High-speed laser confocal microscopy imaging system, method and scanning head

Abstract

The present invention belongs to the field of microscopic imaging, and discloses a high-speed laser confocal microscopic imaging system, method and scanning head. The imaging system includes a high-speed laser confocal microscopic imaging scanning head, a microscope objective lens module and a control system panel; the high-speed laser confocal microscopic imaging scanning head is composed of a multi-wavelength laser light source, a relay optical group, a first MEMS scanning galvanometer, a second MEMS scanning galvanometer, a fluorescence image acquisition module and a synchronous drive control module; the resonance frequency and phase synchronization of the first MEMS scanning galvanometer and the second MEMS scanning galvanometer are controlled by the synchronous drive control module. With the first MEMS scanning galvanometer and the second MEMS scanning galvanometer as the core, the scanning efficiency is improved by synchronous scanning, and high-speed and high-resolution confocal fluorescence imaging is realized, providing a new solution for high-speed fluorescence imaging and detection of living cells.

Description

Technical Field

[0001] The invention relates to the technical field of microscopic imaging systems, in particular to a high-speed laser confocal microscopic imaging system. Background Art

[0002] At present, the main technologies that can perform laser confocal fluorescence imaging of living cells are: resonant mechanical swing mirror laser confocal microscope, line scanning laser confocal microscope, and rotating disk laser confocal microscope. The resonant mechanical swing mirror laser confocal microscope adopts point-by-point scanning, which can realize the true confocal optical path and has the best imaging quality, but the scanning speed is limited and the acquisition efficiency is low. The laser confocal microscope using line scanning usually uses a long strip of aperture to replace the pinhole to obtain high-speed scanning, but because it has confocal characteristics in only one direction, the imaging quality is worse than the former. The rotating disk laser confocal microscope was proposed to solve the confocal detection problem of rapidly changing processes, but the rotating disk laser confocal microscope technology is complex and the system is complicated, and the unit price of the equipment is very high. Therefore, it is very necessary to provide a new set of low-cost high-speed confocal microscopy imaging technology. Summary of the invention

[0003] The object of the present invention is to provide a high-speed laser confocal microscopy imaging system to solve the problems raised in the above background technology.

[0004] To achieve the above-mentioned purpose, the present invention provides the following technical solutions: a high-speed laser confocal microscopy imaging system, comprising a high-speed laser confocal microscopy imaging scanning head, a microscope objective lens module, and a control system panel, wherein the microscope objective lens module and the high-speed laser confocal microscopy imaging scanning head are assembled in a detachable manner; the microscope objective lens module is composed of an inverted microscopy imaging system, a bright field illumination light source, and a Z-axis displacement stage, wherein the bright field illumination light source is used to provide uniform illumination for a sample to be tested, and the sample to be tested is placed on the Z-axis displacement stage after being attached with a fluorescent material; the high-speed laser confocal microscopy imaging scanning head comprises: a multi-wavelength laser light source, a relay optical group, A first MEMS scanning galvanometer, a second MEMS scanning galvanometer, a fluorescence image acquisition module, and a synchronous drive control module, wherein the synchronous drive control module controls the resonance frequency and phase synchronization of the first MEMS scanning galvanometer and the second MEMS scanning galvanometer; the relay optical group is sequentially arranged along the optical axis direction of the multi-wavelength laser light source as follows: a dichroic filter, a collimating beam reducer; the fluorescence image acquisition module includes an imaging lens and a surface array detector, and the surface array detector is arranged at the image focal plane of the imaging lens; the control system panel is used to control the switches of the first MEMS scanning galvanometer, the second MEMS scanning galvanometer, and the multi-wavelength laser light source, and control the surface array The detector performs image acquisition, data transmission, data storage, image processing and display; the control system panel controls the multi-wavelength laser light source to emit laser, and the laser is incident on the first MEMS scanning galvanometer after being collimated and reduced by the relay optical group. The laser reflected by the first MEMS scanning galvanometer is reflected to the inverted microscopic imaging system in the microscope objective module. The inverted microscopic imaging system converges the parallel light into an excitation light spot, and scans the sample to be tested point by point under the drive of the first MEMS scanning galvanometer; the excited fluorescent material on the sample to be tested undergoes energy level transition to generate a fluorescent signal, and the fluorescent signal is returned to the relay optical group through the original optical path, and is transmitted to the relay optical group through the relay optical group. After being reflected by the dichroic filter, the light is incident on the second MEMS scanning galvanometer. The light reflected by the second MEMS scanning galvanometer enters the imaging lens in the fluorescent image acquisition module, and the surface array detector transmits the acquired image to the control system panel; the first MEMS scanning galvanometer and the second MEMS scanning galvanometer scan synchronously, and the excitation light spots form surface scanning light under the drive of the first MEMS scanning galvanometer; the second MEMS scanning galvanometer synchronously scans the fluorescent signal onto the surface array detector; the Z-axis displacement stage carrying the sample to be tested moves up and down according to a fixed step length, and performs surface scanning layer by layer, and finally forms a three-dimensional image after image processing by the control system panel.

