Optical imaging device and optical imaging method

Abstract

The application provides an optical imaging device and an optical imaging method. The optical imaging device comprises a light source module, a first galvanometer module, a second galvanometer module, a switching module and a main mirror body module. The second galvanometer module controls the two-dimensional deflection angle of the incident laser beam, realizes point-by-point scanning of the laser beam, adopts a two-photon excitation form of point-by-point scanning, improves the spatial resolution, increases the accuracy of optical stimulation, and accurately stimulates a single neuron. In addition, the holographic optical stimulation technology in the related art disperses laser light to multiple regions. Since two-photon excitation is proportional to the square of light intensity, a more expensive high-power femtosecond laser is often required. In the application, the laser beam is scanned point by point, so that each region receives sufficient stimulation energy, and a high-energy optical stimulation laser is not required, thereby reducing the cost and being more easily obtained.

Description

Technical Field

[0001] This application relates to the field of optical imaging technology, specifically to an optical imaging device and an optical imaging method. Background Technology

[0002] Two-photon imaging, as an advanced optical imaging method, has become an important tool for observing neuronal activity in neuroscience research due to its high spatial resolution and ability to penetrate deep tissues. In recent years, researchers have attempted to combine two-photon imaging with optogenetic stimulation in order to achieve integrated observation and manipulation within the same system.

[0003] Related technologies combine two-photon imaging with holographic light stimulation, but holographic light stimulation requires extremely high laser power, carries the risk of thermal damage, and is more sensitive to tissue scattering. Summary of the Invention

[0004] To address the aforementioned problems, this application provides an optical imaging device and an optical imaging method.

[0005] In a first aspect, embodiments of this application provide an optical imaging device, comprising: a light source module, a first galvanometer module, a second galvanometer module, a switching module, and a main mirror module; the light source module includes at least two lasers for emitting laser beams of different wavelengths; the switching module is disposed in the optical path between the light source module and the first galvanometer module and the second galvanometer module, and the switching module is used to dynamically guide a portion of the laser beam from the light source module to the first galvanometer module and / or the second galvanometer module; the first galvanometer module is used to control the two-dimensional deflection angle of the incident laser beam; the second galvanometer module is used to control the two-dimensional deflection angle of the incident laser beam; the main mirror module is used to irradiate a target sample with the laser beam from the first galvanometer module to image the target sample, and / or irradiate a target area of ​​the target sample with the laser beam from the second galvanometer module to stimulate the target area.

[0006] In conjunction with the first aspect, in some implementations of the first aspect, the switching module is used to dynamically guide a portion of the laser beam from the light source module to the first galvanometer module and / or the second galvanometer module, including: controlling all the laser beam to enter the first galvanometer module, or controlling a portion of the laser beam to enter the first galvanometer module and a portion of the laser beam to enter the second galvanometer module.

[0007] In conjunction with the first aspect, in some implementations of the first aspect, the switching module includes a first dichroic mirror and a first reflecting mirror.

[0008] In conjunction with the first aspect, in some implementations of the first aspect, the primary mirror module includes a beam combiner, a second dichroic mirror, and an objective lens. The beam combiner is used to combine laser beams from the first galvanometer module and the second galvanometer module; the second dichroic mirror is used to transmit the combined laser beam; the objective lens is used to irradiate the target sample with the combined laser beam; the second dichroic mirror is also used to reflect the first fluorescence emitted by the target sample when irradiated by the laser beam; the beam combiner, the second dichroic mirror, and the objective lens form a two-photon imaging optical path.

[0009] In conjunction with the first aspect, in some implementations of the first aspect, the main mirror module further includes a light source component, a third dichroic mirror, and an image sensor. The light source component is used to emit excitation light, the third dichroic mirror is used to reflect the excitation light emitted by the light source component and transmit the excitation light emitted by the light source component to the objective lens, the objective lens is used to irradiate the target sample with the excitation light emitted by the light source component, and the third dichroic mirror is also used to transmit the second fluorescence emitted by the target sample under the illumination of the light and transmit the second fluorescence to the image sensor; the light source component, the third dichroic mirror, the objective lens, and the image sensor form a large field of view imaging optical path; the main mirror module further includes: a second reflecting mirror, which is located between the beam combining structure and the second dichroic mirror, and between the third dichroic mirror and the objective lens, and is used to control the laser beam released by the laser or the excitation light emitted by the light source component to irradiate the target sample.

[0010] In conjunction with the first aspect, some implementations of the first aspect also include a collection module, which is used to collect the first fluorescence emitted by the target sample when irradiated by the laser beam and convert the first fluorescence into an electrical signal.

[0011] Secondly, one embodiment of this application provides an optical imaging method applied to any of the aforementioned optical imaging devices, comprising: configuring a first optical path in an imaging mode and a second optical path in a stimulation mode, wherein the first optical path passes through a first galvanometer module and the second optical path passes through a second galvanometer module; controlling a laser beam emitted by a first laser to enter the first galvanometer module to image a target sample through the first optical path; and controlling a laser beam emitted by a second laser to enter the second galvanometer module to stimulate a target area through the second optical path.

