Optical system for performing imaging, stimulation and laser ablation on neuron culture
Through the integrated optical systems of fluorescence imaging, optogenetic stimulation and patterned laser ablation, the problem of unchangeable neuron connections in in vitro cultured neural networks is solved, and the precise imaging, stimulation and ablation of neurons is achieved, supporting the construction of modular structures.
Patent Information
- Application Number
- CN202510411660.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-02
- Publication Date
- 2025-07-08
AI Technical Summary
The prior art cannot achieve physical connection changes between neurons in biological neural networks cultured in vitro, resulting in the inability to form a modular structure, and optogenetic stimulation cannot last for long-term changes in the connection intensity between neurons.
An optical system integrating fluorescence imaging, optogenetic stimulation and patterned laser ablation was designed. Through the fluorescence imaging module, optogenetic stimulation module and patterned laser ablation module, a femtosecond laser is used to perform precise ablation of neurons, and combined with a digital micromirror device and a spatial light modulator for fine control of light.
The integration of neuron imaging, stimulation and ablation functions is achieved, and the neuron connection changes can be made with submicron accuracy, providing experimental basis and realizing the modular structure of neurons, supporting a variety of experimental needs.
Smart Images
Figure CN120275348A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of neuroscience, and in particular, to an optical system for imaging, stimulating, and laser ablating neuron cultures. Background Art
[0002] Biological neural networks cultured in vitro are biological models between the scales of neurons and the brain, and are widely used in the research of neural development, neurological diseases, and neural dynamics. In recent years, biological neural networks cultured in vitro have been proven to be able to complete some simple pattern recognition tasks. To achieve these tasks, it is necessary to monitor the activities of neurons in the network and simultaneously stimulate related neurons to generate corresponding activities.
[0003] Using optical methods to record and control the activities of neurons has the advantages of high spatial resolution and strong regulation flexibility. The current mainstream neuron activity detection technology is calcium fluorescence imaging technology, and the neuron activity regulation technology is optogenetic technology. Calcium fluorescence imaging technology labels neurons through gene encoding or chemical calcium indicators. When action potentials trigger calcium influx, the fluorescence intensity is positively correlated with neuron activity, realizing population activity recording with millisecond-level time resolution. Optogenetic technology expresses light-sensitive channel proteins (such as ChR2) in neurons through gene editing, and uses blue light to precisely activate neuron firing.
[0004] There have been literature reports on building an optical system that simultaneously performs optogenetic and calcium fluorescence imaging to regulate the activities of neural networks. However, since optogenetic stimulation can only change the plasticity of biological neurons and cannot change the physical connections between neurons, this means that once optogenetic stimulation is removed, the connection strength between neurons may return to the original state, and it is impossible to make neuron cultures form modular structures. Summary of the Invention
[0005] In view of this, embodiments of the present invention provide an optical system for imaging, stimulating, and laser ablating neuron cultures to eliminate or improve one or more defects existing in the prior art.
[0006] One aspect of the present invention provides an optical system for imaging, stimulating, and laser ablating neuron cultures, the optical system including a fluorescence imaging module, an optogenetic stimulation module, and a patterned laser ablation module:
[0007] The optogenetic stimulation module includes a first light source, a first filter, a total reflection prism, a digital micromirror device, a first dichroic mirror, a first lens, a second dichroic mirror, and an objective lens arranged in sequence. The light emitted by the first light source is modulated by the digital micromirror device into a structured beam after passing through the total reflection prism and is irradiated onto the sample;
[0008] The fluorescence imaging module includes a second light source, a second filter, a first lens, a first plane mirror, a tube lens, a third filter and a camera. The light emitted by the second light source is reduced in beam diameter through the first lens, the second dichroic mirror and the objective lens of the optogenetic stimulation module, irradiated on the sample, and recorded through the optical path of the camera.
[0009] The patterned laser ablation module includes a femtosecond laser, a half-wave plate, a polarization beam splitter, a spatial light modulator and a third dichroic mirror. The light emitted by the femtosecond laser is reduced in beam diameter through the second dichroic mirror and the objective lens of the optogenetic stimulation module to ablate the neurons on the sample.