[0005] Preferably, the multi-wavelength laser light source is a multi-color laser, covering a wavelength of 300-1700nm.

[0006] The image refresh frequency of the area array detector is consistent with the resonance frequency of the first MEMS scanning galvanometer and the second MEMS scanning galvanometer, both of which are MEMS two-dimensional scanning galvanometers.

[0007] The displacement accuracy of the Z-axis displacement stage is 1nm-100μm.

[0008] A fluorescence filter is arranged between the dichroic filter and the second MEMS scanning galvanometer.

[0009] The control system panel is respectively connected to the multi-wavelength laser light source, the surface array detector, the first MEMS scanning galvanometer, and the second MEMS scanning galvanometer through wires or wireless signals, and is used to control the laser frequency of the multi-wavelength laser light source, the resonant frequency and phase of the first MEMS scanning galvanometer and the second MEMS scanning galvanometer, and the reception of surface array detector data.

[0010] Preferably, the collimating beam reducer is composed of a first converging lens, a pinhole diaphragm, and a second converging lens; the object focus of the second converging lens is set at the image focus of the first converging lens, and the pinhole diaphragm is placed at the image focus of the first converging lens. The multi-wavelength laser light source is focused at its image focus by the first converging lens after passing through the dichroic filter, and is then filtered by the pinhole diaphragm and then beam-reduced into collimated parallel light by the second converging lens.

[0011] A high-speed laser confocal microscopy method is also provided based on the high-speed laser confocal microscopy system.

[0012] Based on the above-mentioned high-speed laser confocal microscopy imaging system, a high-speed laser confocal microscopy imaging scanning head is also provided, comprising: a multi-wavelength laser light source, a relay optical group, a first MEMS scanning galvanometer, a second MEMS scanning galvanometer, a fluorescence image acquisition module, and a synchronous drive control module; the synchronous drive control module controls the resonance frequency and phase synchronization of the first MEMS scanning galvanometer and the second MEMS scanning galvanometer; the multi-wavelength laser light source emits laser light, which is incident on the first MEMS scanning galvanometer after passing through the relay optical group, and is reflected by the laser light of the first MEMS scanning galvanometer out of the high-speed laser confocal microscopy imaging scanning head; the sample to be tested is scanned point by point under the drive of the first MEMS scanning galvanometer; the fluorescence signal on the sample to be tested returns to the relay optical group through the original optical path, and is incident on the second MEMS scanning galvanometer after being reflected by the relay optical group; the first MEMS scanning galvanometer and the second MEMS scanning galvanometer scan synchronously, and the excitation light spot forms a surface scanning light under the drive of the first MEMS scanning galvanometer; the second MEMS scanning galvanometer synchronously scans the fluorescence signal into the fluorescence image acquisition module.