[0012] In conjunction with the second aspect, in some implementations of the second aspect, before configuring the first optical path to be in imaging mode and the second optical path to be in stimulation mode, the method further includes: configuring both the first and second optical paths to be in imaging mode; controlling the first laser and the second laser to emit laser beams so that the laser beams irradiate the target sample; and calibrating the scanning parameters of the first galvanometer module and / or the second galvanometer module based on the laser beams.

[0013] In conjunction with the second aspect, in some implementations of the second aspect, calibrating the scanning parameters of the first galvanometer module and / or the second galvanometer module based on the laser beam includes: obtaining a first calibration image corresponding to the laser beam passing through the first galvanometer module and a second calibration image corresponding to the laser beam passing through the second galvanometer module based on the third and fourth fluorescence emitted by the target sample after being irradiated by the laser; calibrating the scanning parameters of the first galvanometer module and / or the second galvanometer module based on the first calibration image and the second calibration image, so that the first calibration image coincides with the second calibration image.

[0014] In conjunction with the second aspect, in some implementations of the second aspect, controlling the laser beam emitted by the second laser to enter the second galvanometer module to stimulate the target area through the second optical path includes: determining the target area and stimulation parameters of the target area; and based on the stimulation parameters, controlling the laser beam emitted by the second laser to enter the second galvanometer module to stimulate the target area through the second optical path.

[0015] In related technologies, two-photon imaging technology is combined with single-photon optical stimulation technology. However, single-photon optical stimulation technology has poor precision, activating fluorescent proteins in all neurons along the entire optical path, but failing to precisely stimulate the fluorescent proteins of a single neuron. To address this issue, this application provides an optical imaging device, including: a light source module, a first galvanometer module, a second galvanometer module, a switching module, and a main mirror module. The second galvanometer module controls the two-dimensional deflection angle of the incident laser beam, realizing point-by-point scanning of the laser beam, and adopting a point-by-point scanning two-photon excitation method to improve spatial resolution and increase the precision of optical stimulation, thereby precisely stimulating a single neuron. In addition, the holographic optical stimulation technology in related technologies disperses the laser beam into multiple regions. Since two-photon excitation is proportional to the square of the light intensity, holographic optical stimulation technology requires more expensive high-power femtosecond lasers, resulting in extremely high costs. In contrast, the point-by-point scanning of the laser beam in this application ensures sufficient stimulation energy to each region, and eliminates the need for high-energy optical stimulation lasers, making it lower in cost and more readily available. Attached Figure Description

[0016] Figure 1 This is a schematic diagram of the structure of an optical imaging device provided in an embodiment of this application.

[0017] Figure 2 This is a schematic diagram of the structure of an optical imaging device provided in another embodiment of this application.

[0018] Figure 3 This is a schematic diagram of the structure of a collection module provided in another embodiment of this application.

[0019] Figure 4 This is a schematic diagram of the main mirror module provided in another embodiment of this application.

[0020] Figure 5 This is a path diagram of the light rays in the primary mirror module and the collection module of the two-photon imaging optical path provided in another embodiment of this application.

[0021] Figure 6 This is a path diagram of light transmission in the main mirror module for a large field-of-view imaging optical path provided in another embodiment of this application.

[0022] Figure 7 This is a photostimulation result diagram provided in another embodiment of this application.

[0023] Figure 8 This is a schematic flowchart of an optical imaging method provided in an embodiment of this application.

[0024] Explanation of reference numerals in the attached figures 100 Optical imaging device; 110 Light source module; 111 Laser; 120 First galvanometer module; 130 Second galvanometer module; 140 Switching module; 141 First dichroic mirror; 142 First reflecting mirror; 150 Main mirror module; 151 Beam combiner structure; 152 Second dichroic mirror; 153 Objective lens; 154 Light source assembly; 155 Third dichroic mirror; 156 Image sensor; 157 Second reflecting mirror; 160 Collection module; 161 Beam splitter structure; 162 Photodetector; 163 Filter. Detailed Implementation

[0025] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0026] In the accompanying drawings, the dimensions of layers and regions may be exaggerated for clarity. It is understood that when a structure is referred to as being "on" or "below" another structure, the structure may be directly on or below the other structure, or there may be intermediate structures. The same reference numerals always indicate the same structure. Structures referred to herein include any of the following: membrane, element, device, component, assembly.

[0027] When a structure is referred to as being “connected” to another structure, it can be directly connected to the other structure or indirectly connected to the other structure by means of one or more intermediate structures placed between them.

