054] In one embodiment, the control system controls the deflection of the beam deflection system so that the position where the light signal converges switches between different photoelectric sensors in the photoelectric sensor system and different sub-areas on the sensor's photosensitive surface, and the switching time between the two positions is less than the time resolution required by the system.

[0055] In one embodiment, the light source system is turned on when the pointing of the beam deflection system is stable and is turned off during switching between different positions.

[0056] In one embodiment, the beam deflection system includes a light deflector, and the control system controls the light deflector to deflect at different angles, so that the output light signal is sequentially focused on each photoelectric sensor and different sub-areas of each photoelectric sensor. Specifically, the control system controls the light deflector to rotate at different angles according to the number of photoelectric sensors so that the light signal is collected by the first photoelectric sensor, the second photoelectric sensor, ... the Mth photoelectric sensor.

[0057] The light beam deflection system also includes a focusing lens. The light signal output by the light deflector is sequentially focused on each photoelectric sensor and different sub-areas of each photoelectric sensor after passing through the focusing lens.

[0058] The light deflector is any one of a galvanometer oscillator, a piezoelectric resonant oscillator and a polygonal mirror, or other devices that can deflect light, and those skilled in the art can set it according to actual needs.

[0059] Figure 3 A specific structural schematic diagram of a high-speed, large-field-of-view microscopic imaging device is provided. The light source system 1 includes a laser 1-1 and a lens 1-2; the microscopic imaging system 3 includes a microscope objective 3-1, a dichroic mirror 3-2, a first focusing lens 3-3, a reflector 3-4, an aperture 3-5, and a second focusing lens 3-6; the beam deflection system 4 includes a galvanometer galvanometer, a piezoelectric resonant galvanometer, a multi-faceted rotating mirror, and other devices that can rapidly deflect the light beam. Figure 3 In the figure, the beam deflection system 4 includes a galvanometer galvanometer mirror 4-1; in addition, the beam deflection system 4 also includes a third focusing lens 4-2 and a triangular beam splitter 4-3.

[0060] The number of sensors and the number of sub-images on each sensor can be set according to actual needs. Figure 3 In the embodiment, the photoelectric sensor system 5 includes two photoelectric sensors, namely a first photoelectric sensor 5-1 and a second photoelectric sensor 5-2, and four sub-images are arranged on the target surface of each photoelectric sensor. The first photoelectric sensor 5-1 and the second photoelectric sensor 5-2 both include SCMOS devices.

[0061] like Figure 3 As shown, a lens 1-2 and a dichroic mirror 3-2 are sequentially arranged on the optical path of the laser 1-1. The dichroic mirror 3-2 is arranged at a certain angle so that the laser beam is incident on the dichroic mirror 3-2 at a certain incident angle. The microscope objective lens 3-1 and the sample 2 are arranged on the reflection optical path of the dichroic mirror 3-2 so that the light emitted by the laser is converged by the lens 1-2 and then output. The light output from the lens 1-2 is reflected by the dichroic mirror 3-2 to the microscope objective lens 3-1 and then irradiated onto the sample 2, thereby stimulating the sample 2 to emit a light signal, which sequentially passes through the microscope objective lens 3-1 and the dichroic mirror 3-2 and is then emitted from the dichroic mirror 3-2. 2 transmits a light signal, a first focusing lens 3-3 and a reflector 3-4 are sequentially arranged on the transmission light path of the dichroic mirror 3-2, an aperture 3-5, a second focusing lens 3-6 and a galvanometer galvanometer 4-1 are sequentially arranged on the reflection light path of the reflector 3-4; a third focusing lens 4-2 and a triangular beam splitter 4-3 are sequentially arranged on the deflection light path of the galvanometer galvanometer 4-1, a first photoelectric sensor 5-1 and a second photoelectric sensor 5-2 are symmetrically arranged on both sides of the triangular beam splitter 4-3, so that the first photoelectric sensor 5-1 and the second photoelectric sensor 5-2 can collect the light signal emitted by the sample 2.

[0062] In one embodiment, the dichroic mirror 3 - 2 is arranged at a certain angle so that the laser beam is incident on the dichroic mirror 3 - 2 at a certain incident angle. The incident angle may be greater than or equal to 45°, and in one embodiment, may be 45°.

[0063] Figure 3 The working principle of the high-speed and large-field microscopic imaging device is combined with Figure 4 The timing diagram analysis is as follows:

[0064] When the voltage of the Y galvanometer mirror 4-1 is voltage 1, the Y axis of the galvanometer mirror 4-1 is deflected to the target surface of the first photoelectric sensor 5-1. When the voltage of the Y galvanometer mirror 4-1 is voltage 2, the Y axis of the galvanometer mirror 4-1 is deflected to the target surface of the second photoelectric sensor 5-2.