[0010] With the above solution, the system of this solution can integrate the modules for imaging, stimulating and ablating neurons, and can output light to the sample through a common path (i.e., the second dichroic mirror and the objective lens), with a more compact structure. This solution can simultaneously have the functions of stimulating and ablating neurons, and in the actual use process, it can provide image basis for users through the imaging of neurons. Users can select neurons according to the actual needs of the experiment and apply light stimulation using the optogenetic stimulation module, or ablate the selected neurons on the sample using the patterned laser ablation module.
[0011] In some embodiments of the present invention, the central wavelength of the first filter is 470 nm and the bandwidth is 40 nm. The light emitted by the first light source is wavelength-limited to 450 nm to 490 nm after passing through the first filter; the central wavelength of the second filter is 560 nm and the bandwidth is 50 nm. The light emitted by the second light source is wavelength-defined to 535 nm to 585 nm after passing through the second filter.
[0012] In some embodiments of the present invention, during the light propagation process of the optogenetic stimulation module, the light emitted by the first light source is wavelength-limited to 450 nm to 490 nm after passing through the first filter, modulated by the digital micromirror device into a customized structured light beam after passing through the total reflection prism, and then reflected by the total reflection prism to the first dichroic mirror. Subsequently, the beam is irradiated on the sample by the beam reduction system composed of the first lens and the objective lens.
[0013] In some embodiments of the present invention, the fluorescence imaging module includes an output light optical path and a received light optical path. In the output light optical path, the light emitted by the second light source is wavelength-defined to 535 nm to 585 nm after passing through the second filter, reduced in beam diameter through the first lens and the objective lens of the optogenetic stimulation module, and irradiated on the sample; in the received light optical path, the light emitted by the sample is recorded by the microscopic imaging optical path composed of the objective lens, the first plane mirror, the tube lens, the third filter and the camera.
[0014] In some embodiments of the present invention, the patterned laser ablation module further includes a light barrier, which cooperates with the polarization beam splitter to receive the light reflected by the polarization beam splitter.
[0015] In some embodiments of the present invention, the patterned laser ablation module further includes a second plane mirror, a second lens, and a third lens. The light passing through the polarization beam splitter is expanded by the beam expander system composed of the second lens and the third lens after passing through the second plane mirror.
[0016] In some embodiments of the present invention, the patterned laser ablation module further includes a third plane mirror, which reflects the expanded light emitted by the third lens to the spatial light modulator for phase modulation.
[0017] In some embodiments of the present invention, the patterned laser ablation module further includes a fourth lens and a fifth lens. The light beam emitted by the spatial light modulator is condensed by the fourth lens and the fifth lens and propagates to the third dichroic mirror.
[0018] In some embodiments of the present invention, the third dichroic mirror outputs light to the second dichroic mirror.
[0019] In some embodiments of the present invention, the spatial light modulator is composed of a liquid crystal pixel array, and each pixel is used to individually control the phase of the reflected light, so as to be able to finely control the light.
[0020] Additional advantages, objects, and features of the present invention will be partially described below and will become partially apparent to those of ordinary skill in the art after studying the following text, or can be learned from the practice of the present invention. The objects and other advantages of the present invention can be pointed out and obtained specifically in the specification and the drawings.
[0021] Those skilled in the art will understand that the objects and advantages that can be achieved by the present invention are not limited to the above specifically described, and the above and other objects that the present invention can achieve will be more clearly understood according to the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] The drawings described herein are used to provide a further understanding of the present invention, form a part of this application, and do not limit the present invention.
[0023] Figure 1 It is a schematic diagram of the architecture of the optical system for imaging, stimulating, and laser ablating neuron cultures in this solution;
[0024] Figure 2 It is a schematic diagram of calcium ion fluorescence imaging data;
[0025] Figure 3 Schematic diagram of multi-point optogenetic stimulation;
[0026] Figure 4 Schematic diagram of the effect of serial single-point optogenetic stimulation;
[0027] Figure 5 Schematic diagram of a photo of using femtosecond laser to ablate neuron process clusters;
[0028] Figure 6 Schematic diagram of a photo of using an optical system to ablate a single neuron axon;
[0029] Figure 7 Schematic diagram of the result of an optical system simultaneously generating two effective cutting light spots;
[0030] Figure 8 Schematic diagram of a sequential laser ablation operation;
[0031] Figure 9 Schematic diagram of the experimental process of using an optical system to pattern ablate an in vitro biological neural network;
[0032] Figure 10 Schematic diagram of the calcium fluorescence signal response of a single neuron and its surrounding neurons when using an optogenetic stimulation module to stimulate a single neuron in the visual field;
[0033] Figure 11 Schematic diagram of the change in the calcium fluorescence intensity of neurons in the visual field when using a laser ablation module to ablate an in vitro neural network. Detailed implementation manners
[0034] To make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below in combination with the implementation manners and the accompanying drawings. Herein, the illustrative implementation manners and descriptions of the present invention are used to explain the present invention, but do not limit the present invention.