[0028] Figure 1 This is a schematic diagram of the structure of an optical imaging device provided in one embodiment of this application. Figure 1As shown, this application provides an optical imaging device 100, including: a light source module 110, a first galvanometer module 120, a second galvanometer module 130, a switching module 140, and a main mirror module 150.

[0029] The light source module 110 includes at least two lasers 111 for emitting laser beams of different wavelengths. A switching module 140 is disposed in the optical path between the light source module 110 and the first galvanometer module 120 and the second galvanometer module 130. The switching module 140 is used to dynamically guide the laser beam from the light source module 110 to the first galvanometer module 120 and / or the second galvanometer module 130. The first galvanometer module 120 is used to control the two-dimensional deflection angle of the incident laser beam. The second galvanometer module 130 is used to control the two-dimensional deflection angle of the incident laser beam. The main mirror module 150 is used to irradiate the target sample with the laser beam from the first galvanometer module 120 to image the target sample, and / or irradiate the target area of ​​the target sample with the laser beam from the second galvanometer module 130 to stimulate the target area.

[0030] The target region is located on the target sample and includes a single neuron.

[0031] It is worth noting that the laser beam passing through the first galvanometer module 120 is used to image neurons in the target sample, and the laser beam passing through the second galvanometer module 130 is used to image or stimulate neurons in the target sample. The imaging includes two-photon imaging.

[0032] Understandably, the optical imaging device 100 in the related technology includes a light source module 110, a first galvanometer module 120, and a main mirror module 150. The laser beam is irradiated onto the target sample through the first galvanometer module 120 and the main mirror module 150 to achieve two-photon imaging. The optical imaging device 100 provided in this embodiment adds a second galvanometer module 130 to the optical imaging device 100 provided in the related technology and reuses the first galvanometer module 120. Moreover, the second galvanometer module 130 and the first galvanometer module 120 can share the same light source module 110, without the need to add a new light source module 110, thus reducing costs.

[0033] In some embodiments, a first galvanometer module 120 includes a first scanning galvanometer and a second scanning galvanometer. The first scanning galvanometer is used to control the propagation of the laser passing through the first galvanometer module 120 along a first direction, and the second scanning galvanometer is used to control the propagation of the laser passing through the first galvanometer module 120 along a second direction. The first direction and the second direction do not overlap, for example, the first direction and the second direction are at 90°. A second galvanometer module 130 includes a third scanning galvanometer and a fourth scanning galvanometer. The third scanning galvanometer is used to control the propagation of the laser passing through the second galvanometer module 130 along the first direction, and the fourth scanning galvanometer is used to control the propagation of the laser passing through the second galvanometer module 130 along the second direction. In this embodiment, the first scanning galvanometer and the second scanning galvanometer can control the transmission direction of the laser beam entering the first galvanometer module 120 to achieve point-by-point scanning on the target sample; similarly, the third scanning galvanometer and the fourth scanning galvanometer can control the transmission direction of the laser beam entering the second galvanometer module 130 to achieve point-by-point scanning on the target sample, thereby accurately stimulating the target area, that is, achieving accurate stimulation of a single neuron.

[0034] In this embodiment, an optical imaging device 100 is provided, including: a light source module 110, a first galvanometer module 120, a second galvanometer module 130, a switching module 140, and a main mirror module 150. The second galvanometer module 130 controls the two-dimensional deflection angle of the incident laser beam to achieve point-by-point scanning of the laser beam. It employs a point-by-point scanning two-photon excitation method to improve spatial resolution and increase the accuracy of optical stimulation, thereby precisely stimulating individual neurons. Furthermore, related holographic optical stimulation techniques disperse the laser beam across multiple regions. Since two-photon excitation is proportional to the square of the light intensity, it often requires more expensive high-power femtosecond lasers. In contrast, the point-by-point scanning of the laser beam in this application ensures sufficient stimulation energy for each region, eliminating the need for high-energy optical stimulation lasers, resulting in lower costs and greater availability.

[0035] In some embodiments, the switching module 140 is used to dynamically guide a portion of the laser beam from the light source module 110 to the first galvanometer module 120 and / or the second galvanometer module 130, including: controlling all the laser beam to enter the first galvanometer module 120, or controlling a portion of the laser beam to enter the first galvanometer module 120 and a portion of the laser beam to enter the second galvanometer module 130.

[0036] Specifically, the switching module 140 includes a first dichroic mirror and a first reflecting mirror, meaning that the first dichroic mirror and the first reflecting mirror are switchable.

[0037] For example, the light source module 110 includes two lasers 111 that emit a laser beam of a first wavelength and a laser beam of a second wavelength, respectively.