[0065] When the Y axis of the galvanometer galvanometer mirror 4-1 is deflected to the target surface of the first photoelectric sensor 5-1, the voltage of the X galvanometer mirror is set to voltage 1, voltage 2, voltage 3 and voltage 4 respectively, corresponding to the X axis of the galvanometer galvanometer mirror 4-1 being deflected to the position of the first sub-image, the position of the second sub-image, the position of the third sub-image and the position of the fourth sub-image on the target surface of the first photoelectric sensor 5-1 (corresponding to Figure 4 P1, P2, P3 and P4 in the above table.

[0066] When the Y axis of the galvanometer galvanometer mirror 4-1 is deflected to the target surface of the second photoelectric sensor 5-2, the voltage of the X axis of the galvanometer galvanometer mirror 4-1 is set to voltage 4, voltage 3, voltage 2 and voltage 1 respectively, corresponding to the X axis of the galvanometer galvanometer mirror 4-1 being deflected to the position of the first sub-image, the position of the second sub-image, the position of the third sub-image and the position of the fourth sub-image on the target surface of the second photoelectric sensor 5-2 (corresponding to Figure 4 P5, P6, P7 and P8 in the figure).

[0067] The time for each voltage on the X-ray galvanometer to be maintained includes the light signal convergence time and the switching time. The light signal convergence time refers to the time when the light signal converges to the position of a sub-image of the first photoelectric sensor 5-1 or the second photoelectric sensor 5-2, and the switching time refers to the time for switching between two convergence positions. The light source remains on during the light signal convergence time and remains off during the switching time. Therefore, the light source is in a process of constantly turning on and off, and the control system controls the on and off of the light source.

[0068] Specifically, the Y axis of the galvanometer galvanometer mirror 4-1 is deflected to the target surface of the first photoelectric sensor 5-1, and the X axis is deflected to the position of the first sub-image on the target surface of the first photoelectric sensor 5-1. After the galvanometer galvanometer mirror 4-1 is stabilized, the light source system 1 is turned on, and the illumination light emitted by the laser 1-1 is irradiated on the sample 2 through the lens 1-2, the dichroic mirror 3-2 and the microscope objective lens 3-1. The sample 2 emits a light signal, which is collected by the microscopic imaging system 3. The light signal passes through the microscope objective lens 3-1, the dichroic mirror 3-2, the first focusing lens 3-3, the reflector 3-4, the aperture 3-5, the second focusing lens 3-6, the galvanometer galvanometer mirror 4-1, and the third focusing lens 4-2, and then is split and focused on the first photoelectric sensor 5-1 and the second photoelectric sensor 5-2 by the triangular beam splitter 4-3, and the light signal of the sample 2 is collected.

[0069] When the acquisition time arrives, the X-axis of the galvanometer mirror 4-1 deflects to the position of the second sub-image on the target surface of the first photoelectric sensor 5-1. After the galvanometer mirror 4-1 is stable, the laser 1-1 is turned on and the optical signal of the sample 2 is acquired.

[0070] When the acquisition time arrives, the X-axis of the galvanometer mirror 4 - 1 deflects to the position of the third sub-image on the target surface of the first photoelectric sensor 5 - 1 , and after the galvanometer mirror 4 - 1 is stabilized, the laser 1 - 1 is turned on to acquire the optical signal of the sample 2 .

[0071] When the acquisition time arrives, the X-axis of the galvanometer mirror 4 - 1 deflects to the position of the fourth sub-image on the target surface of the first photoelectric sensor 5 - 1 , and after the galvanometer mirror 4 - 1 is stabilized, the laser 1 - 1 is turned on to acquire the optical signal of the sample 2 .

[0072] When the first photoelectric sensor 5 - 1 collects light signals, the second photoelectric sensor 5 - 2 converts the collected signals and digitally transmits the signals of its four sub-images to the control system.

[0073] The Y-axis of the galvanometer galvanometer 4-1 is deflected to the target surface of the photoelectric sensor 5-2, and the X-axis of the galvanometer galvanometer 4-1 is deflected to the position of the first sub-image on the target surface of the second photoelectric sensor 5-2. After the galvanometer galvanometer 4-1 is stable, the light source system 1 is turned on and the light signal of the sample 2 is collected.

[0074] When the acquisition time arrives, the X-axis of the galvanometer mirror 4 - 1 deflects to the position of the second sub-image on the target surface of the second photoelectric sensor 5 - 2 . After the position of the galvanometer mirror 4 - 1 stabilizes, the light source system 1 is turned on and the light signal of the sample 2 is acquired.