[0013] Beneficial Effects

[0014] The high-speed laser confocal microscopy system provided by the present invention is based on the first MEMS scanning galvanometer and the second MEMS scanning galvanometer. It improves the scanning efficiency through synchronous scanning, realizes high-speed and high-resolution confocal fluorescence imaging, and provides a new solution for high-speed fluorescence imaging and detection of living cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is a schematic diagram of the working principle of the present invention;

[0016] Figure 2 It is a schematic diagram of the optical path of the present invention;

[0017] Figure 3 Schematic diagram of the scanning path of the present invention.

[0018] Among them, 1-multi-wavelength laser light source, 2-dichroic filter, 3-first converging lens, 4-pinhole aperture, 5-second converging lens, 6-first MEMS scanning galvanometer, 7-microscope imaging system, 8-fluorescence filter, 9-second MEMS scanning galvanometer, 10-imaging lens, 11-surface array detector. DETAILED DESCRIPTION

[0019] The following are specific embodiments of the present invention and the accompanying drawings to further describe the technical solution of the present invention, but the present invention is not limited to these embodiments.

[0020] Example

[0021] like Figure 1 and Figure 2 As shown, a high-speed laser confocal microscopy system includes a high-speed laser confocal microscopy scanning head, a microscope objective module, and a control system panel, wherein the microscope objective module and the high-speed laser confocal microscopy scanning head are assembled in a detachable manner; the microscope objective module is composed of an inverted microscopy system 7, a bright field illumination light source, and a Z-axis displacement stage, wherein the bright field illumination light source is used to provide uniform illumination for a sample to be tested, and the sample to be tested is placed on the Z-axis displacement stage after being attached with a fluorescent material;

[0022] The high-speed laser confocal microscopy imaging scanning head comprises: a multi-wavelength laser light source 1, a relay optical group, a first MEMS scanning galvanometer, a second MEMS scanning galvanometer, a fluorescence image acquisition module, and a synchronous drive control module, wherein the synchronous drive control module controls the resonance frequency and phase synchronization of the first MEMS scanning galvanometer and the second MEMS scanning galvanometer; the relay optical group is sequentially arranged along the optical axis direction of the multi-wavelength laser light source as: a dichroic filter 2, a first converging lens 3, a pinhole diaphragm 4, and a second converging lens 5, wherein the object focus of the second converging lens is arranged at the first The image focus of a converging lens, the pinhole diaphragm is placed at the image focus of the first converging lens; the fluorescence image acquisition module includes an imaging lens 10 and a surface array detector 11, and the surface array detector is arranged at the image focal plane of the imaging lens; the control system panel is used to control the switch of the first MEMS scanning galvanometer 6, the second MEMS scanning galvanometer 9, and the multi-wavelength laser light source, and control the execution of image acquisition, data transmission, data storage, image processing and display of the surface array detector; a fluorescence filter 8 is arranged between the dichroic filter and the second MEMS scanning galvanometer;

[0023] The control system panel controls the multi-wavelength laser light source to emit laser, and the laser is incident on the first MEMS scanning galvanometer after being collimated and reduced by the relay optical group. The laser is reflected by the first MEMS scanning galvanometer to the inverted microscopic imaging system in the microscope objective module. The inverted microscopic imaging system converges the parallel light into an excitation light spot, and scans the sample to be tested point by point under the drive of the first MEMS scanning galvanometer. The excited fluorescent material on the sample to be tested undergoes energy level transition to generate a fluorescent signal, and the fluorescent signal returns to the relay optical group through the original optical path, and is incident on the second MEMS after being reflected by the dichroic filter in the relay optical group. The light reflected by the second MEMS scanning galvanometer enters the imaging lens in the fluorescence image acquisition module, and the surface array detector transmits the acquired image to the control system panel; the first MEMS scanning galvanometer and the second MEMS scanning galvanometer scan synchronously, and the excitation light spot forms a surface scanning light under the drive of the first MEMS scanning galvanometer; the second MEMS scanning galvanometer synchronously scans the fluorescence signal to the surface array detector; the Z-axis displacement stage carrying the sample to be tested moves up and down according to a fixed step length, and performs surface scanning layer by layer, and finally forms a three-dimensional image after image processing by the control system panel.

