An intelligent visual projection method and system suitable for optical imaging

Abstract

The application discloses an intelligent visual projection method and system suitable for optical imaging, wherein the system comprises a visual stimulation control module for setting a visual stimulation control signal; an image output module for generating full-spectrum visual stimulation and light intensity according to the visual stimulation control signal; a spectrum switching module for receiving the full-spectrum visual stimulation and the light intensity and outputting first specific spectrum visual stimulation; an infrared light source module for outputting infrared spectrum visual stimulation; a spectrum processing module for filtering preset green spectrum signals of the first specific spectrum visual stimulation and integrating the infrared spectrum visual stimulation to output second specific spectrum visual stimulation; and an image stimulation focusing module for fine-tuning the projection area and light intensity of the second specific spectrum visual stimulation and projecting the second specific spectrum visual stimulation to a visual tissue region. The application can solve the problem that the prior art cannot meet the research demand of visual neural optical signals and has the characteristics of intelligence, automation, precision and no optical pollution.

Description

Technical Field

[0001] This invention relates to the field of visual neuroscience technology, and more specifically, to an intelligent visual projection method and system suitable for optical imaging. Background Technology

[0002] The detection of group activity of various types of neurons in the visual circuit, such as retinal ganglion cells, can effectively assess the functional differences of different neuronal subtypes and map the functional classification of neurons, forming the visual foundation for understanding neural circuits, neural coding mechanisms, and disease models. In recent years, scientists have increasingly emphasized non-invasive optical imaging techniques to study visual functional coding mechanisms, such as two-photon calcium imaging, which has become an indispensable cutting-edge technique in visual neuroscience. As a novel nonlinear optical imaging technique, two-photon calcium imaging differs from conventional visual electrophysiology techniques. It is primarily based on the strict correspondence between neuronal activity and changes in calcium ion concentration. Using a special calcium ion indicator, the concentration of calcium ions in neurons is displayed in real time as fluorescence intensity. Simultaneously, a fluorescence microscope is used to capture, track, and collect the firing activity of neurons, thereby achieving visualization and characterization of neuronal activity. This technique mainly involves emitting a laser to neurons at specific locations to collect the fluorescence signals generated by the neurons, enabling long-term dynamic detection and functional analysis of hundreds of neurons. Therefore, two-photon calcium imaging itself is a non-invasive method for analyzing the optical signal activity of neuronal cell bodies and dendrites.

[0003] Visual function coding research is based on the premise that "various types of visual stimuli can stimulate the neural firing activity of neurons". Therefore, two-photon calcium imaging systems must be combined with a comprehensive and diverse range of visual stimulation systems in order to realize the analysis of the functional coding of visual-related neuronal populations and the assessment of the differences among various subtypes of neurons.

[0004] The existing technology has the following shortcomings and defects:

[0005] 1. Optical imaging technologies (such as two-photon calcium imaging) are not limited to detecting the action potential signals of neurons like conventional electrophysiological systems. Instead, they detect the optical signals of neurons. Therefore, the stimulus signals of conventional visual systems will be coupled and interfere with the spectral signals of neuronal activity, making it impossible to accurately analyze neuronal activity. In addition, conventional visual stimulus signals can further oversaturate the photomultiplier tubes of the optical signal acquisition system (such as the two-photon calcium imaging system), causing serious damage to the system.

[0006] 2. The fluorescence signal activity of vision-related neurons (such as retinal ganglion cells) exhibits a high response delay. The short delay between consecutive visual stimuli in the conventional visual system severely interferes with the high resolution of neuronal signals. For example, in a typical complex White Noise stimulus, each visual stimulus frame lasts 33ms or 17ms, but the neuronal evoked signal response delay is greater than 33ms. This significantly interferes with the correlation between the neuronal optical response and the visual stimulus, leading to resolution failure.

[0007] 3. In conventional visual systems, color-related visual stimuli are selected from screen stimuli without spectral characteristic evaluation and specific ultra-wide spectrum LED stimuli. The former cannot accurately assess the optimal spectral range of color stimuli, while the latter has a wide and inaccurate spectral range for specific LED spectra. For example, the 565nm LED stimulus source (565L3) produced by Thorlab, a top LED manufacturer, has a spectral output range of 490-680nm, which would cover the optimal sensitive spectral range of M-type and L-type cone cells and rod cells in the retina, thus causing deviations in color resolution.

[0008] 4. Conventional visual stimulation systems lack infrared imaging capabilities, while the interpretation of neuronal visual function encoding requires an infrared imaging system. Otherwise, conventional visible light neuronal imaging can easily cause photobleaching and phototoxicity in retinal neurons, leading to disordered visual encoding activity and functional interpretation deviations. Furthermore, optical signal acquisition systems (such as two-photon calcium imaging systems) also require an infrared spectroscopy system to identify and locate retinal tissue and select the acquisition area.

[0009] 5. Conventional visual stimuli require a relatively long duration of stimulation to successfully resolve the visual function encoding of neurons. This can cause phototoxicity to the optical signal acquisition system (such as a two-photon calcium imaging system) and the retinal tissue within the platform, and significantly prolong the total experimental time for each tissue, thereby reducing the activity of neurons in the tissue. For example, in complex White Noise stimuli, each frame has a brightness ranging from 0 to 255, requiring approximately one hour to resolve the spatiotemporal encoding characteristics of retinal neurons.

[0010] 6. Conventional visual stimuli also lack real-world images or videos, making it impossible to resolve the encoding of natural or video stimuli by neuronal populations; conventional visual stimuli and their subtypes are output in a fixed order, which introduces errors into the original strategy of visual encoding; in addition, the parameters of each stimulus cannot be stored each time it runs, which will significantly increase the time for visual stimulus resolution and introduce certain errors.

[0011] In summary, how to invent a visual stimulation method and system suitable for visual neurooptic imaging research is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0012] To address the problem that existing technologies cannot meet the needs of visual neuroscience research, this invention provides an intelligent visual projection method and system suitable for optical imaging. It can meet the needs of visual function encoding and parsing and functional classification map construction for optical imaging of visual-related neurons, and has the characteristics of intelligence, automation, precision and no optical pollution.

[0013] To achieve the above-mentioned objectives of this invention, the technical solution adopted is as follows:

[0014] A smart visual projection method suitable for optical imaging includes the following specific steps:

[0015] Set visual stimulation parameters to induce fluorescence signal responses in several neurons within the target visual tissue region;

[0016] Based on the visual stimulus parameters, the optimal control of the initial specific spectral visual stimulus and the corresponding light intensity is generated;

[0017] Based on the visual stimulus parameters, the initial specific spectral visual stimulus and the light intensity, the specific spectral output range is switched, the residual green spectral signal in the first specific spectral visual stimulus is filtered twice and the optimal light intensity output range is selected to generate the first specific spectral visual stimulus.

