Multi-focus and multi-band channel automatic focusing confocal microscopic imaging system

Abstract

The invention provides a multi-focus and multi-band channel auto-focusing confocal microscopic imaging system, and relates to the technical field of microscopic imaging, the system comprises an excitation end, a detection end and a control module, the control module is connected with the excitation end and the detection end; according to the invention, through integration of advanced optical design and an automatic control algorithm, rapid and accurate focusing is realized, and imaging under multiple wavebands is supported at the same time.

Description

Technical Field

[0001] The present invention relates to the field of microscopic imaging technology, and more particularly to an automatic focusing confocal microscopic imaging system with multiple focal points and multiple waveband channels. Background Art

[0002] Currently, confocal microscopy, as an advanced imaging tool, is widely used in fields such as cell biology, tissue engineering and material research due to its superior spatial resolution and imaging capabilities.

[0003] However, traditional confocal microscope systems usually only support imaging of a single focus and wavelength, which imposes certain limitations on the observation of complex samples. For example, in biological samples, single focus scanning greatly limits the speed and detection efficiency of imaging. In addition, different cell structures and tissue components often need to be imaged at different wavelengths in order to obtain complete information. Although existing multi-band imaging technologies have the ability to acquire images at multiple wavelengths, they often require manual adjustment of the focus. This not only increases the complexity of the operation, but also leads to a further reduction in the imaging speed. When processing multi-layer samples, traditional technologies are difficult to focus quickly and accurately, often resulting in blurred images, affecting subsequent data analysis and interpretation. In addition, with the increase in sample complexity, the requirements for data processing capabilities and image quality are also constantly increasing, and the performance of existing systems in this regard often cannot meet the needs. In the biomedical field, especially in cell imaging and tissue section analysis, researchers are often faced with the challenge of how to efficiently obtain high-quality images at different depths and wavelengths. Due to the diversity of biological samples, many experiments need to be performed at high throughput to obtain sufficient data to support scientific research.

[0004] Therefore, how to realize a confocal microscopy system that automatically focuses and supports multi-band imaging is an urgent problem that needs to be solved by those skilled in the art. Summary of the invention

[0005] In view of this, the present invention provides an automatic focusing confocal microscopy imaging system with multiple focus points and multiple band channels, which realizes fast and precise focusing by integrating advanced optical design and automatic control algorithms, and supports imaging in multiple bands at the same time.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] An automatic focusing confocal microscopic imaging system with multiple focal points and multiple waveband channels comprises an excitation end, a detection end and a control module, wherein the control module is connected to the excitation end and the detection end.

[0008] Preferably, the excitation end comprises a laser, a laser adjustment device, a short-pass dichroic mirror, a two-dimensional galvanometer and a scanning device which are arranged in sequence.

[0009] Preferably, the laser adjustment device comprises a spectroscopic positioner, a polarizer, a correction lens and a first filter which are sequentially arranged on the output light path of the laser.

[0010] Preferably, the scanning device includes a scanning lens, a tube lens, a microscope objective lens, a three-dimensional electric displacement platform and a focusing detection device which are sequentially arranged on the output light path of the two-dimensional galvanometer, and a microscopic imaging sample is placed on the three-dimensional electric displacement platform.

[0011] Preferably, the focus detection device comprises a focus detection end filter and a focus detection camera which are arranged in sequence.

[0012] Preferably, the detection end comprises a second filter, a calibration lens, short-pass dichroic mirrors of multiple wavelength bands and a plurality of light intensity detector arrays which are sequentially arranged on the output light path of the short-pass dichroic mirror.

[0013] Preferably, the control module includes: a processing module, an autofocus control module, a light intensity acquisition switching module, a PMT array acquisition module, an electric galvanometer motor control module, a laser light intensity control module and a sample motion module;

[0014] The processing module is connected to the laser light intensity control module, the PMT array acquisition module, the autofocus control module, and the electric galvanometer motor control module; the autofocus control module is connected to the sample motion module and the light intensity acquisition switching module;

[0015] The laser light intensity control module and the light intensity acquisition switching module are both connected to the laser, the PMT array acquisition module is connected to the light intensity detector array, the electric galvanometer motor control module is connected to the two-dimensional galvanometer, and the sample motion module is connected to the three-dimensional electric displacement platform.