[0035] Herein, it should also be noted that in order to avoid obscuring the present invention due to unnecessary details, only the structures and / or processing steps closely related to the solution of the present invention are shown in the drawings, while other details less related to the present invention are omitted.
[0036] As Figure 1 shown, the present invention provides an optical system for imaging, stimulating and laser ablating neuron cultures, and the optical system includes a fluorescence imaging module, an optogenetic stimulation module and a patterned laser ablation module:
[0037] The optogenetic stimulation module comprises a first light source, a first filter, a total reflection prism, a digital micromirror device, a first dichroic mirror, a first lens, a second dichroic mirror and an objective lens which are arranged in sequence. The light emitted by the first light source passes through the total reflection prism and is modulated by the digital micromirror device into a structured light beam, which is irradiated onto the sample.
[0038] In a specific implementation process, the focal length of the first lens (L1) is 200 mm, the cut-off wavelength of the first dichroic mirror (DM1) is 505 nm, and the cut-off wavelength of the second dichroic mirror (DM2) is 588 nm.
[0039] The fluorescence imaging module comprises a second light source, a second filter, a first lens, a first plane reflector, a tube lens, a third filter and a camera, wherein the light emitted by the second light source is beam-contracted by the first lens, the second dichroic mirror and the objective lens of the optogenetic stimulation module, irradiated on the sample, and recorded through the optical path of the camera;
[0040] In the specific implementation process, the first light source (LED1) and the second light source (LED2) are both LED light sources; the focal length of the tube lens (TL) is 200 mm; the third filter (F3) is a high-pass filter with a starting wavelength of 590 nm; the pixel size of the camera is 6.5 μm, and the sensor target surface size is 16.6×14.0 mm.
[0041] The patterned laser ablation module includes a femtosecond laser, a half-wave plate (HWP), a polarization beam splitter (PBS), a spatial light modulator (SLM) and a third dichroic mirror (DM3). The light emitted by the femtosecond laser is beam-contracted by the second dichroic mirror and the objective lens of the optogenetic stimulation module to ablate neurons on the sample.
[0042] In the specific implementation process, the femtosecond pulse laser emitted by the femtosecond laser, with its ultra-short pulse width (up to 10-15 seconds) and high peak power, triggers the ionization process inside the material through nonlinear optical effects such as multiphoton absorption and photoionization, and achieves tissue cutting with submicron precision. In the field of neuroscience, its high spatial selectivity is used for non-thermal ablation of neural tissue, such as cutting off specific nerve fibers or reshaping neural network connections. Femtosecond lasers can accurately remove target neurons through plasma-induced ablation without causing significant thermal damage to surrounding tissues.
[0043] In a specific implementation process, the wavelength of the femtosecond laser is 1032 nm, the pulse width is 320 fs, and the cutoff wavelength of the third dichroic mirror is 805 nm.
[0044] Adopting the above solution, the system of this solution can integrate modules for neuron imaging, stimulation, and ablation, and can output light to the sample through a common path (i.e., the second dichroic mirror and the objective lens), with a more compact structure. This solution can simultaneously possess the functions of neuron stimulation and ablation, and during actual use, it can provide image basis for users through neuron imaging. Users can select neurons based on the actual needs of the experiment and apply light stimulation using the optogenetic stimulation module, or ablate the selected neurons on the sample using the patterned laser ablation module.
[0045] In some embodiments of the present invention, the central wavelength of the first filter (F1) is 470 nm and the bandwidth is 40 nm. The light emitted by the first light source is wavelength-limited to 450 nm to 490 nm after passing through the first filter; the central wavelength of the second filter (F2) is 560 nm and the bandwidth is 50 nm. The light emitted by the second light source is wavelength-defined to 535 nm to 585 nm after passing through the second filter.