[0018] Set up infrared spectral visual stimulation;

[0019] The system uses a triple-filtering technique to extract residual green spectral information from the first specific spectral visual stimulus and integrates infrared spectral visual stimulus. It then redirects and directs the output to a second specific spectral visual stimulus, perpendicular to the direction of the visual tissue.

[0020] By focusing a second specific spectral visual stimulus onto the visual tissue area with a specific projection area, neurons can be visualized in real time, inducing the light response characteristics of neurons without visual signal contamination.

[0021] An intelligent visual projection system suitable for optical imaging, the system comprising a visual stimulus control module, an image output module, a spectrum switching module, a control center, a spectrum processing module, an infrared light source module, an infrared light source control module, and an image stimulus focusing module;

[0022] The visual stimulus control module is used to output a specified visual stimulus type or batch type and set control signals for various visual stimuli according to the user's experimental needs.

[0023] The control center is used to generate visual stimulus control signals, including but not limited to visual stimulus initiation, type marking, key node marking, and continuous marking parameters, to the image output module, the spectrum switching module, and the optical imaging system, and to feed them back to the visual stimulus control module to store various visual stimulus parameters in a timely manner.

[0024] The image output module is used to generate specific visual stimuli without green spectral emission sources and control the corresponding light intensity based on the received visual stimulus control signal.

[0025] The spectral switching module is used to select a specified spectral range module, perform double filtering, and generate a first specific spectral visual stimulus based on the visual stimulus control signal, a specific spectral visual stimulus without a green emission source, and light intensity control.

[0026] The infrared light source module is used to output infrared spectral visual stimulation to the spectral processing module in parallel, thereby realizing the infrared imaging function of retinal neurons.

[0027] The infrared light source control module is used to control the switch of the infrared light source module output and the intensity of its infrared light, so as to ensure clear infrared imaging of retinal neurons.

[0028] The spectral processing module is used to perform triple filtering on the specific green spectral signal remaining from the first specific spectral visual stimulus output by the spectral switching module, and then integrate the infrared spectral visual stimulus to synchronously output the second specific spectral visual stimulus without green spectrum.

[0029] The image stimulus focusing module is used to select a specified projection range and resolution for the second specific spectral visual stimulus output by the spectral processing module according to the needs of scientific research, and to clearly focus the visual stimulus without green spectrum on the visual tissue area, thereby inducing the effective light response characteristics of neurons.

[0030] Preferably, the visual stimulation control module includes an input device and a stimulation workstation; it includes a stimulation control interface and a stimulation workstation; the stimulation control interface has a variety of visual stimulation options, including but not limited to WhiteLight stimulation, ColorFlash stimulation, Bar stimulation, Chirp stimulation, WhiteNoise stimulation, LuminanceGradient stimulation, NaturalScenes stimulation, NaturalMovie stimulation and ipRGCs stimulation and control of their visual parameters; the stimulation workstation is a computer workstation used to regulate the signal output of the visual control center based on the visual parameter information input by the user through the stimulation control interface.

[0031] Furthermore, the image output module includes an image stimulator without a green emission source; the image stimulator is used to generate various flashing stimuli, image stimuli, and movie stimuli based on visual stimulus control signals. The image stimulator has a spectral output without a green emission source, thereby minimizing noise interference to the neuronal signals acquired by the optical imaging system and damage to the optical recording system at the stimulation source.

[0032] Furthermore, the gamma value of the image stimulator without a green emission source is calibrated according to the specific spectral range of the output to ensure that the intensity of the visual stimulus output light is linearly related to the analog control value.

[0033] The parameters such as the number of cycles of the visual stimulus, duration, interval between cycles, and dark adaptation time of the image stimulus can be adjusted.

[0034] The flashing stimuli include WhiteLight stimulation, ColorFlash stimulation, LuminanceGradient stimulation, and ipRGCs stimulation.

[0035] The image stimuli include Bar stimulation, Chirp stimulation, White Noise stimulation, and NaturalScenes stimulation;

[0036] The movie stimuli include NaturalMovie stimuli;

[0037] Among them, the brightness value distribution of each grid of the WhiteNoise stimulus is Gaussian, while the neuron signal acquired by the optical imaging system is optical information with a large time delay. Both ensure the effective analysis of the spatiotemporal characteristics of the neuron.

[0038] For the assessment of neuronal orientation selectivity and directional selection, Bar stimulation was used for analysis; for the assessment of neuronal frequency and contrast, Chirp stimulation was used for analysis, wherein Chirp stimulation presented stimulation patterns of different frequencies with uniform brightness and the same frequency with gradually increasing brightness; for the assessment of neuronal light sensitivity, LuminanceGradient stimulation was used for analysis; for the analysis of the group encoding of neurons to natural visual images and movie stimuli, NaturalScenes stimulation and NaturalMovie stimulation were used for analysis.

[0039] In addition to selecting a single narrow-spectrum stimulus that deviates from the green detection band of the optical imaging system for the ColorFlash stimulus, the DarkAdaptation time for each visual stimulus is set to ensure that it is incubated with extremely low background light before operation, thereby enabling further analysis of the changes in neuronal signals acquired by the optical imaging system.

[0040] Furthermore, the spectral switching module includes an optical wheel module, an optical wheel switching control center, an optical wheel power supply, and a neutral density filter group;

[0041] The optical wheel module includes a targeted narrow-band filter for ColorFlash stimulation and dual-band spectral filters for other visual stimuli. The former aims to target and activate the spectral stimulation corresponding to the S-, M-, and L-type cone cells in the retina while acquiring the green fluorescence signal of neurons, thereby activating the color-related circuitry in the retina. The latter is a dual-band spectral filter that selects a more comprehensive spectral stimulation output while avoiding optical noise interference. In addition, the optical wheel module can optionally add more filter modules as needed.

[0042] The light wheel switching control center receives the visual stimulus spectrum switching signal sent by the visual stimulus control module through the control center, and then automatically switches the light wheel module to the specified filter group according to the visual stimulus type. Then, it further processes the input specific spectrum visual stimulus without green emission source and outputs the first specific spectrum visual stimulus.

[0043] The neutral density filter group is used to control the light intensity of the first specific spectral visual stimulus output. The light intensities filtered by the various targeted narrow-band filters of the ColorFlash stimulus are not equal to those filtered by the dual-band spectral filters of the other visual stimuli. By adding different neutral density filter groups, the intensity of all visual stimulus spectra is ensured to be equal, and the optimal light intensity range for retinal neuron visual response is guaranteed. The stimulus control interface in the visual stimulus control module can also optionally be configured to further set the light intensity of various visual stimuli output by inputting analog control values ​​from the image stimulator. The light wheel switching control center, the neutral density filter group, and the stimulus control interface work together to ensure that the final image output intensity is within the optimal sensitive light intensity range of the visual neurons.