[0038] In some embodiments, when it is not necessary to stimulate the neurons of the target sample, the switching module 140 is configured as a first reflector, which reflects a first wavelength laser beam and a second wavelength laser beam so that both the first wavelength laser beam and the second wavelength laser beam enter the first galvanometer module 120 to achieve two-photon imaging; when it is necessary to observe the potential change of the neurons of the target sample after stimulation, the switching module 140 is configured as a first dichroic mirror, which separates the first wavelength laser beam and the second wavelength laser beam, so that the first wavelength laser beam enters the first galvanometer module 120 to achieve two-photon imaging, and the second wavelength laser beam enters the second galvanometer module 130 to achieve stimulation.

[0039] It is worth noting that in some embodiments, the optical imaging device 100 needs to be calibrated before observing the potential changes of neurons in the target sample after stimulation. Specifically, the target sample is replaced with a target sample, the switching module 140 is configured as a first dichroic mirror, the first dichroic mirror separates the first wavelength laser beam and the second wavelength laser beam, so that the first wavelength laser beam enters the first galvanometer module 120 to achieve two-photon imaging, and the second wavelength laser beam enters the second galvanometer module 130 to achieve two-photon imaging. The first calibration image presented by the first wavelength laser beam through the first galvanometer module 120 and the second calibration image presented by the second wavelength laser beam through the second galvanometer module 130 are superimposed to achieve calibration of the optical imaging device 100.

[0040] Understandably, optical imaging devices can be used solely to achieve multicolor imaging, or they can be used to achieve precise stimulation of neurons based on multicolor imaging.

[0041] For example, the first wavelength can be 920 nm and the second wavelength can be 1030 nm.

[0042] Figure 2 This is a schematic diagram of the structure of an optical imaging device provided in another embodiment of this application. For example... Figure 2 As shown, in some embodiments, the optical imaging device 100 further includes a collection module 160, which is used to collect the first fluorescence emitted by the target sample when irradiated by the laser beam and convert the first fluorescence into an electrical signal to achieve image display.

[0043] Figure 3 This is a schematic diagram of the structure of a collection module provided in another embodiment of this application. For example... Figure 3As shown, in some embodiments, the collection module 160 includes a beam splitting structure 161 and at least two photomultiplier tubes (PMTs). The beam splitting structure 161 is used to split the first fluorescence into sub-fluorescences, and the sub-fluorescences enter their respective corresponding photodetectors 162. The photodetectors 162 convert the sub-fluorescences into electrical signals.

[0044] Specifically, a first-wavelength laser beam irradiates the target sample, exciting it to emit a first sub-fluorescence. A second-wavelength laser beam irradiates the target sample, exciting it to emit a second sub-fluorescence. The first and second sub-fluorescences mix and enter the collection module 160. A beam-splitting structure 161 in the collection module 160 separates the first and second sub-fluorescences, transmitting the first sub-fluorescence to a first photodetector and the second sub-fluorescence to a second photodetector. It is worth noting that this embodiment uses two photodetectors 162 (i.e., a first photodetector and a second photodetector), but this application does not limit the number of photodetectors 162.

[0045] In some embodiments, the collection module 160 further includes a filter 163, which is located before the beam splitting structure 161 along the propagation direction of the first fluorescence. The filter 163 is used to filter the laser beam emitted by the laser 111. It is understood that the energy of the laser beam is higher than the energy of the first fluorescence. If a small amount of laser beam leaks into the collection module 160, the photodetector 162 will preferentially detect the higher-energy laser beam rather than the lower-energy first fluorescence, interfering with the imaging of the first fluorescence. Therefore, by setting the filter 163, this embodiment can effectively filter out stray light that may leak from the laser beam into the collection module 160, ensuring that the signal received by the photodetector 162 is mainly the first fluorescence emitted by the target sample, thereby improving the signal-to-noise ratio and accuracy of the imaging.

[0046] Figure 4 This is a schematic diagram of the main mirror module provided in another embodiment of this application. Figure 4As shown, in some embodiments, the primary mirror module 150 includes a beam combiner structure 151, a second dichroic mirror 152, and an objective lens 153. The beam combiner structure 151 is used to combine the laser beams from the first galvanometer module 120 and the second galvanometer module 130. The second dichroic mirror 152 is used to transmit the combined laser beam, and the objective lens 153 is used to irradiate the target sample with the combined laser beam. The second dichroic mirror 152 is also used to reflect the first fluorescence emitted by the target sample when irradiated by the laser beam. The beam combiner structure 151, the second dichroic mirror 152, and the objective lens 153 form a two-photon imaging optical path. That is, the two-photon imaging optical path includes the laser beams emitted by the first galvanometer module 120 and the second galvanometer module 130 passing sequentially through the beam combiner structure 151, the second dichroic mirror 152, and the objective lens 153. The first fluorescence emitted by the target sample is transmitted to the collection module 160 through the second dichroic mirror 152.

[0047] Understandably, the first fluorescence is transmitted to the collection module 160 to convert the first fluorescence into an electrical signal for image display.