[0046] In some embodiments of the present invention, during the light propagation process of the optogenetic stimulation module, the light emitted by the first light source is wavelength-limited to 450 nm to 490 nm after passing through the first filter, and after being modulated by the digital micromirror device into a customized structured light beam through the total internal reflection prism (TIR), it is then reflected by the total internal reflection prism to the first dichroic mirror, and subsequently, the beam is irradiated onto the sample by the beam reduction system composed of the first lens and the objective lens.
[0047] During the specific implementation process, the central wavelength of the first light source is 470 nm and the bandwidth is 28 nm.
[0048] During the specific implementation process, the light emitted by the first light source is reduced into a beam of corresponding size after passing through the first filter and the objective lens and then irradiated onto a sample containing the jRCaMP1b
[12] calcium ion protein, and the sample emits fluorescence with a central wavelength of approximately 595 nm.
[0049] Figure 2 shows the calcium ion fluorescence imaging data of neurons in the sample on the 16th day after culturing, Figure 2 (a) and on the 22nd day, as Figure 2 (b) shows. The left figure is a photo of the calcium ion fluorescence imaging of the sample collected by the optical system. The white circular object in the figure is the neuron (with a diameter of about 15 μm). The right figure is the calcium ion fluorescence curve graph of four neurons at the same position in the photo on different days (ΔF / F0: relative change in calcium ion fluorescence intensity, ΔF = F - F0, F is the real-time fluorescence intensity value of the neuron, and F0 is the background fluorescence intensity value of the neuron when there is no activity), indicating that the time resolution of the optical system for recording calcium ion fluorescence imaging meets the experimental requirements.
[0050] This optical system can generate diverse optical stimulation patterns. Figure 3 The experimental process of using the optical system for multi-point optogenetic stimulation of a sample is shown, mainly divided into three steps: First, after performing calcium fluorescence imaging on the sample, select the neurons that need to be optogenetically stimulated; second, create a stimulation pattern suitable for a digital micromirror device (DMD) according to the positions of the selected neurons; third, activate the optogenetic module and run the DMD to generate the corresponding structured light beam. From the actual effect, the optical system can accurately perform multi-point stimulation. In addition to multi-point simultaneous stimulation of the sample, this optical system can also perform single-point serial stimulation. As shown in Figure 4 (a) and 4(b), after creating a serial single-point stimulation pattern, the optical system can stimulate the neurons in the sample point by point. The optical system can limit the stimulation spot to the single neuron cell body, reducing the influence on the stimulation of other neurons.
[0051] In some embodiments of the present invention, the fluorescence imaging module includes an output light optical path and a receiving light optical path. In the output light optical path, the light emitted by the second light source is wavelength-limited to 535 nm to 585 nm after passing through the second filter, and is reduced in beam size by the first lens and the objective lens of the optogenetic stimulation module and then irradiated on the sample; in the receiving light optical path, the light emitted by the sample is recorded by the microscopic imaging optical path composed of the objective lens, the first plane mirror (M1), the tube lens, the third filter, and the camera.
[0052] In some embodiments of the present invention, the patterned laser ablation module further includes a light stop (OB), and the light stop cooperates with the polarization beam splitter to receive the light reflected by the polarization beam splitter.
[0053] In some embodiments of the present invention, the patterned laser ablation module further includes a second plane mirror (M2), a second lens, and a third lens. The light passing through the polarization beam splitter is expanded by the beam expander system composed of the second lens and the third lens after passing through the second plane mirror.
[0054] In the specific implementation process, the focal length of the second lens is 50 mm, and the focal length of the third lens is 80 mm.
[0055] In some embodiments of the present invention, the patterned laser ablation module further includes a third plane mirror (M3), and the third plane mirror reflects the expanded light emitted by the third lens to the spatial light modulator for phase modulation.
[0056] In some embodiments of the present invention, the patterned laser ablation module further includes a fourth lens and a fifth lens. The light beam emitted by the spatial light modulator is reduced in beam size by the fourth lens and the fifth lens and then propagates to the third dichroic mirror.
[0057] In a specific implementation process, the focal length of the fourth lens is 250 mm, and the focal length of the fifth lens is 200 mm.