[0044] Furthermore, the final output visual stimulus control spectrum range deviates from the excitation and emission spectrum of the neuronal fluorescence signal, thereby avoiding the overlap and misalignment between the direct induction of visual stimulus and the autonomous induction of the excitation spectrum of the neuronal fluorescence signal. In other words, the visual stimulus spectrum cannot directly induce the artifact response of abnormal visual excitation produced by the neuronal excitation spectrum, otherwise the study of neuronal visual function encoding will fail.

[0045] Furthermore, the spectral processing module includes a light filtering system and a projection integration system, namely a green light filtering system for triple filtering of residual green spectral signals and a projection integration system for integrating the filtered spectral visual stimuli and infrared spectral visual stimuli.

[0046] Among them, the green light filtering system has a wider spectral range than that detected by the optical imaging system. By selecting a dichroic mirror, the residual green spectral signal in the first specific spectral visual stimulus can be triple-filtered, thereby further reducing the contamination of the neuronal optical signal. The projection integration system can integrate the triple-filtered visual stimulus signal with the infrared spectral visual stimulus signal in parallel and turn it 90 degrees to form a vertical output of visual stimulus, thus generating a second specific spectral visual stimulus.

[0047] Furthermore, the image stimulation focusing module includes a focusing system and an outer ring clutter filtering and waterproofing system;

[0048] The focusing system is used to control the size of the projection area of ​​the image stimulus output. Due to the inconsistent needs of neural tissue research, some require a larger projection area while others require higher resolution and a smaller projection area. The focusing system can controllably change the projection area to the required size by adjusting the lens group while ensuring that the image stimulus is clearly projected onto the neural tissue area.

[0049] The output port of the image stimulation focusing module is also equipped with an outer ring clutter filtering and waterproofing system, including a black light shield with a transparent aperture glass slide and a water flow system; the former ensures clutter filtering of the outer ring of the image stimulation and shields the system from damage caused by the overflow of the solution from the vertical top; the latter further ensures that the overflowing liquid flows out in time to avoid affecting the accuracy of the visual stimulus projection.

[0050] Furthermore, the control center includes a visual control center and a visual feedback center; the former is mainly used to accurately identify the start time of various visual stimuli and the time of their internal key nodes; the latter is responsible for saving all visual stimulus parameters according to the recording time and feeding them back to the control center and the visual stimulus control module in a timely manner.

[0051] The visual control center implements four different coded control strategies by synchronously outputting visual stimulus control signals through a digital output channel, as follows:

[0052] Low-latency, high-speed signal markers are used to mark the key nodes responsible for the start time of each frame of WhiteNoise stimulus, the start time of each random direction of Bar stimulus, the start and end time of flicker stimulus, frequency stimulus and contrast stimulus in Chirp stimulus, the time of light stimulation and light removal stimulus in flicker stimulus, the stimulus initiation time after dark adaptation, and the end time of each type of stimulus.

[0053] Precision type signal labeling is used to label each type of visual stimulus. That is, the same number of waves with the same number as the visual stimulus type are automatically input for automatic identification of stimulus type during subsequent data analysis. The output of this stimulus type matches and offsets the spectral range acquired by the optical imaging system to avoid signal contamination caused by visual stimuli.

[0054] Color signal markers, used for markers of flashing stimulus types, have inconsistent numbers of repeating markers;

[0055] The light wheel switching signal is used for targeted switching of the light wheel switching control center in the spectrum switching module. It switches to the specified light wheel sequence by switching digital signals, and at the same time identifies the current sequence number of the light wheel module in real time to ensure the accuracy of light wheel switching.

[0056] The control center also includes a visual feedback center, which is used to promptly feed back and retain the visual stimulus type and parameters output by the image stimulus focusing module each time to the stimulus workstation;

[0057] Specifically, this includes, but is not limited to, various visual stimulus parameters, the brightness of each square in each frame of WhiteNoise, the random direction sequence of Bar stimulation, and the random sequence marker information of colors, and then storing this visual information in the stimulation workstation in real time according to the stimulation time.

[0058] The beneficial effects of this invention are as follows:

[0059] The present invention provides an intelligent visual projection system suitable for optical imaging, which realizes the intelligence, high precision, high accuracy, real-time performance, high resolution, infrared imaging capability, projection randomness, high feedback and high compatibility of projection of specific spectral visual stimuli.

[0060] Unlike conventional visual stimulation systems, the intelligent visual projection system of this invention can intelligently output visual stimuli within a specific spectral range without optical signal contamination. This does not overlap with the spectral detection range of optical imaging systems (such as two-photon calcium imaging systems), ensuring that the optical imaging system can only acquire the specific fluorescence spectrum induced by visual stimulation of visual neurons (such as the retina).

[0061] The intelligent visual projection system of this invention has a controllable image stimulation area size and a resolution up to the nanometer level, realizing high-precision, high-area, and low-latency functional accurate analysis of visual neurons, and reducing phototoxicity to visual tissues and optical imaging systems.

[0062] Unlike conventional stimulation systems, this invention also integrates an infrared light source module, enabling clear imaging of visual neurons in dark, light-avoiding conditions and synchronous localization of the visual stimulation area by the optical imaging system.

[0063] The full-spectrum visual stimulation in this invention is compatible with systems such as two-photon calcium imaging, optical imaging systems of neurons in live-cell workstations, and non-optical action potential signal acquisition systems of neurons such as multi-electrode array systems. Compared with conventional visual stimulation systems, it has higher compatibility and wider application range. Attached Figure Description

[0064] Figure 1 This is a flowchart illustrating an intelligent visual projection method applicable to optical imaging according to the present invention.

[0065] Figure 2 This is a block diagram illustrating the principle of an intelligent visual projection system suitable for optical imaging according to the present invention.

[0066] Figure 3 This is a schematic diagram of the stimulation control interface of the present invention.

[0067] Figure 4 This is a block diagram illustrating the principle of the spectral switching module of this invention.

[0068] Figure 5 This is a block diagram illustrating the principle of the spectral processing module and the image stimulus focusing module of the present invention. Detailed Implementation

[0069] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0070] Example 1

[0071] like Figure 1 As shown, an intelligent visual projection method suitable for optical imaging includes the following specific steps:

[0072] Set visual stimulation parameters to induce fluorescence signal responses in tens of thousands of neurons within the target visual tissue region;

[0073] Based on the visual stimulus parameters, the optimal control of the initial specific spectral visual stimulus and the corresponding light intensity is generated;

[0074] Based on the visual stimulus parameters, the initial specific spectral visual stimulus and the light intensity, the specific spectral output range is switched, the residual green spectral signal in the first specific spectral visual stimulus is filtered twice and the optimal light intensity output range is selected to generate the first specific spectral visual stimulus.

[0075] Set up infrared spectral visual stimulation;

[0076] The system uses a triple-filtering technique to extract residual green spectral information from the first specific spectral visual stimulus and integrates infrared spectral visual stimulus. It then redirects and directs the output to a second specific spectral visual stimulus, perpendicular to the direction of the visual tissue.