[0048] In some embodiments, the main lens module 150 further includes a light source assembly 154, a third dichroic mirror 155, and an image sensor 156. The light source assembly 154 emits excitation light, the third dichroic mirror 155 reflects the excitation light emitted by the light source assembly 154 and transmits the excitation light emitted by the light source assembly 154 to the objective lens 153. The objective lens 153 illuminates the target sample with the excitation light emitted by the light source assembly 154. The third dichroic mirror 155 also transmits the second fluorescence emitted by the target sample when illuminated by the light and transmits the second fluorescence to the image sensor 156. The light source assembly 154, the third dichroic mirror 155, the objective lens 153, and the image sensor 156 form a large field-of-view imaging optical path, that is, the large field-of-view imaging optical path includes the excitation light emitted by the light source assembly 154 passing sequentially through the third dichroic mirror 155 and the objective lens 153, and the second fluorescence emitted by the target sample being transmitted to the image sensor 156 through the third dichroic mirror 155. Exemplarily, the light source assembly 154 includes an LED light source assembly.

[0049] In some embodiments, the main mirror module 150 further includes a second reflector 157 removable from the optical path. The second reflector 157 is located between the beam combiner 151 and the second dichroic mirror 152, and between the third dichroic mirror 155 and the objective lens 153, for controlling the laser beam released by the laser 111 or the excitation light emitted by the light source assembly 154 to illuminate the target sample. For example, when two-photon imaging and / or precise light stimulation are required, the second reflector 157 is moved into the optical path, at which time the laser beam from the beam combiner 151 can be reflected by the second reflector 157 to the second dichroic mirror 152, and then illuminate the target sample through the objective lens 153; while when large-field imaging is required, the second reflector 157 is moved out of the optical path, so that the excitation light directly illuminates the target sample through the objective lens 153. In this embodiment, by moving the second reflector 157 in and out, different optical path modes are switched to ensure that the corresponding light beam can reach the target sample with the optimal path under different imaging or stimulation requirements, thereby improving the integration and ease of operation of the optical imaging device 100. Figure 4 The dashed box indicates that the optical path can be removed.

[0050] Figure 5 This is a path diagram of the light rays in the primary mirror module and the collection module of the two-photon imaging optical path provided in another embodiment of this application. For example... Figure 5 As shown, a first-wavelength laser beam and a second-wavelength laser beam are combined by a beam combiner 151. The combined laser beam is reflected by a second reflector 157 to a second dichroic mirror 152. The second dichroic mirror 152 transmits the combined laser beam, allowing it to be transmitted to an objective lens 153. The objective lens 153 illuminates the target sample with the combined laser beam. The target sample emits a first fluorescence upon being illuminated by the laser beam. The first fluorescence is transmitted through the objective lens 153 to the second dichroic mirror 152, which reflects the first fluorescence to a collection module 160. When both the first-wavelength and second-wavelength laser beams are used for imaging, the first fluorescence includes a first sub-fluorescence emitted by the excitation of the first-wavelength laser beam and a second sub-fluorescence emitted by the excitation of the second-wavelength laser beam. Within the collection module 160, the first and second sub-fluorescences are transmitted through the filter 163 to the beam splitter 161. The beam splitter 161 reflects the first sub-fluorescence and transmits the second sub-fluorescence, so that the first and second sub-fluorescences are respectively transmitted to two photodetectors 162 (e.g., ...). Figure 5 (As shown). When a laser beam of the first wavelength is used for imaging and a laser beam of the second wavelength is used for stimulation, the first fluorescence consists only of the first sub-fluorescence emitted by the excitation of the laser beam of the first wavelength. Within the collection module 160, the first sub-fluorescence is transmitted to the photodetector 162 (not shown) via a filter 163 and a beam splitter 161.

[0051] Understandably, the bundle structure 151 includes a fourth dichroic mirror.

[0052] In this embodiment, the beam combiner 151, the second dichroic mirror 152, and the objective lens 153 form a two-photon imaging optical path. Two-photon imaging based on the first sub-fluorescence allows observation of the potential changes in the stimulated neuron, or the potential changes in neurons surrounding the stimulated neuron. These potential changes include action potentials, resting potentials, etc.

[0053] Figure 6 This is a path diagram of light transmission in the main mirror module for a large field-of-view imaging optical path provided in another embodiment of this application. For example... Figure 6 As shown, the light source assembly 154 emits excitation light, which is transmitted to the third dichroic mirror 155. The third dichroic mirror 155 reflects the excitation light so that it is transmitted to the objective lens 153. The objective lens 153 illuminates the target sample with the excitation light, and the target sample emits second fluorescence when illuminated by the excitation light. The second fluorescence is transmitted through the objective lens 153 to the third dichroic mirror 155, and the third dichroic mirror 155 transmits the second fluorescence so that it is transmitted to the image sensor 156 to achieve imaging of the second fluorescence.