[0058] In some embodiments of the present invention, the third dichroic mirror outputs light to the second dichroic mirror.
[0059] In some embodiments of the present invention, the spatial light modulator is composed of a liquid crystal pixel array, and each pixel is used to individually control the phase of the reflected light, so as to be able to finely control the light.
[0060] Specifically, the HWP and PBS constitute a laser attenuation system. Rotating the HWP can adjust the light intensity of the laser after passing through the PBS, and a light baffle is used to receive the light reflected by the PBS. The light passing through the PBS will be expanded by an expanding system composed of a second lens (L2) and a third lens (L3) after passing through M2. The third plane mirror (M3) reflects the expanded light to the SLM for phase modulation. After that, the light beam passes through a beam contraction system composed of a fourth lens (L4) and a fifth lens (L5), and finally the objective lens focuses the light beam onto the sample surface. By controlling the power of the femtosecond laser, neurons at specific positions can be ablated; Figure 5 Shown is a photograph of using a femtosecond laser to ablate a cluster of neuron protrusions. It can be seen that the optical system can ablate the connection between two nerve clusters. This optical system can also achieve more precise laser ablation. Figure 6 Shown is a photograph of the optical system ablating a single neuron axon. Figure 6 (a) and Figure 6 (b) respectively represent microscopic pictures of the neuron axon before and after ablation. It can be seen that the axon of the neuron is cut, but the neuron cell body remains intact. By editing the phase map of the spatial light modulator, the optical system can also generate two ablation light spots simultaneously. The spatial light modulator (SLM) in this solution is composed of a liquid crystal pixel array, and each pixel can individually control the phase of the reflected light, so as to be able to finely control the light and allow the user to create a two-dimensional patterned light field with controllable features. In the patterned laser ablation module, the user can make the target stimulation position into a stimulation pattern, use the Gerchberg Saxton (GS) algorithm to convert the stimulation pattern into a phase map, and input the phase map into the SLM, then a patterned stimulation light field can be formed on the sample surface. Figure 7 (a) and Figure 7 (b) show the light intensity distribution diagrams of the two ablation light spots generated by the system. Figure 7(c) shows the normalized intensity distribution maps of two light spots in the x and y directions. According to the full width at half maximum rule, the diameter of each light spot is calculated to be approximately 5 μm, which is smaller than the diameter of the neuron cell body, indicating that the optical system has the potential for sub-neuronal level laser ablation operations at multiple positions. This optical system also supports sequential laser ablation operations. Figure 8 Shows a schematic diagram of the sequential laser ablation operation, which is mainly divided into three parts. One is to determine the ablation area. The second is to create a phase map that needs to be imported into the SLM according to the ablation position. The third is to start the optical system, adjust the output power of the laser, and irradiate the patterned ablation light field onto the sample surface in sequence to ablate the neurons at the corresponding positions. Figure 9 Shows the experimental process of using the optical system to perform patterned ablation on an in vitro biological neural network. The structured laser starts ablating from the bottom of the neuron sample until a square area is ablated in the central position of the neuron sample. Specifically, Figure 9 (a - d) The neuron cultures in the orange frames are ablated in sequence until an independent square area remains in the middle of the neural network. The red arrows represent the ablation directions. Figure 9 The enlarged view of the area corresponding to the red dashed box in the figure shows the situation before, during, and after ablation of this area.