[0077] By focusing a second specific spectral visual stimulus onto the visual tissue area with a specific projection area, neurons can be visualized in real time, and the light response characteristics of neurons without visual signal contamination can be precisely induced.

[0078] Example 2

[0079] To achieve the combined application of comprehensive visual function encoding and analysis of visual neurons (such as the retina) and optical imaging technologies (such as two-photon calcium imaging), such as... Figure 2 As shown, this embodiment provides an intelligent visual projection system suitable for optical imaging. The system includes a visual stimulus control module, an image output module, a spectrum switching module, a control center, a spectrum processing module, an infrared light source module, an infrared light source control module, and an image stimulus focusing module.

[0080] The visual stimulus control module is used to output a specified visual stimulus type or batch type and set control signals for various visual stimuli according to the user's experimental needs.

[0081] The control center is used to generate visual stimulus control signals with parameters such as visual stimulus initiation, type labeling, key node labeling, and duration labeling to the image output module, the spectrum switching module, and the optical imaging system, and to feed them back to the visual stimulus control module to store various visual stimulus parameters in a timely manner.

[0082] The image output module is used to generate specific visual stimuli without green spectral emission sources and control the corresponding light intensity based on the received visual stimulus control signal.

[0083] The spectral switching module is used to select a specified spectral range module, perform double filtering, and generate a first specific spectral visual stimulus based on the visual stimulus control signal, a specific spectral visual stimulus without a green emission source, and light intensity control.

[0084] The infrared light source module is used to output infrared spectral visual stimulation to the spectral processing module in parallel, thereby realizing the infrared imaging function of retinal neurons.

[0085] The infrared light source control module is used to control the switch of the infrared light source module output and the intensity of its infrared light, so as to ensure clear infrared imaging of retinal neurons.

[0086] The spectral processing module is used to perform triple filtering on the specific green spectral signal remaining from the first specific spectral visual stimulus output by the spectral switching module, and then integrate the infrared spectral visual stimulus to synchronously output the second specific spectral visual stimulus without green spectrum.

[0087] The image stimulus focusing module is used to select a specified projection range and resolution for the second specific spectral visual stimulus output by the spectral processing module according to the needs of scientific research, and to clearly focus the visual stimulus without green spectrum on the visual tissue area, thereby inducing the effective light response characteristics of neurons.

[0088] The present invention provides an intelligent visual projection system suitable for optical imaging, which realizes the projection of specific spectral visual stimuli without optical pollution, and features intelligence, high precision, high accuracy, real-time performance, high resolution, infrared imaging, projection randomness, high feedback and high compatibility.

[0089] Unlike conventional visual stimulation systems, the intelligent visual projection system of this invention employs a stimulator without a green emission source and undergoes dual and triple spectral filtering to intelligently output specific spectral visual stimuli free from optical signal contamination. Its output spectral range does not overlap with the detection range of the optical imaging system, thereby avoiding signal crosstalk and ensuring that the optical imaging system can only acquire the pure fluorescence spectrum of visual neurons induced by the stimulus.

[0090] The system of this invention features a controllable image projection area and high resolution, reaching 3.15 micrometers, and supports an ultra-large field of view of 6048*3402 micrometers. This not only achieves high-precision analysis of neuronal function but also meets the needs of large-scale population coding research on special structures such as the macula of the retina in higher species. Furthermore, it significantly reduces phototoxicity through efficient algorithms.

[0091] Unlike conventional stimulation systems, this invention also incorporates an infrared light source module, enabling clear imaging of neurons in dark, light-protected conditions, as well as synchronous localization of the scanning area of ​​the optical imaging system and the visual stimulation area.

[0092] The specific spectral visual stimulation in this invention is not only compatible with optical imaging systems such as two-photon calcium imaging systems and live-cell workstations, but also with non-optical electrophysiological signal acquisition systems such as multi-electrode arrays, patch clamps, and in vivo multi-channel recording. Compared with conventional visual stimulation systems, it has significantly higher compatibility and a wider range of applications.

[0093] In one specific embodiment, the visual stimulation control module includes a stimulation control interface and a stimulation workstation;

[0094] The stimulation control interface includes control over nine types of visual stimuli (WhiteLight stimulation, ColorFlash stimulation, Bar stimulation, Chirp stimulation, WhiteNoise stimulation, LuminanceGradient stimulation, NaturalScenes stimulation, NaturalMovie stimulation, and ipRGCs stimulation) and their visual parameters; the stimulation workstation is a computer workstation used to regulate the signal output of the visual control center based on the visual parameter information input by the user through the stimulation control interface.

[0095] like Figure 3As shown, this is the stimulus control interface. In this embodiment, the Visual Stimulus System (LF, Two-photon) software can automatically identify other modules within the system and acquire visual parameter information. The visual stimulus control module mainly connects to and regulates the central control unit in real time, controlling the visual parameters of the image output module and the visual stimulus control signals of the spectral switching module, thereby automatically projecting the specified visual stimulus and ensuring the accuracy of the visual stimulus output. The output parameters within the stimulus control interface can be adjusted at any time according to actual needs and the limitations of the total stimulus time. Furthermore, to reduce the discomfort and interference for researchers operating in dark environments, the intelligent visual projection system can control the intelligent output of multiple visual stimuli in specific sequences; its built-in stimulus types are all output according to random sequences.

[0096] In one specific embodiment, the image output module is primarily an image stimulator without a green emission source;

[0097] The image stimulator is used to generate various flashing stimuli, image stimuli and movie stimuli based on visual stimulus control signals. The image stimulator has no spectral output of green emission source, thereby avoiding noise interference to the neuronal signals collected by the optical imaging system and damage to the optical recording system at the source of stimulation.

[0098] The flashing stimuli include WhiteLight stimulation, ColorFlash stimulation, LuminanceGradient stimulation, and ipRGCs stimulation.

[0099] The image stimuli include Bar stimulation, Chirp stimulation, White Noise stimulation, and NaturalScenes stimulation;

[0100] The movie stimuli include NaturalMovie stimuli;

[0101] Of course, more types of visual stimulation can be added as needed.

[0102] The image output module described in this embodiment is mainly responsible for generating specific spectral visual stimuli and controlling the intensity of their output light.

[0103] Currently, these nine types of visual stimuli meet almost all the stimulation types required for retinal visual function research. They satisfy the needs of visual function analysis and functional subtype classification research, while maintaining low phototoxicity and high neuronal survival rate due to shorter visual stimulation time.

[0104] In this embodiment, the image stimulator without a green emission source is also calibrated with gamma value according to the specific spectral range of the output, so that the intensity of the visual stimulus output light is linearly related to the analog control value, thus ensuring the accuracy of the linear output of light intensity.