[0016] It can be seen from the above technical solutions that, compared with the prior art, the present invention discloses an automatic focusing confocal microscopy imaging system with multi-focus and multi-band channels. By introducing automatic focusing and multi-focus technology, it can effectively overcome the limitations of traditional systems, thereby achieving efficient and fast real-time confocal imaging. The automatic focusing function can automatically adjust the focus according to the height change of the sample, greatly reducing the need for manual adjustment and improving the efficiency of imaging. At the same time, multi-focus technology allows the information of multiple focuses to be obtained simultaneously in one scan, greatly improving the speed and quality of image acquisition, and significantly enhancing the dynamic imaging capability. In addition, the multi-band design of the system enables it to image at multiple wavelengths at the same time, broadening its scope of application. Researchers can obtain a variety of information through fluorescence signals of different wavelengths in one experiment, promoting a deep understanding of complex biological systems. This flexibility makes the system have broad application prospects in biomedical research, materials science and other related fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0018] Figure 1 A structural principle block diagram of an automatic focusing confocal microscopic imaging system with multiple focal points and multiple waveband channels provided by the present invention;

[0019] Figure 2 A schematic diagram of the structure of an autofocus confocal microscopy imaging system with multiple focal points and multiple waveband channels provided by an embodiment of the present invention;

[0020] Figure 3 A structural principle block diagram of a control module provided by the present invention;

[0021] Figure 4 A control logic diagram of a control module provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0022] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] See also Figure 1As shown, an embodiment of the present invention discloses an automatic focusing confocal microscopic imaging system with multiple focal points and multiple waveband channels, including: an excitation end, a detection end and a control module, wherein the control module is connected to the excitation end and the detection end.

[0024] See also Figure 2 As shown, in a specific embodiment, the excitation end includes a laser 1, a laser adjustment device, a short-pass dichroic mirror 6, a two-dimensional galvanometer, and a scanning device arranged in sequence, wherein the two-dimensional galvanometer may include an x-axis galvanometer 7 and a y-axis galvanometer 8;

[0025] Laser 1 is used to emit laser beams, and can use conventional industrial multi-band fiber lasers to emit narrow linewidth lasers, and the specific coverage wavelengths use common excitation wavelengths such as 488nm, 532nm, 635nm, 658nm, and 808nm;

[0026] The laser adjustment device is arranged on the output light path of the laser, and is used to split and adjust the laser beam to obtain corresponding multiple beams;

[0027] The short-pass dichroic mirror 6 needs to be customized according to the output band of the laser 1 to ensure that the laser output band is transmitted and other bands are reflected. The short-pass dichroic mirror 6 can be set as a multi-band window transmission beam splitter for a multi-band laser, or can be set as a turntable switching structure of multiple short-pass dichroic mirrors to switch for different output bands.

[0028] The two-dimensional galvanometer is arranged on the output light path of the laser adjustment device, and is used to scan multiple light beams. The scanning device is arranged on the output light path of the two-dimensional galvanometer, and is used to use multiple light beams to achieve multi-point focusing and simultaneously excite the fluorescence signal. The x-axis galvanometer 7 and the y-axis galvanometer 8 can adopt two galvanometers or one galvanometer and one resonant galvanometer to achieve two-dimensional scanning with one fast axis and one slow axis.

[0029] In a specific embodiment, the laser adjustment device includes a spectroscopic positioner 2, a polarizer 3, a correction lens 4 and a first filter 5 which are sequentially arranged on the output light path of the laser, wherein the spectroscopic positioner 2 can be replaced by a diffraction optical element, a digital micromirror array, a spatial light modulator, a free-form surface element, etc. to generate multiple focal points, wherein the multi-focal point arrangement is not limited to a square matrix arrangement such as 3*3, 5*5*, 7*7, etc., and can also be a circular arrangement.