[0061] Specifically, the three modules of this solution can not only operate independently but also cooperate with each other to study the changes in network activities of in vitro neural networks when stimulated and when their topological structures change. Figure 10 Shows the calcium fluorescence signal responses of a single neuron and its surrounding neurons in the visual field when stimulated using the optogenetic stimulation module. When the optical system only stimulates neuron No. 4 in the picture, there are obvious differences in the responses of the neurons close to it before and after stimulation. For example, the firing frequency of neural activities of neuron No. 1 during the time periods before and after neuron No. 4 is stimulated is quite different, and the neural activity frequency of neuron No. 3 significantly decreases during the time period when neuron No. 4 is stimulated. These indicate that the optical system has a direct impact on neuron No. 4, but the impact on its neighboring neurons is indirect. Figure 11 Shows the change in the calcium fluorescence intensity of neurons in the visual field when using the laser ablation module to ablate the in vitro neural network. Figure 11 (a) Shows the calcium fluorescence imaging map (pictures I - IX) of the in vitro biological neural network during ablation. For easy display, the original image has been pseudocolored. To understand the fluorescence intensity changes at each position in the visual field during ablation, the differences between all pixels in the calcium fluorescence intensity distribution maps of the 2nd and 9th images and the corresponding pixels in the calcium fluorescence intensity distribution map of the first image were calculated, namely ΔF 21 and ΔF 91 , as Figure 11(b), it can be found that as the ablation process progresses, more and more affected neurons (with significant changes in fluorescence intensity) will appear. Select 5 of these neurons, Figure 11 (c) shows the plot of the relative fluorescence intensity change rate of these neurons over time. The arrows and pentagrams in the figure indicate that there are obvious activity changes (rapid increase in the relative fluorescence intensity change rate) in the neurons at that moment. From Figure 11 (c), it can be seen that the relative change rate of neuron 1 basically remains near 0 throughout the ablation process, indicating that it is hardly affected during the whole process. While neurons 2 and 3 are affected in the early stage of ablation, and neurons 4 and 5 are affected in the later stage. This shows that the patterned laser ablation module can effectively control the influence range of ablation and flexibly regulate the neuronal activities within the region.
[0062] The optical system proposed in this patent can meet the requirements of calcium fluorescence imaging, structured optogenetic stimulation, and patterned laser ablation for in vitro cultured neural networks. After testing, when using a 10× objective lens (numerical aperture NA = 0.3), this system can achieve the detection of calcium fluorescence signal activities of neurons within a field of view of up to 2.32 mm 2 and can simultaneously perform optogenetic stimulation on neurons within a range of 1.73 mm 2 When using a 20× objective lens (numerical aperture NA = 0.5), the range that can be ablated by the laser is approximately 0.40 mm 2 , and this range is sufficient to ensure the ablation requirements for hundreds of neurons within the field of view. This system can help researchers comprehensively understand the dynamic characteristics of in vitro cultured neural networks and manipulate the functional and physical connections of neural networks. The system proposed in this patent can also be used for fluorescence imaging and laser ablation operations of other types of in vitro cell cultures.
[0063] In summary, the system of this solution is divided into three modules, namely the fluorescence imaging module, the optogenetic stimulation module, and the patterned laser ablation module. The fluorescence imaging module can be used to detect in vitro cultured biological neural networks and indirectly reflect the neural activity firing conditions of the network. The optogenetic stimulation module can perform customized structured light field stimulation on the network, control neurons at corresponding positions to rapidly generate neural activities, and basically maintain the original activity of neurons. The patterned laser ablation module can ablate neurons at corresponding positions in the biological neural network without moving the biological sample and can be used to artificially construct a network with a special topological structure. Through appropriate optical path design, these three modules can operate independently or be combined with each other, with high flexibility. This system can be widely applied in the fields of neurobiology, computational neuroscience, neuropathology, etc.
[0064] Those of ordinary skill in the art should understand that the various exemplary components, systems, and methods described in connection with the embodiments disclosed herein can be implemented in hardware, software, or a combination of both. Specifically, whether to implement in hardware or software depends on the specific application and design constraints of the technical solution. Professional technicians can use different methods to implement the described functions for each specific application, but such implementation should not be considered to exceed the scope of the present invention. When implemented in hardware, it can be, for example, an electronic circuit, an application-specific integrated circuit (ASIC), appropriate firmware, a plug-in, a functional card, and so on. When implemented in software, the elements of the present invention are programs or code segments used to perform the required tasks. The program or code segment can be stored in a machine-readable medium or transmitted via a data signal carried in a carrier wave over a transmission medium or a communication link.
[0065] It should be clear that the present invention is not limited to the specific configurations and processes described above and illustrated in the figures. For the sake of brevity, detailed descriptions of known methods are omitted here. In the above embodiments, several specific steps are described and illustrated as examples. However, the method process of the present invention is not limited to the specific steps described and illustrated. Those skilled in the art can make various changes, modifications, and additions, or change the order between steps after understanding the spirit of the present invention.
[0066] In the present invention, the features described and / or exemplified for one embodiment can be used in the same or a similar manner in one or more other embodiments, and / or combined with the features of other embodiments or replace the features of other embodiments.