[0105] In this embodiment, the resolution of the image stimulus can be refined to the nanometer level as needed, and the stimulation field of view can also be adjusted according to scientific research requirements. It is controlled by the focusing system in the image stimulus focusing module. Currently, the intelligent visual projection system provided in this embodiment mainly uses a resolution of 3.15 micrometers and a field of view size of 6048*3402 micrometers. This satisfies both the precise analysis of the functional encoding of retinal neurons and the analysis of a large field of view of the retinal population. In particular, the unique macular structure of the retina of higher species has a large deviation between the positions of photoreceptor cells and ganglion cells in this region. Visual stimulation with a small field of view cannot achieve the photostimulation-induced response of retinal neurons.

[0106] To optimize the optical response characteristics of visually related neurons, the number of cycles, duration, interval between cycles, and projection area of ​​the image stimulus for various visual stimuli can all be adjusted.

[0107] Each type of visual stimulus and the markers for the start, end, and key time points of each cycle are generated by the visual control center of the regulatory center. The time delay is precisely controlled within 1.5 milliseconds, and some are accurate to 0.5 milliseconds, which reduces the total duration of visual stimuli and improves the resolution accuracy between visual stimuli and neuronal optical evoked responses.

[0108] WhiteNoise stimulation, in which the brightness of each square in each frame is 0 or 255, and the brightness value of each square is distributed in a Gaussian distribution, can ensure the rapid resolution of the spatiotemporal receptive field characteristics of visual neurons, and can also greatly reduce the duration of stimulation. Currently, the spatiotemporal receptive field of neurons can be effectively mapped in 5 minutes, significantly reducing the phototoxicity caused by the long-term average brightness of 127.5 per frame. In contrast, the neuronal signals collected by the optical imaging system are optical information with a large time delay. Therefore, the time interval between each frame of WhiteNoise stimulation is relatively large, i.e., 0.2s. Both of these factors ensure the effective resolution of the spatiotemporal characteristics of neurons.

[0109] For the assessment of neuronal orientation selectivity and pointing selectivity, Bar stimulation was used for analysis. The Bar stimulation provides 8+1 random orientation sequences, a width of 300 micrometers, a black background light, and a short duration of stimulation mode, which can effectively avoid human error and phototoxicity. 1 is a blank stimulus.

[0110] For the assessment of neuronal frequency and contrast characteristics, Chirp stimulation was used for analysis. The Chirp stimulation presented different stimulation patterns with sinusoidal variations but uniform brightness, and the same frequency but gradually increasing brightness. The precise control and marking of the start and end times of flicker, frequency and contrast ensured the accuracy of the analysis.

[0111] To assess the photosensitivity of neurons, Luminance Gradient stimulation with 4+2 light intensity gradients was used for analysis. The 4 gradients were four light intensity gradients in the Luminance Gradient stimulation with random sequence initiation, and the 2 gradients were the blinking stimuli in WhiteLight stimulation and Chirp stimulation, respectively. By evaluating the relationship between the intensity of the light response and the magnitude of the light intensity, the photosensitivity curve of the neuron could be fitted and calculated.

[0112] For the evaluation of neuronal color selection, precise stimulation within a narrow spectral range of approximately 405nm, 570nm, and 605nm is selected. ColorFlash stimulation is used to analyze the light response intensity of neurons under various colors, thereby realizing the analysis of color encoding.

[0113] For the analysis of the group encoding of neurons to natural visual images and movie stimuli, NaturalScenes stimuli and NaturalMovie stimuli were used. NaturalScenes stimuli consisted of 37 image stimuli, involving animals, plants, graphics, color, moderate myopia, and high myopia in the natural world. They were randomly cycled 10 times, and the image stimuli in each frame were randomly distributed. The average brightness of the gray image stimuli contained therein was similar to that of the gray-scale blank stimuli, and the standard deviation of the pixels was 50% of the average brightness. NaturalMovie stimuli, on the other hand, were real-world scenes of multiple people moving, analyzing the neurons' perception of the real world.

[0114] To screen for the unique characteristics of visual function in autonomous photosensitive ganglion cells (iRGCs), ipRGCs were stimulated with spectral stimulation other than the green spectrum, ensuring the robustness of the visual evoked response of ipRGCs.

[0115] By setting the DarkAdaptation time for each visual stimulus, and ensuring that the system is incubated under extremely low background light before operation, the changes in neuronal signals acquired by the optical imaging system can be further analyzed more accurately.

[0116] In a specific embodiment, such as Figure 4 As shown, the spectral switching module includes an optical wheel module, an optical wheel switching control center, an optical wheel power supply, and a neutral density filter group module;

[0117] The spectral switching module is mainly responsible for the targeted narrow-band filter for the ColorFlash stimulus in visual stimulation and the dual-band spectral filters for other visual stimuli. The former aims to target and activate the 405nm, 570nm, and 605nm spectral stimuli corresponding to the S-type, M-type, and L-type cone cells in the retina while collecting the green fluorescence signal of neurons, thereby activating the color-related circuit in the retina. The latter is a 59003xv2 dual-band spectral filter (380-410nm and 560-592nm transmission, Chroma) to select a more comprehensive spectral stimulus output while avoiding optical noise interference. In addition, the optical wheel module can continue to add the required filter modules to meet the needs of scientific research.

[0118] The optical wheel module includes a 59003xv2 filter (380-410nm and 560-592nm transmittance, chroma), a 405nm / 20bp filter (peak 405nm, bandwidth 10nm, chroma), a 570nm / 20bp filter (peak 570nm, bandwidth 20nm, chroma), and a 605nm / 70bp filter (peak 605nm, bandwidth 70nm, chroma), while reserving two blank areas for additional research needs;

[0119] Among them, the 59003xv2 is a dual-band spectral filter, which ensures that the output of visual stimulation covers blue and red spectral information at the same time, and is mainly responsible for the needs of stimulation other than ColorFlash stimulation; while the 405nm / 20bp filter, 570nm / 20bp filter, and 605nm / 70bp filter are all ColorFlash stimuli, which target and activate the S-type, M-type, and L-type cone cells of the retina respectively, and are single narrow spectral stimuli that deviate from the green detection band of the optical imaging system; both ensure that the final output visual stimulation does not interfere with the neuronal signal and does not damage the recording system.

[0120] The ipRGCs stimulation is a long-term scintillation stimulus with a dual-band spectrum of 59003xv2, used to activate retinal autonomic photosensitive ganglion cells, and then to study their functional coding and functional classification characteristics.

[0121] The working principle of the spectral switching module is as follows:

[0122] The light wheel switching control center receives the visual stimulus spectrum switching signal sent by the visual stimulus control module through the control center, and then automatically switches the light wheel module to the specified filter group according to the visual stimulus type. Then, it further processes the input specific spectrum visual stimulus without green emission source and outputs the first specific spectrum visual stimulus.