[0030] In a specific embodiment, the scanning device includes a scanning lens 9, a tube lens 10, a microscope objective lens 11, a three-dimensional electric displacement platform and a focus detection device, which are sequentially arranged on the output light path of the two-dimensional galvanometer, and a microscopic imaging sample 12 is placed on the three-dimensional electric displacement platform;

[0031] Through the scanning lens 9, the tube lens 10 and the microscope objective lens 11, multi-point focusing can be achieved on the microscopic imaging sample 12, that is, multi-point fluorescence signals can be excited.

[0032] In a specific embodiment, the focus detection device includes a focus detection end filter 13 and a focus detection camera 14, which are arranged in sequence, and are used to obtain bright field image information, wherein the focus detection camera 14 can adopt the information recording of a traditional scientific research CMOS camera, and according to the information obtained by the camera, image background noise removal, image light intensity adaptive adjustment, and subsequent image enhancement and other image post-processing algorithms can be performed.

[0033] In a specific embodiment, the detection end includes a second filter 15, a calibration lens 16, a plurality of short-pass dichroic mirrors and a plurality of light intensity detector arrays which are sequentially arranged on the output light path of the short-pass dichroic mirror 6, wherein the second filter 15 can be set as a filter for a plurality of specific fluorescence bands, or can be set as a plurality of filter turntable switching structures to switch for different output bands;

[0034] The multiple-band short-pass dichroic mirror may include a dichroic mirror 2 17 and a dichroic mirror 3 18, which can realize the separation of optical signals of different bands, and the cut-off wavelength of the dichroic mirror 2 17 is smaller than the cut-off wavelength of the dichroic mirror 3 18;

[0035] The number of light intensity detector arrays can be 3, namely detector array 19, detector array 20 and detector array 3 21, which are used to obtain the corresponding band light intensity information. The light intensity detector array can use a high-sensitivity scientific research camera or a PMT array. The front end of the detector array needs to add a filter of the corresponding excitation fluorescence band; if a PMT array is used, the number of PMTs needs to be consistent with the number of focal points. In addition, if necessary, an array pinhole (the size of the pinhole is generally set to the half-height full width of the focal plane spot of the objective lens) needs to be set at the front end of the photomultiplier tube array to filter out the out-of-focus light, thereby improving the imaging contrast of the system.

[0036] See also Figure 3-4 As shown, in a specific embodiment, the control module includes: a processing module, an autofocus control module, a light intensity acquisition switching module, a PMT array acquisition module, an electric galvanometer motor control module, a laser light intensity control module and a sample motion module;

[0037] The processing module is connected to the laser light intensity control module, the PMT array acquisition module, the autofocus control module, and the electric galvanometer motor control module; the autofocus control module is connected to the sample motion module and the light intensity acquisition switching module;

[0038] The laser light intensity control module and the light intensity acquisition switching module are connected to the laser, the PMT array acquisition module is connected to the light intensity detector array, the electric galvanometer motor control module is connected to the two-dimensional galvanometer, and the sample motion module is connected to the three-dimensional electric displacement platform.

[0039] The specific control process of the processing module provided by the embodiment of the present invention is as follows: first, select the laser output band, and switch the band through the light intensity acquisition switching module. After the switching is completed, the displacement stage is moved by the sample motion module and the focusing is monitored in real time. During the focusing process, the displacement stage will be adjusted multiple times according to the detection information. After the automatic focus is completed, the scanning motor of the galvanometer can be controlled by the electric galvanometer motor control module to scan, and the light intensity information is collected through the PMT array acquisition module during the scanning process. After the detection galvanometer scanning cycle is completed, a single-band multi-focus channel imaging process is completed, and a single-band microscopic image can be fused. After completing a single band, you can continue to switch the laser output band and repeat the above operation until the imaging of multiple bands is completed.