[0067] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, various changes and modifications can be made to the embodiments of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.
Claims
1. An optical system for imaging, stimulating, and laser ablating neuronal cultures, characterized in that, The optical system includes a fluorescence imaging module, an optogenetic stimulation module, and a patterned laser ablation module: The optogenetic stimulation module includes a first light source, a first filter, a total reflection prism, a digital micromirror device, a first dichroic mirror, a first lens, a second dichroic mirror, and an objective lens arranged in sequence. The light emitted by the first light source is modulated by the digital micromirror device into a structured light beam after passing through the total reflection prism and then irradiates the sample. The fluorescence imaging module includes a second light source, a second filter, a first lens, a first plane mirror, a tube lens, a third filter, and a camera. The light emitted by the second light source is focused by the first lens, the second dichroic mirror, and the objective lens of the optogenetic stimulation module and irradiates the sample, and is recorded through the optical path of the camera. The patterned laser ablation module includes a femtosecond laser, a half-wave plate, a polarization beam splitter, a spatial light modulator, and a third dichroic mirror. The light emitted by the femtosecond laser is focused by the second dichroic mirror and the objective lens of the optogenetic stimulation module to ablate the neurons on the sample.
2. The optical system for imaging, stimulating, and laser ablating a neuronal culture according to claim 1, wherein The central wavelength of the first filter is 470 nm and the bandwidth is 40 nm. The light emitted by the first light source is wavelength-limited to 450 nm to 490 nm after passing through the first filter; the central wavelength of the second filter is 560 nm and the bandwidth is 50 nm. The light emitted by the second light source is wavelength-defined to 535 nm to 585 nm after passing through the second filter.
3. The optical system for imaging, stimulating, and laser ablating a neuronal culture according to claim 2, wherein During the light propagation process of the optogenetic stimulation module, the light emitted by the first light source is wavelength-limited to 450 nm to 490 nm after passing through the first filter, is modulated by the digital micromirror device into a customized structured light beam after passing through the total reflection prism, and is then reflected by the total reflection prism to the first dichroic mirror. Subsequently, the beam is irradiated onto the sample by the focusing system composed of the first lens and the objective lens.
4. The optical system for imaging, stimulating, and laser ablating a neuronal culture according to claim 2, wherein The fluorescence imaging module includes an output light optical path and a received light optical path. In the output light optical path, the light emitted by the second light source is wavelength-defined to 535 nm to 585 nm after passing through the second filter, and is focused by the first lens and the objective lens of the optogenetic stimulation module and irradiates the sample. In the received light optical path, the light emitted by the sample is recorded by the microscopic imaging optical path composed of the objective lens, the first plane mirror, the tube lens, the third filter, and the camera.
5. The optical system for imaging, stimulating, and laser ablating a neuronal culture according to any one of claims 1-4, characterized in that The patterned laser ablation module further includes a light stop, and the light stop cooperates with the polarization beam splitter to receive the light reflected by the polarization beam splitter.
6. The optical system for imaging, stimulating, and laser ablating a neuronal culture according to claim 5, wherein The patterned laser ablation module further includes a second plane mirror, a second lens, and a third lens. The light passing through the polarization beam splitter is expanded by the beam expansion system composed of the second lens and the third lens after passing through the second plane mirror.
7. The optical system for imaging, stimulating, and laser ablating a neuronal culture according to claim 6, wherein The patterned laser ablation module further includes a third plane mirror, and the third plane mirror reflects the expanded light emitted by the third lens to the spatial light modulator for phase modulation.
8. The optical system for imaging, stimulating, and laser ablating a neuronal culture according to claim 7, wherein The patterned laser ablation module further includes a fourth lens and a fifth lens. The beam emitted by the spatial light modulator is focused by the fourth lens and the fifth lens and propagates to the third dichroic mirror.
9. The optical system for imaging, stimulating, and laser ablating a neuronal culture according to claim 8, wherein The third dichroic mirror outputs light to the second dichroic mirror.
10. The optical system for imaging, stimulating, and laser ablating a neuronal culture according to claim 1, wherein The spatial light modulator consists of a liquid crystal pixel array, and each pixel is used to individually control the phase of the reflected light, so that the light can be finely controlled.