[0123] The neutral density filter group controls the output light intensity of the first specific spectral visual stimulus and the final image stimulator. Because retinal neurons have low light sensitivity to intensity and photomultiplier tubes in optical imaging systems (such as two-photon calcium imaging systems) are easily damaged by high light intensity, it is necessary to strictly control the output light intensity to ensure efficient resolution of neuronal functional encoding without compromising the optical imaging system. Specifically, the light intensities filtered by the various targeted narrow-band filters for the ColorFlash stimulus are not equal to those filtered by the dual-band spectral filters for the other visual stimuli. By adding different neutral density filter groups, the intensity of all visual stimulus spectra is ensured to be equal, with a maximum spectral brightness of 6.0 log₂. 10 photons / μm 2 / s ensures the optimal light intensity range for visual responses induced by retinal neurons. The stimulation control interface within the visual stimulation control module can also be configured by inputting simulated control values ​​from the image stimulator to further set the light intensity of various visual stimuli. The light wheel switching control center, neutral density filter group, and stimulation control interface work together to ensure that the final image output intensity is within the optimal sensitive light intensity range of the visual neurons.

[0124] In this embodiment, the output light intensity of the ColorFlash stimulus is controllable. The specific value (ranging from 0 to 255) can be input on the stimulus control interface as needed. Combined with the neutral density filter module, the gamma-value-corrected image stimulator can output stimulus intensity with equal light intensity of each spectrum through the light wheel switching control center. It also provides precise stimulation of the color spectrum with extremely narrow bandwidth, thus achieving accurate analysis of color encoding.

[0125] The spectral switching module, based on the visual stimulus control signal output by the control center, synchronously controls and switches according to the different filters in the light wheel module and their corresponding codes via digital signals.

[0126] Specifically, the output of the light wheel modules 1 to 6 is precisely controlled and intelligently switched according to the same sequence of 1-6 digital signals. The filter information and position of the light wheel modules are displayed in real time to ensure the accuracy and randomness of the visual stimulus output.

[0127] In this embodiment, the spectral switching module can also manually adjust the position of the targeting light wheel module based on the position information in the stimulus control interface. Therefore, according to different visual stimulus control signals, it intelligently, automatically, and randomly outputs spectral color stimuli and dual-spectral non-color image stimuli with equal light intensity.

[0128] In a specific embodiment, such as Figure 5As shown, the spectral processing module includes a filtering system and a projection integration system. The green light filtering system is used for triple filtering of residual green spectral signals, and the projection integration system integrates the filtered spectral visual stimuli and infrared spectral visual stimuli. The green light filtering system has a wider spectral range than that detected by the optical imaging system. By selecting a dichroic mirror, the residual green spectral signals in the first specific spectral visual stimulus can be triple filtered, thereby further reducing the contamination of the neuronal optical signals. The projection integration system can integrate the triple-filtered visual stimulus signals with the infrared spectral visual stimulus signals in parallel and turn them 90 degrees to form a vertical output of visual stimulus, generating a second specific spectral visual stimulus.

[0129] Because the green fluorescence signal of visual neurons (Oregon Green 488 BAPTA dye) ranges from 500 to 580 nm with a maximum spectral value of 521 nm, and the photomultiplier tubes in optical imaging systems (such as two-photon calcium imaging systems) detect light signals within the spectral range of 508-530 nm (Chroma; ET520 / 20m), if the light signal in the visual stimulus contains light within the spectral range detected by the optical imaging system, there are two major application limitations: First, it will be connected in series with the spectral signal of light-induced neural activity in visual neurons (such as the retina), causing distortion of the neural signal, which will severely interfere with the resolution of neural activity signals and lead to resolution failure; second, the visual signal will severely damage the photomultiplier tube in the optical imaging system, rendering its signal detection ineffective. Therefore, special intervention is needed in the spectral signal of the visual stimulus to ensure successful projection of the visual stimulus.

[0130] Therefore, this embodiment will adopt a green light filtering system, which uses a laser-grade notch dichroic mirror (Semrock; NFD01-532; 350-508nm and 562-1600nm transmit, while the 508-550nm (full) and 550-562nm (partial) spectra are reflected and filtered at 90 degrees). This system can filter out the first specific spectral visual stimulus of red, green and blue spectra from the spectral range that the optical imaging system can detect, that is, filter out the residual green component in the first specific spectral visual stimulus and allow the red and blue components to continue to transmit, thereby solving the two major drawbacks of optical signals.

[0131] However, optical imaging systems operate in dark environments, making it impossible to use visible light sources for the localization and focusing of visual neurons. Otherwise, severe phototoxicity to visual tissues (such as the retina) and the optical imaging system would lead to experimental failure. Therefore, the projection integration system needs to further integrate visual stimuli filtered from residual green light and infrared spectral visual stimuli. The projection integration system described in this embodiment uses a broadband dichroic mirror (T760lpxxr, which reflects spectral stimuli below 760nm at a 90-degree angle and transmits infrared stimuli above 760nm), which integrates and projects visual and infrared stimuli, while further filtering clutter spectral signals, ultimately outputting visual stimuli of a specific spectrum.

[0132] In summary, the spectral processing module can output a second specific spectral visual stimulus that is not connected in series with the detection spectral range of the fluorescence signal of the visual neuron. Ultimately, this enables the optical imaging system to both acquire the fluorescence signal of the visual neuron (such as retinal calcium signal) and simultaneously provide comprehensive visual stimulation.

[0133] In a specific embodiment, such as Figure 2 , 5 As shown, the image stimulation focusing module includes a focusing system and an outer ring clutter filtering and waterproofing system;

[0134] The focusing system controls the projection area of ​​the image stimulus output. Due to varying needs in neural tissue research, some studies require a larger projection area while others require higher resolution and a smaller projection area. This focusing system, while ensuring clear projection of the image stimulus onto the neural tissue region, can controllably adjust the projection area by changing the lens group. This embodiment selects a biconvex lens group, which can reduce the final image stimulus and accurately project it onto the retinal tissue, achieving a resolution of up to 3.15 micrometers and a projection field of view of 6048*3402 micrometers. This simultaneously satisfies the requirements for precise analysis of retinal neuron functional encoding and analysis of a large retinal population, especially considering the unique macular structure of the retina in higher species, ensuring high accuracy in image stimulus analysis.

[0135] The output port of the image stimulation focusing module is also equipped with an outer ring clutter filtering and waterproofing system.

[0136] The outer ring clutter filtering and waterproofing system can eliminate interference from stray light scattering from the outer ring, prevent liquid from leaking downwards from the recording port of the optical imaging system, and drain the liquid out in time to prevent interference from the uneven surface of the water mirror.

[0137] This embodiment achieves precise control of light intensity through a multi-light control system, ultimately ensuring that the image output intensity is within the optimal light intensity range of 3.5-6.0 log for visual induction. 10 photons / μm2 / s can effectively stimulate the light response characteristics of neurons.

[0138] In one specific embodiment, the control center includes a visual control center for accurately identifying the start time, end time, and key node times of various visual stimuli.