[0054] It is worth noting that the imaging achieved through the wide-field-of-view imaging optical path is single-photon imaging, which can be used to quickly acquire overall structural information of the target sample, such as observing the distribution of neurons or the macroscopic morphology of tissue sections, providing regional localization reference for subsequent two-photon imaging and precise stimulation. In practice, users can first perform a preliminary scan of the target sample through the wide-field-of-view imaging optical path to determine the region of interest, and then switch to the two-photon imaging optical path for high-resolution fine imaging and neuronal stimulation experiments, thereby achieving multi-scale observation and research from macroscopic to microscopic levels.

[0055] Figure 7 This is a diagram showing the result of photostimulation provided in one embodiment of this application. For example... Figure 7 As shown, the area stimulated by light will undergo photobleaching, appearing black. The area outlined is the light stimulation trajectory. Understandably, the outlined area is the target area.

[0056] Figure 8 This is a schematic flowchart of an optical imaging method provided in an embodiment of this application. Figure 8 As shown, one embodiment of this application provides an optical imaging method, applied to any of the optical imaging devices mentioned above. The optical imaging method includes: Step S110: Configure the first optical path to be in imaging mode and the second optical path to be in stimulation mode.

[0057] The first optical path passes through the first galvanometer module, and the second optical path passes through the second galvanometer module.

[0058] Step S120: Control the laser beam emitted by the first laser to enter the first galvanometer module to image the target sample through the first optical path; control the laser beam emitted by the second laser to enter the second galvanometer module to stimulate the target area through the second optical path.

[0059] It is worth noting that the number of first lasers can be one or more, and the laser beams emitted by multiple first lasers have different wavelengths; similarly, the number of second lasers can be one or more, and the laser beams emitted by multiple second lasers have different wavelengths. That is, multiple wavelength laser beams can be used for imaging, and multiple wavelength laser beams can be used for stimulation.

[0060] As can be understood, the imaging mode refers to the mode in which the laser beam is used to image the target sample; the stimulation mode refers to the mode in which the laser beam is used to stimulate the target region. In the first optical path, the laser beam propagates along the first direction and the second direction respectively to achieve point-by-point scanning of the target sample; in the second optical path, the laser beam propagates along the first direction and the second direction respectively to achieve point-by-point scanning of the target sample, thereby precisely stimulating the target region, i.e., achieving precise stimulation of a single neuron. The first direction and the second direction do not overlap.

[0061] For example, a first laser emits a laser beam of a first wavelength, and a second laser emits a laser beam of a second wavelength. The first wavelength laser beam enters a first galvanometer module and illuminates the target sample through a first optical path. The second wavelength laser beam enters a second galvanometer module and illuminates the target sample through a second optical path. After being illuminated by the first wavelength laser beam, the target sample emits first fluorescence, achieving two-photon imaging of the target sample. When neurons in the target region are illuminated by the second wavelength laser beam (i.e., stimulated by the second wavelength laser beam), the potential of the neurons themselves or surrounding neurons changes. The potential changes of the stimulated neurons or the potential changes of neurons surrounding the stimulated neurons can be observed based on two-photon imaging using the first fluorescence.

[0062] In this embodiment, by configuring the first optical path to imaging mode and the second optical path to stimulation mode, the laser beam emitted by the first laser is controlled to enter the first galvanometer module to image the target sample through the first optical path; the laser beam emitted by the second laser is controlled to enter the second galvanometer module to stimulate the target area through the second optical path, achieving point-by-point scanning of the laser beam. The point-by-point scanning two-photon excitation method improves spatial resolution and increases the accuracy of optical stimulation, enabling precise stimulation of individual neurons. Furthermore, related holographic optical stimulation techniques disperse the laser across multiple regions. Since two-photon excitation is proportional to the square of the light intensity, it often requires more expensive high-power femtosecond lasers. In contrast, the point-by-point scanning of the laser beam in this application ensures sufficient stimulation energy to each region, eliminating the need for high-energy optical stimulation lasers, resulting in lower cost and easier availability.

[0063] In some embodiments, controlling the laser beam emitted by the second laser to enter the second galvanometer module to stimulate the target region through the second optical path includes: determining the target region and stimulation parameters of the target region; and, based on the stimulation parameters, controlling the laser beam emitted by the second laser to enter the second galvanometer module to stimulate the target region through the second optical path. The stimulation parameters include stimulation duration, stimulation power, and stimulation mode. In this embodiment, based on the characteristics of a preset fluorescent protein on a neuron in the target region, the stimulation power, stimulation duration, and stimulation mode are set. The optical imaging device can precisely control the deflection angle and deflection speed of the second galvanometer module based on these parameters, thereby guiding the laser beam emitted by the second laser to perform point-by-point scanning stimulation within the target region according to a preset trajectory. It is understood that the stimulation parameters for different target regions can be the same or different.