[0024] The microscopic imaging system includes a scanning lens, a lens, and an objective lens in sequence; the image focus of the scanning lens coincides with the object focus of the lens, and the light reflected by the first MEMS scanning galvanometer enters the scanning lens in sequence and is converged at the focal plane by the objective lens.

[0025] Figure 1-2 It only illustrates the positional relationship between the components; the size of the figure and the proportional relationship between the various components do not constitute a specific limitation on this solution. Figure 3 Schematic diagram of the scanning path of the present invention; the first MEMS scanning galvanometer and the second MEMS scanning galvanometer swing at angles α and β in the horizontal and vertical directions respectively, wherein α is between 0° and ±25°, and β is between 0° and ±15°, and the first MEMS scanning galvanometer and the second MEMS scanning galvanometer are used to realize scanning of the laser light source in the horizontal and vertical directions.

[0026] The scanning method of the first MEMS scanning galvanometer and the second MEMS scanning galvanometer is: after scanning an angle α from left to right along the horizontal direction at a fixed vertical position, the horizontal direction is quickly rotated in the opposite direction by an angle -α, and the vertical direction is rotated by an angle γ, γ<β, and then the vertical position is scanned from left to right along the horizontal direction by an angle α, and the above steps are repeated in sequence.

[0027] The surface array detector is composed of a high-speed COMS or sCOMS surface array detector unit arrangement.

[0028] In the above scheme, when the first MEMS scanning galvanometer and the second MEMS scanning galvanometer realize the same frequency and the same phase scanning, a distortion-free fluorescence image is obtained on the area array detector, and automatic correction of the sinusoidal scanning timing is realized. At the same time, the amplitudes of the first MEMS scanning galvanometer and the second MEMS scanning galvanometer can be different. As long as the frequency and phase are kept the same, fluorescence imaging with a magnification different from that of the objective lens image plane is obtained on the area array detector, ensuring that the objective lens NA adjusts the imaging magnification to achieve the best imaging effect.

[0029] A high-speed laser confocal microscopic imaging system of the present invention uses a first MEMS scanning galvanometer as a scanning mechanism, and a control system panel controls a multi-wavelength laser light source to emit laser light, and the light emitted by the laser passes through, thereby improving the optical quality of the laser reaching the sample surface;

[0030] After being split by the dichroic filter, the excitation light becomes collimated parallel light with a small divergence angle and concentrated energy after being collimated and focused by the collimating beam reducer. The first MEMS scanning galvanometer is used as a scanning mechanism to reflect the light spot emitted by the collimation, and then the sample to be tested is scanned point by point through the microscope objective module to form a surface scanning light source; taking the sample to be tested as a cell sample as an example, after the surface scanning light source reaches the cell surface, it stimulates the fluorescent material in the cell sample to undergo energy level transition to produce fluorescence of another wavelength, which returns through the original light path, and then continues to reach the dichroic filter through the original light path. After being reflected by the dichroic filter, it is reflected by the second MEMS scanning galvanometer to the fluorescence image acquisition module; the Z-axis stage carrying the sample moves up and down according to a fixed step size, and performs surface scanning layer by layer, and finally forms a three-dimensional image after image processing by the control system panel.

[0031] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the invention content within the protection scope of the present invention.