[0139] The visual control center synchronously outputs visual stimulus control signals through a digital output channel to implement four different coded control strategies, exhibiting extremely high accuracy and low latency, as detailed below:

[0140] 1) High-speed signal markers with low latency of 0.5ms are used to mark key nodes such as the start time of each WhiteNoise stimulus, the start time of each random direction of Bar stimulus (movement direction codes 1-9 are equal to the number of high-speed signal markers), the start and end times of flicker stimulus, frequency stimulus and contrast stimulus in Chirp stimulus, the time of light stimulation and light removal stimulus in flicker stimulus, the stimulus initiation time after dark adaptation, and the end time of each stimulus.

[0141] 2) Accurate 1ms type signal labeling is used to label each type of visual stimulus. That is, according to the visual stimulus type number (1-WhiteLight stimulus, 2-ColorFlash stimulus, 3-Bar stimulus, 4-Chirp stimulus, 5-WhiteNoise stimulus, 6-LuminanceGradient stimulus, 7-NaturalScenes stimulus, 8-NaturalMovie stimulus and 9-ipRGCs stimulus), an equivalent number of 1ms square waves are automatically input for automatic identification of stimulus type during subsequent data analysis. The output of these 9 stimulus types is not a conventional full-spectrum visual stimulus, but rather a spectrum range matched and offset from that acquired by the optical imaging system to avoid signal contamination caused by visual stimuli.

[0142] 3) 1.5ms color signal marking is used for marking flashing stimulus types (including 1-WhiteLight stimulus, 2-ColorFlash stimulus and 6-LuminanceGradient stimulus), and the number of repeats of the markings is not consistent; the number of 1-WhiteLight markings is 1, the number of spectral stimuli markings for 405nm, 570nm and 605nm in ColorFlash are 2-4 respectively, and the number of four gradient markings for 6-LuminanceGradient stimulus is 1-4 from weak to strong, which facilitates intelligent evaluation during subsequent analysis;

[0143] 4) Digital type light wheel switching signal is used for targeted switching of the light wheel switching control center in the spectrum switching module. The switching digital signal 1 to 6 switches to the specified light wheel sequence, and at the same time identifies the current light wheel module's serial number and its filter information in real time, further ensuring the accuracy of the light wheel position.

[0144] In this embodiment, the control center also includes a visual feedback center, which is used to promptly feed back the type of visual stimulus and its parameters output by the image stimulus focusing module each time to the control center and the visual stimulus control module and retain them in the stimulus workstation.

[0145] Specifically, this includes various visual stimulus parameters, the brightness of each square in each frame of WhiteNoise, the random direction sequence of Bar stimulation, and the random sequence markers of colors, and then this visual information is stored in the stimulation workstation in real time according to the stimulation time.

[0146] Therefore, the regulatory center precisely switches and controls specific types or sequences of visual stimuli according to different coding strategies, and stores the visual parameters in the stimulus workstation in a timely, intelligent and automatic manner.

[0147] In one specific embodiment, the infrared light source module mainly includes an infrared light source and a collimation system. The infrared light source module is responsible for the output of the infrared light source, thereby realizing the infrared imaging function of retinal neurons. In order to locate the detection area through infrared imaging of visual neurons (such as the retina) and avoid the influence of photobleaching, this embodiment selects an 850nm infrared light source (Thorlab; M850L3) to output spectral stimulation of a specific infrared wavelength. Combined with the collimation system, this ensures the uniformity of infrared stimulation output and the high definition of the imaging effect.

[0148] In one specific embodiment, the intelligent visual projection system further includes an infrared control module, which mainly comprises an infrared light intensity controller and an infrared power supply. This module is primarily responsible for switching the infrared light source output on and off and controlling the light intensity. To reduce the absorption energy of infrared photons on retinal tissue and the photomultiplier tube in the optical imaging system, this embodiment employs a manually modulated infrared light intensity controller to precisely control the output and intensity of the infrared light.

[0149] The intelligent visual projection system described in this embodiment can achieve the following technical effects:

[0150] This invention employs a triple green light filtering system to present visual stimuli with a specific spectrum devoid of green light, avoiding the range of the green fluorescence signal spectrum generated by neurons and the detection spectrum of the two-photon calcium imaging system. The final output visual stimulus deviates from the excitation and emission bands of the neuronal fluorescence signal spectrum, thereby avoiding the overlap and misalignment caused by direct induction of visual stimulation and the autonomous induction of the excitation band of the neuronal fluorescence signal. In other words, the visual stimulus spectrum cannot directly induce abnormal visual excitation artifacts generated by the neuronal excitation spectrum; otherwise, the study of neuronal visual function encoding would fail.

[0151] Therefore, this invention reduces the visual delay time to within 1.5 milliseconds, and in some cases to 0.5 milliseconds, through an encoding control strategy, which significantly reduces the total time of visual stimulation and its time delay interference.

[0152] Therefore, this invention selects a combination of a filter group with a light wheel module for a specific spectral range to optimize the output range of each spectrum and ensure consistent output light intensity. It is also highly sensitive to various types of neurons in the retina and can accurately analyze visual color codes.

[0153] Therefore, the present invention employs a projection integration system with a built-in infrared imaging system with controllable light intensity to meet the requirements of retinal tissue localization and clear infrared imaging in dark environments.

[0154] Therefore, all stimulus types in this invention are projected in a random sequence, and each stimulus can be given individually or in batches. The visual parameters are stored in the stimulus workstation in a timely manner according to the running time for subsequent efficient visual decoding.

[0155] Unlike conventional visual stimulation systems, the intelligent visual projection system described in this embodiment can intelligently output visual stimuli without optical contamination within a specific spectral range. This does not overlap with the spectral detection range of optical imaging systems (such as two-photon calcium imaging systems), ensuring that the optical imaging system can only acquire specific fluorescence spectra of visual neuron (such as retinal) activity, rather than optical signal contamination from visual stimuli.

[0156] This visual projection system features a controllable image stimulation area size and resolution up to the nanometer level, significantly reducing stimulation time. Compared with conventional visual stimulation systems, it achieves high-precision and rapid functional resolution of visual neurons and reduces phototoxicity to visual tissues and optical imaging systems.

[0157] When continuously outputting stimuli at the millisecond level, conventional visual systems cannot accurately output low-latency continuous stimulus signals. For example, in WhiteNoise stimuli, the stimulus time per frame is 33 or 17 milliseconds, while the delay time of each frame stimulus is higher than 20 milliseconds; at the same time, the delay time of neuronal optical signal response is greater than 33 ms, which will significantly interfere with the correlation between neuronal optical response and visual stimulus, thus causing resolution failure. This invention has achieved continuous stimulus output with low latency within 0.5 milliseconds, thereby significantly reducing the total duration of visual stimulus and the phototoxicity of visual tissues and optical imaging systems.