[0064] In some embodiments, after controlling the laser beam emitted by the first laser to enter the first galvanometer module to image the target sample through the first optical path; and controlling the laser beam emitted by the second laser to enter the second galvanometer module to stimulate the target area through the second optical path, the method further includes: acquiring an image of the target sample based on the first fluorescence emitted by the target sample irradiated by the laser beam emitted by the first laser; acquiring the stimulation result of the target sample irradiated by the laser beam emitted by the first laser, and saving the image and the light stimulation result.

[0065] In some embodiments, before configuring the first optical path to be in imaging mode and the second optical path to be in stimulation mode, the method further includes: configuring both the first and second optical paths to be in imaging mode; controlling the first and second lasers to emit laser beams so that the laser beams irradiate the target sample; and calibrating the scanning parameters of the first and / or second galvanometer modules based on the laser beams. The scanning parameters include scanning range, scanning speed, scanning accuracy, and the mapping relationship between the galvanometer deflection angle and the actual scanning position.

[0066] Specifically, the scanning parameters of the first galvanometer module and / or the second galvanometer module are calibrated based on the laser beam, including: obtaining a first calibration image corresponding to the laser beam passing through the first galvanometer module and a second calibration image corresponding to the laser beam passing through the second galvanometer module based on the third and fourth fluorescence emitted by the target sample after being irradiated by the laser; calibrating the scanning parameters of the first galvanometer module and / or the second galvanometer module based on the first calibration image and the second calibration image, so that the first calibration image coincides with the second calibration image.

[0067] For example, a first laser emits a laser beam of a first wavelength, and a second laser emits a laser beam of a second wavelength. The first wavelength laser beam enters a first galvanometer module and illuminates a target sample through a first optical path. The second wavelength laser beam enters a second galvanometer module and illuminates the target sample through a second optical path. After being illuminated by the first wavelength laser beam, the target sample emits a third fluorescence, achieving two-photon imaging of the target sample. After being illuminated by the second wavelength laser beam, the target sample emits a fourth fluorescence. A first calibration image is obtained based on the third fluorescence, and a second calibration image is obtained based on the fourth fluorescence. The first and second calibration images are then overlapped to calibrate the scanning parameters of the first galvanometer module and / or its scanning parameters. In this embodiment, calibration using a target sample effectively eliminates system errors that may occur during mechanical installation and optical path transmission between the first and second galvanometer modules. When the first and second calibration images completely overlap, it indicates that the scanning positions of the first and second optical paths on the target sample are precisely corresponding, and the scanning parameters of the first and second galvanometer modules have reached an optimal matching state. Subsequently, when imaging and stimulating the target sample, it can be ensured that the spatial positions of the imaging area and the stimulation area correspond precisely, avoiding the problem of mismatch between the stimulation position and the observation position due to optical path calibration deviation, thereby further improving the accuracy and reliability of the experimental results. Understandably, the imaging mode and stimulation mode can be freely switched.

[0068] The basic principles of this application have been described above with reference to specific embodiments. However, it should be noted that the advantages, benefits, and effects mentioned in this application are merely examples and not limitations, and should not be considered as essential features of each embodiment of this application. Furthermore, the specific details disclosed above are for illustrative and facilitative purposes only, and are not limitations. These details do not limit the application to the necessity of employing the aforementioned specific details for implementation.

[0069] The block diagrams of devices, apparatuses, devices, and systems involved in this application are merely illustrative examples and are not intended to require or imply that they must be connected, arranged, or configured in the manner shown in the block diagrams. As those skilled in the art will recognize, these devices, apparatuses, devices, and systems can be connected, arranged, and configured in any manner. Words such as “comprising,” “including,” “having,” etc., are open-ended terms meaning “including but not limited to,” and are used interchangeably with them. The terms “or” and “and” as used herein refer to the terms “and / or,” and are used interchangeably with them unless the context clearly indicates otherwise. The term “such as” as used herein refers to the phrase “such as but not limited to,” and is used interchangeably with it.

[0070] It should also be noted that in the apparatus, equipment, and methods of this application, the components or steps can be disassembled and / or recombined. These disassemblies and / or recombinations should be considered as equivalent solutions of this application.

[0071] The above description of the disclosed aspects is provided to enable any person skilled in the art to make or use this application. Various modifications to these aspects will be readily apparent to those skilled in the art, and the general principles defined herein can be applied to other aspects without departing from the scope of this application. Therefore, this application is not intended to be limited to the aspects shown herein, but rather to be accorded the widest scope consistent with the principles and novel features disclosed herein.

[0072] The above description has been given for purposes of illustration and description. Furthermore, this description is not intended to limit the embodiments of this application to the forms disclosed herein. Although numerous exemplary aspects and embodiments have been discussed above, those skilled in the art will recognize certain variations, modifications, alterations, additions, and sub-combinations thereof.

[0073] The specific embodiments described above do not constitute a limitation on the scope of protection of this invention. Those skilled in the art should understand that various modifications, combinations, sub-combinations, and substitutions can be made according to design requirements and other factors. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this invention should be included within the scope of protection of this invention.