[0040] The specific working principle of the system provided by the present invention is as follows: the laser 1 is fixed to the spectroscopic positioner 2 after being split, and multiple light beams are obtained, which pass through the polarizer 3, the correction lens 4, the filter 5 and the short-pass dichroic mirror 6 in sequence, and reach the two-dimensional galvanometer (composed of the x-axis 7 and the y-axis 8 galvanometers). The light beam passes through the two-dimensional galvanometer to realize the light beam scanning control process. At the same time, the filter 15 and the short-pass dichroic mirror 6 use a turntable control to perform corresponding band switching according to the output band of the laser 1.

[0041] The light beam after the galvanometer is then passed through the scanning lens 9, the tube lens 10 and the microscope objective lens 11, and can realize multi-point focusing on the microscopic sample 12, that is, it can excite multi-point fluorescence signals; wherein the image information of the bright field is obtained through the focus detection end filter 13 and the focus detection camera 14, and before imaging, the information of the bright field imaging is used to determine whether the best focus position is reached. If the best focus position is not reached, the sample is moved until the best focus position is reached. On the other hand, the multi-focus fluorescence signal returns to the surface of the dichroic mirror 16 by the original path. Since the fluorescence signal has a longer wavelength than the excitation light, it is reflected on the surface of the dichroic mirror 16. After the reflected fluorescence signal passes through the filter 15 and the calibration lens 16, the light signals of different bands can be separated through the dichroic mirror 2 17 and the dichroic mirror 3 18, and then the corresponding band light intensity information is obtained through three detector arrays, the detector array 19, the detector array 20, and the detector array 3 21.

[0042] In this specification, each embodiment is described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the embodiments can be referred to each other. For the device disclosed in the embodiment, since it corresponds to the method disclosed in the embodiment, the description is relatively simple, and the relevant parts can be referred to the method part.

[0043] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An automatic focusing confocal microscopy imaging system with multiple focal points and multiple wavelength channels, characterized in that: include: An excitation end, a detection end and a control module, wherein the control module is connected to the excitation end and the detection end.

2. The multi-focus, multi-band channel autofocus confocal microscopy imaging system according to claim 1, characterized in that: The excitation end comprises a laser, a laser adjustment device, a short-pass dichroic mirror, a two-dimensional galvanometer and a scanning device which are arranged in sequence.

3. The multi-focus, multi-band channel autofocus confocal microscopy imaging system according to claim 2, characterized in that: The laser adjustment device comprises a beam splitter positioner, a polarizer, a correction lens and a first filter which are sequentially arranged on the output light path of the laser.

4. The multi-focus, multi-band channel autofocus confocal microscopy imaging system according to claim 2, characterized in that: The scanning device comprises a scanning lens, a tube lens, a microscope objective lens, a three-dimensional electric displacement platform and a focusing detection device which are sequentially arranged on the output light path of the two-dimensional galvanometer, and a microscopic imaging sample is placed on the three-dimensional electric displacement platform.

5. The multi-focus, multi-band channel autofocus confocal microscopy imaging system according to claim 3, characterized in that: The focus detection device comprises a focus detection end filter and a focus detection camera which are arranged in sequence.

6. The multi-focus, multi-band channel autofocus confocal microscopy imaging system according to claim 4, characterized in that: The detection end comprises a second filter, a calibration lens, short-pass dichroic mirrors of multiple wavelength bands and a plurality of light intensity detector arrays which are sequentially arranged on the outgoing light path of the short-pass dichroic mirror.

7. The multi-focus, multi-band channel autofocus confocal microscopy imaging system according to claim 6, characterized in that: The control module includes: a processing module, an autofocus control module, a light intensity acquisition switching module, a PMT array acquisition module, an electric galvanometer motor control module, a laser light intensity control module and a sample motion module; The processing module is connected to the laser light intensity control module, the PMT array acquisition module, the autofocus control module, and the electric galvanometer motor control module; the autofocus control module is connected to the sample motion module and the light intensity acquisition switching module; The laser light intensity control module and the light intensity acquisition switching module are both connected to the laser, the PMT array acquisition module is connected to the light intensity detector array, the electric galvanometer motor control module is connected to the two-dimensional galvanometer, and the sample motion module is connected to the three-dimensional electric displacement platform.

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