[0075] When the acquisition time arrives, the X-axis of the galvanometer mirror 4 - 1 deflects to the position of the third sub-image on the target surface of the second photoelectric sensor 5 - 2 . After the position of the galvanometer mirror 4 - 1 stabilizes, the light source system 1 is turned on and the light signal of the sample 2 is acquired.

[0076] After the acquisition time arrives, the X-axis of the galvanometer galvanometer mirror 4-1 deflects to the position of the fourth sub-image on the target surface of the second photoelectric sensor 5-2. After the position of the galvanometer galvanometer mirror 4-1 is stable, the light source system 1 is turned on and the light signal of the sample 2 is acquired.

[0077] While the second photoelectric sensor 5 - 2 is collecting light signals, the first photoelectric sensor 5 - 1 converts the collected signals and digitally transmits the signals of its four sub-images to the control system.

[0078] Repeat the above optical signal collection process until all data collection is completed.

[0079] like Figure 4As shown, during the time when the X-axis deflects to the target surface of the first photoelectric sensor 5-1, the first photoelectric sensor 5-1 collects light signals, and the second photoelectric sensor 5-2 performs data conversion on the signals it has collected. During the time when the X-axis deflects to the target surface of the second photoelectric sensor 5-2, the first photoelectric sensor 5-1 performs data conversion on the signals it has collected, and the second photoelectric sensor 5-2 collects light signals. That is, the first photoelectric sensor 5-1 and the second photoelectric sensor 5-2 alternately integrate light signals and convert signals, which can separate the two processes of light signal integration and integrated signal readout of the photoelectric sensor, thereby preventing the readout process from affecting the integration process, ensuring the synchronization of image exposure, and also increasing the image acquisition speed.

[0080] The time for switching between sub-regions in each sensor is the sum of the switching time and the exposure time. The time for switching between sub-regions is equivalent to the temporal resolution, e.g. Figure 3 The switching time between P1 and P2 plus the exposure time of P2 is the time resolution. The time for the galvanometer galvanometer mirror 4-1 to switch to a small angle is less than 400us. Therefore, when the sampling time is 1ms, each sampling has an exposure time of more than 600us. At the same time, there is a data conversion time of about 4ms for the two photoelectric sensors. According to 10us, one line of conversion is completed. Two lines of calculation are converted at the same time, which can achieve image acquisition with a maximum height of 800 pixels. Figure 5 As shown, in this embodiment, the size of each sub-image is set to 500*500 pixels, and the image size obtained by the first SCMOS image sensor and the second SCMOS image sensor (corresponding to the first photoelectric sensor and the second photoelectric sensor) is 2048*2048 pixels, thereby being able to provide a larger imaging size during high-speed imaging; the conversion time is sufficient for reading the sub-image, and the acquisition process can also ensure that the exposure time of each row in each sub-image is consistent.

[0081] Those skilled in the art should know that Figure 3 The middle beam deflection system includes a galvanometer galvanometer 4-1, a third focusing lens 4-2 and a triangular beam splitter 4-3, and the photoelectric sensor system includes two photoelectric sensors. The photosensitive surface of each photoelectric sensor includes four sub-areas, which is only one embodiment and does not constitute a limitation of the embodiments of the present invention. Those skilled in the art can set other light deflectors according to actual needs, set M photoelectric sensors, and set N sub-areas for each photoelectric sensor. It is only necessary to control the light deflector to rotate at different angles according to the number of photoelectric sensors, M≥2, N≥2, for example, M can be 4, 6, 8, etc., and N can be 2, 6, 8, etc.

[0082] The present invention proposes a high-speed, large-field-of-view microscopic imaging device and a working method thereof, which solves the shortcomings of slow imaging speed and small imaging area in traditional technologies, not only improves the imaging speed, but also makes the imaging area larger, and also solves the problem of inconsistent exposure time of the same image. It can be applied to the field of biological research, such as high-speed imaging in cell membrane potential measurement.

[0083] The above is a specific description of the preferred implementation of the present invention, but the present invention is not limited to the above-mentioned implementation mode. Technical personnel familiar with the field can also make various equivalent deformations or substitutions without violating the spirit of the present invention. These equivalent deformations or substitutions are all included in the scope defined by the claims of the present invention.

Claims

1. A high-speed, large-field-of-view microscopic imaging device, characterized in that: include: A light source system, used to provide the light source required for microscopic imaging; A microscopic imaging system, used to receive and focus light signals emitted by the sample; A beam deflection system, used for receiving the optical signal output by the microscopic imaging system, and controlling the position where the optical signal converges to switch between different photoelectric sensors in the photoelectric sensor system and different sub-areas on the sensor photosensitive surface; A photoelectric sensor system includes at least two photoelectric sensors, each photosensitive surface of each photoelectric sensor includes N sub-areas, the switching time between two positions is less than the time resolution required by the system, each sensor is used to alternately integrate the light signal and convert the signal, after the signal conversion, the digital data of the image of the N sub-areas on each photoelectric sensor is obtained, and the digital data is transmitted to the control system, wherein N 2; The control system is used to synchronously and orderly control the light source system, the beam deflection system, and the photoelectric sensor system, and to obtain a complete time series of images within the entire field of view based on the digitized data of the images of N sub-areas of each photoelectric sensor.