Claims

1. An optical imaging device, characterized by, include: The light source module, the first galvanometer module, the second galvanometer module, the switching module, and the main mirror module; The light source module includes at least two lasers for emitting laser beams of different wavelengths; The switching module is disposed in the optical path between the light source module and the first galvanometer module and the second galvanometer module. The switching module is used to dynamically guide a portion of the laser beam from the light source module to the first galvanometer module and / or the second galvanometer module. The first galvanometer module is used to control the two-dimensional deflection angle of the incident laser beam; The second galvanometer module is used to control the two-dimensional deflection angle of the incident laser beam; The main mirror module is used to irradiate the target sample with a laser beam from the first galvanometer module to image the target sample, and / or irradiate the target region of the target sample with a laser beam from the second galvanometer module to stimulate the target region.

2. The optical imaging device according to claim 1, characterized in that, The switching module is used to dynamically guide a portion of the laser beam from the light source module to the first galvanometer module and / or the second galvanometer module, including: controlling all the laser beams to enter the first galvanometer module, or controlling a portion of the laser beams to enter the first galvanometer module and a portion of the laser beams to enter the second galvanometer module.

3. The optical imaging device according to claim 1, characterized in that, The switching module includes a first dichroic mirror and a first reflecting mirror.

4. The optical imaging device according to claim 1, characterized in that, The main mirror module includes a beam combining structure, a second dichroic mirror, and an objective lens. The beam combining structure is used to combine laser beams from the first galvanometer module and the second galvanometer module. The second dichroic mirror is used to transmit the combined laser beam. The objective lens is used to irradiate the target sample with the combined laser beam. The second dichroic mirror is also used to reflect the first fluorescence emitted by the target sample when irradiated by the laser beam. The beam combining structure, the second dichroic mirror, and the objective lens form a two-photon imaging optical path.

5. The optical imaging device according to claim 4, characterized in that, The main mirror module also includes a light source assembly, a third dichroic mirror, and an image sensor. The light source assembly is used to emit excitation light, the third dichroic mirror is used to reflect the excitation light emitted by the light source assembly and transmit the excitation light emitted by the light source assembly to the objective lens, the objective lens is used to irradiate the target sample with the excitation light emitted by the light source assembly, and the third dichroic mirror is also used to transmit the second fluorescence emitted by the target sample under the irradiation of the excitation light and transmit the second fluorescence to the image sensor. The light source assembly, the third dichroic mirror, the objective lens, and the image sensor form a large field-of-view imaging optical path; The main mirror module further includes a second reflecting mirror, which is located between the beam combining structure and the second dichroic mirror, and between the third dichroic mirror and the objective lens, for controlling the laser beam released by the laser or the excitation light emitted by the light source assembly to irradiate the target sample.

6. The optical imaging device according to claim 1, characterized in that, It also includes a collection module, which is used to collect the first fluorescence emitted by the target sample when irradiated by the laser beam, and convert the first fluorescence into an electrical signal.

7. An optical imaging method, applied to the optical imaging device according to any one of claims 1 to 6, characterized in that, include: The first optical path is configured to be in imaging mode and the second optical path is configured to be in stimulation mode, wherein the first optical path passes through the first galvanometer module and the second optical path passes through the second galvanometer module; The laser beam emitted by the first laser is controlled to enter the first galvanometer module so as to image the target sample through the first optical path; The laser beam emitted by the second laser is controlled to enter the second galvanometer module to stimulate the target area through the second optical path.

8. The optical imaging method according to claim 7, characterized in that, Before configuring the first optical path to be in imaging mode and the second optical path to be in stimulation mode, the method further includes: Both the first optical path and the second optical path are configured to be in imaging mode; The first laser and the second laser are controlled to emit laser beams so that the laser beams irradiate the target sample; The scanning parameters of the first galvanometer module and / or the second galvanometer module are calibrated based on the laser beam.

9. The method according to claim 8, characterized in that, The calibration of the scanning parameters of the first galvanometer module and / or the second galvanometer module based on the laser beam includes: Based on the third and fourth fluorescence emitted by the target sample after being irradiated by laser, a first calibration image corresponding to the laser beam passing through the first galvanometer module and a second calibration image corresponding to the laser beam passing through the second galvanometer module are obtained. Based on the first calibration image and the second calibration image, calibrate the scanning parameters of the first galvanometer module and / or the first galvanometer module so that the first calibration image coincides with the second calibration image.

10. The method according to claim 7, characterized in that, The laser beam emitted by the second laser is controlled to enter the second galvanometer module to stimulate the target region through the second optical path, including: Determine the target region and the stimulation parameters of the target region; Based on the stimulation parameters, the laser beam emitted by the second laser is controlled to enter the second galvanometer module to stimulate the target area through the second optical path.

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