Claims

1. A high-speed laser confocal microscopy imaging system, comprising a high-speed laser confocal microscopy imaging scanning head, a microscope objective lens module, and a control system panel, wherein the microscope objective lens module and the high-speed laser confocal microscopy imaging scanning head are assembled in a detachable manner; the microscope objective lens module is composed of an inverted microscopy imaging system, a bright field illumination light source, and a Z-axis displacement stage, wherein the bright field illumination light source is used to provide uniform illumination for a sample to be tested, and the sample to be tested is placed on the Z-axis displacement stage after being attached with a fluorescent material; Features: The high-speed laser confocal microscopy imaging scanning head comprises: a multi-wavelength laser light source, a relay optical group, a first MEMS scanning galvanometer, a second MEMS scanning galvanometer, a fluorescence image acquisition module, and a synchronous drive control module, wherein the synchronous drive control module controls the resonance frequency and phase synchronization of the first MEMS scanning galvanometer and the second MEMS scanning galvanometer; The relay optical group is arranged in sequence along the optical axis direction of the multi-wavelength laser light source: a dichroic filter, a collimating beam reducer; The fluorescent image acquisition module includes an imaging lens and a surface array detector, wherein the surface array detector is arranged at the image focal plane of the imaging lens; The control system panel is used to control the switches of the first MEMS scanning galvanometer, the second MEMS scanning galvanometer, and the multi-wavelength laser light source, and to control the execution of image acquisition, data transmission, data storage, image processing, and display of the surface array detector; The control system panel controls the multi-wavelength laser light source to emit laser, and the laser is incident on the first MEMS scanning galvanometer after being collimated and reduced by the relay optical group. The laser is reflected by the first MEMS scanning galvanometer to the inverted microscopic imaging system in the microscope objective module. The inverted microscopic imaging system converges the parallel light into an excitation light spot, and scans the sample to be tested point by point under the drive of the first MEMS scanning galvanometer. The excited fluorescent material on the sample to be tested undergoes energy level transition to generate a fluorescent signal, and the fluorescent signal returns to the relay optical group through the original optical path, and is incident on the second MEMS after being reflected by the dichroic filter in the relay optical group. The light reflected by the second MEMS scanning galvanometer enters the imaging lens in the fluorescence image acquisition module, and the surface array detector transmits the acquired image to the control system panel; the first MEMS scanning galvanometer and the second MEMS scanning galvanometer scan synchronously, and the excitation light spot forms a surface scanning light under the drive of the first MEMS scanning galvanometer; the second MEMS scanning galvanometer synchronously scans the fluorescence signal to the surface array detector; the Z-axis displacement stage carrying the sample to be tested moves up and down according to a fixed step length, and performs surface scanning layer by layer, and finally forms a three-dimensional image after image processing by the control system panel.

2. The high-speed laser confocal microscopy imaging system according to claim 1, characterized in that: The first MEMS scanning galvanometer and the second MEMS scanning galvanometer are both MEMS two-dimensional scanning galvanometers, which are used for scanning in two dimensions simultaneously.

3. The high-speed laser confocal microscopy imaging system according to claim 1, characterized in that: The multi-wavelength laser light source is a multi-color laser, covering a wavelength of 300-1700nm.

4. The high-speed laser confocal microscopy imaging system according to claim 1, characterized in that: The image refresh frequency of the planar array detector is consistent with the resonance frequency of the first MEMS scanning galvanometer and the second MEMS scanning galvanometer, both of which are MEMS two-dimensional scanning galvanometers.

5. The high-speed laser confocal microscopy imaging system according to claim 1, characterized in that: The displacement accuracy of the Z-axis displacement stage is 1nm-100μm.

6. The high-speed laser confocal microscopy imaging system according to claim 1, characterized in that: A fluorescence filter is arranged between the dichroic filter and the second MEMS scanning galvanometer.

7. The high-speed laser confocal microscopy imaging system according to claim 1, characterized in that: The control system panel is respectively connected to the multi-wavelength laser light source, the surface array detector, the first MEMS scanning galvanometer, and the second MEMS scanning galvanometer through wires or wireless signals, and is used to control the laser frequency of the multi-wavelength laser light source, the resonant frequency and phase of the first MEMS scanning galvanometer and the second MEMS scanning galvanometer, and the reception of surface array detector data.

8. The high-speed laser confocal microscopy imaging system according to claim 1, characterized in that: The collimating beam reducer is composed of a first converging lens, a pinhole diaphragm and a second converging lens; the object focus of the second converging lens is arranged at the image focus of the first converging lens, and the pinhole diaphragm is arranged at the image focus of the first converging lens.

9. A high-speed laser confocal microscopy imaging method comprising performing imaging using a high-speed laser confocal microscopy imaging system according to any one of claims 1 to 8.

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