2. The high-speed, large-field-of-view microscopic imaging device according to claim 1, characterized in that: Each sensor is used to alternately integrate the light signal and convert the signal including: When the light signal converges to the i-th photoelectric sensor, the i-th photoelectric sensor integrates the light signal, where , M is the number of photoelectric sensors, M 2; In the process of the i-th photoelectric sensor integrating the light signal, the j-th photoelectric sensor that has not yet integrated the light signal converts the light signal that has been integrated to obtain the digital data of the image of the N sub-areas on the j-th photoelectric sensor, where .

3. The high-speed, large-viewing-field microscopic imaging device according to claim 1, characterized in that: The light source system comprises a light source and a lens, and the light emitted by the light source is converged by the lens and then output.

4. The high-speed, large-viewing-field microscopic imaging device according to claim 1, characterized in that: The microscopic imaging system includes a microscope objective, a dichroic mirror, a first focusing lens, a reflector, an aperture and a second focusing lens. Light output from a light source system is reflected by the dichroic mirror to the microscope objective and then irradiated onto a sample. The sample emits a light signal, which passes through the microscope objective and the dichroic mirror in sequence. The light signal transmitted from the dichroic mirror passes through the first focusing lens and the reflector in sequence. The light signal reflected from the reflector passes through the aperture and the second focusing lens in sequence.

5. The high-speed, large-viewing-field microscopic imaging device according to claim 1, characterized in that: The beam deflection system includes an optical deflector and a third focusing lens. The control system controls the optical deflector to deflect at different angles so that the optical signal output from the microscopic imaging system is sequentially focused on each photoelectric sensor and different sub-areas of each photoelectric sensor after passing through the third focusing lens.

6. The high-speed, large-viewing-field microscopic imaging device according to claim 5, characterized in that: The optical deflector is any one of a galvanometer oscillator, a piezoelectric resonant oscillator and a polygonal mirror.

7. The high-speed, large-viewing-field microscopic imaging device according to claim 5, characterized in that: When the light deflector is a galvanometer galvanometer, the light beam deflection system also includes a triangular beam splitter, and the at least two photoelectric sensors include a first photoelectric sensor and a second photoelectric sensor. The light signal output from the microscopic imaging system passes through the galvanometer needle mirror, the third focusing lens and the triangular beam splitter in sequence, and is focused on the first photoelectric sensor and the second photoelectric sensor in sequence after being split by the triangular beam splitter. The first photoelectric sensor and the second photoelectric sensor collect the light signal emitted by the sample.

8. A method for operating a high-speed, large-field-of-view microscopic imaging device, characterized in that: The high-speed, large-viewing-field microscopic imaging device comprises a light source system, a microscopic imaging system, a beam deflection system, a photoelectric sensor system and a control system. The photoelectric sensor system comprises M photoelectric sensors, and the photosensitive surface of each photoelectric sensor comprises N sub-areas, wherein M 2, N 2; The method comprises: Turn on the light source system, the light emitted by the light source system shines on the sample, the sample emits a light signal, and the microscopic imaging system collects the light signal; The control system controls the beam deflection system to deflect, so that the light signal is sequentially converged on each of the M photoelectric sensors, and when the light signal converges on the i-th photoelectric sensor, the control system controls the light signal to converge on the area 1 to the area N of the i-th photoelectric sensor in sequence, and the i-th photoelectric sensor integrates the light signal, wherein ; In the process of the i-th photoelectric sensor integrating the light signal, the j-th photoelectric sensor that has not integrated the light signal converts the light signal that has been integrated to obtain the digital data of the image of the N sub-areas on the j-th photoelectric sensor, and transmits the digital data to the control system, wherein ; The control system obtains a complete time series of images within the entire field of view according to the digitized data of the images of the N sub-areas of each photoelectric sensor.

9. The working method according to claim 8, characterized in that: The control system controls the deflection of the beam deflection system so that the position where the light signal converges switches between different photoelectric sensors in the photoelectric sensor system and different sub-areas on the sensor's photosensitive surface, and the switching time between the two positions is less than the time resolution required by the system.

10. The working method according to claim 8, characterized in that: The light source system is turned on when the pointing direction of the beam deflector is stable and is turned off during the switching process between different positions.

Images