[0158] Conventional visual stimulation systems require a long duration of visual stimulation to analyze the functional characteristics of visual neurons. For example, WhiteNoise stimulation requires continuous output for one hour to accurately analyze the spatiotemporal receptive field characteristics of visual neurons. However, this invention employs a novel visual algorithm that can effectively analyze the functional coding characteristics of neurons in just 5 minutes. This significantly reduces the total stimulation time and phototoxicity, ensuring high resolution of visual stimulation.

[0159] Unlike conventional stimulation systems, this invention also incorporates an infrared light source module, enabling synchronous localization of the visual neurons' imaging and the scanning area of ​​the optical imaging system in dark, light-avoided conditions.

[0160] Conventional visual stimuli lack images or videos of the real natural world, thus failing to analyze the encoding of natural or video stimuli by neuronal populations. This invention also incorporates NaturalScenes stimuli and NaturalMovie stimuli. NaturalScenes stimuli contain 37 image stimuli covering aspects of the natural world, including animals, plants, graphics, color, moderate myopia, and high myopia. NaturalMovie stimuli are real-world scenes of multiple people moving around. Both ensure that this invention can analyze the neurons' perception of the real world.

[0161] In this invention, the output of each type of visual stimulus and each subtype of stimulus within each type is output according to a random sequence, which will more accurately evaluate visual function encoding than conventional visual stimulus systems; the visual stimulus parameter information of each run can be automatically stored in the stimulus workstation in a timely manner, and calling this information during subsequent function analysis can significantly reduce analysis time and improve accuracy.

[0162] The specific spectral visual stimulation in this invention is compatible with optical imaging systems for neurons (such as two-photon calcium imaging systems and live-cell workstations) and non-optical action potential signal acquisition systems for neurons (such as multi-electrode array systems, patch-clamp systems, and in vivo multichannel recording systems). Compared with conventional visual stimulation systems, it has higher compatibility and wider application range.

[0163] Example 3

[0164] Based on Example 2, an intelligent visual projection system suitable for optical imaging is combined with a two-photon calcium imaging system, achieving intelligence, high precision, high accuracy, real-time performance, high resolution, infrared imaging capability, projection randomness, high feedback, and high compatibility in the projection of specific spectral visual stimuli. This effectively resolves the visual functional coding characteristics and classification atlas of retinal neurons. Specifically, as follows:

[0165] The intelligent visual projection system mainly includes a visual stimulus control module, an image output module, a spectrum switching module, a control center, a spectrum processing module, an infrared light source module, an infrared control module, and an image stimulus focusing module.

[0166] The visual stimulus control module is mainly responsible for integrating and communicating in real time with other modules within the visual projection system, such as the image output module, the spectral switching module, and the control center. Through the control software system (Visual Stimulus System (LF, Two-photon)), it can intelligently output visual stimuli of specific types or sequences, and automatically output visual stimuli of specific spectral ranges according to different visual types, ultimately achieving the goal of "effectively identifying and detecting retinal neuron fluorescence spectral signals without interfering with the two-photon calcium imaging system".

[0167] The image output module is mainly responsible for outputting high-precision, diverse visual stimuli with specified light intensity and projection area; the gamma-calibrated spectral image stimulator without green emission source can output complex and simple stimuli with specific spectrum and precise brightness control, and the output is controlled by the central control according to the random sequence generated in order; each type of stimulus adopts a brand-new visual coding algorithm, which can quickly resolve the characteristics of retinal neurons with only a small stimulation time, thereby effectively reducing the total recording time and phototoxicity of each retina.

[0168] The spectral switching module is mainly responsible for providing color stimuli of equal intensity within a specified spectral range and selecting other visual stimuli of the dual-band spectrum, performing dual filtering and outputting the first specific spectral visual stimulus. Following the sequence numbers 1-6 of the light wheel module, the following filters are selected sequentially: 59003xv2 (380-410nm and 560-592nm transmission, Chroma) filter, 405nm / 20bp filter (peak 405nm, bandwidth 10nm, Chroma), 570nm / 20bp filter (peak 570nm, bandwidth 20nm, Chroma), 605nm / 70bp filter (peak 605nm, bandwidth 70nm, Chroma), blank, and blank. These filters sequentially control the specific spectral visual stimulus and color stimulus of equal intensity (405nm, sensitive to S-type cone cells; 570nm, sensitive to M-type cone cells; 605nm, sensitive to L-type cone cells).

[0169] The control center is primarily responsible for marking and controlling information such as the initiation, cessation, key boundary points, stimulus type, and light wheel sequence switching of various types of visual stimulus output. It requires only one control output port to control the entire system's central control, resulting in high efficiency. The control time delay for each type of visual stimulus is extremely short, with marking precisely controlled within 1.5 milliseconds, ensuring accurate output of continuous visual stimuli and the marking of each frame's signal. Controlling the visual stimulus output of various subtype random sequences effectively marks the time of key nodes and promptly stores visual parameter information in the stimulus workstation, facilitating rapid analysis of subsequent neuronal functional encoding. It also identifies and switches spectral switching module sequences in real time, intelligently controlling the output of visual stimuli within a specified spectral range.

[0170] The spectral processing module primarily filters the spectral range of neuronal fluorescence signals and adds infrared spectral information. The module includes a notch dichroic mirror (Semrock; NFD01-532) and a broadband dichroic mirror (Chroma; T760lpxxr). The former filters out the 508-562nm spectral information from the first specific spectral visual stimulus, thus enabling the synchronous acquisition of various types of precise spectral visual stimuli to retinal neurons and the green fluorescence signals emitted by the neurons. The latter integrates the infrared spectrum again after filtering out the visual stimuli with special green spectral wavelengths, achieving synchronous and clear visibility and localization of retinal neurons.

[0171] The infrared light source module and infrared control module are mainly responsible for providing infrared spectral stimulation with real-time controllable intensity and uniformity, ensuring clear infrared imaging while minimizing the phototoxicity of infrared photons to retinal tissue and optical imaging systems.

[0172] The image stimulus focusing module is primarily responsible for clearly focusing the second specific spectral visual stimulus onto the retinal tissue, achieving a resolution down to the nanometer level, which can be adjusted as needed. This project uses a biconvex lens for image focusing, achieving a resolution of up to 3.15 micrometers and a projection field of view of 6048*3402 micrometers, fully meeting the precise resolution requirements for neuronal function within the retinal-specific macular structure of higher species. When used in conjunction with multiple neutral density filter lenses, its light intensity output is controllable, currently maintained within the optimal sensitivity range of 3.5-6.0 log for retinal neurons. 10 photons / μm 2 / s; This module adds light-proof and waterproof features to prevent leakage and corrosion of liquids in the recording tank of the optical imaging system.

[0173] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An intelligent visual projection system suitable for optical imaging, characterized in that: The system includes a visual stimulus control module, an image output module, a spectrum switching module, a control center, a spectrum processing module, an infrared light source module, an infrared light source control module, and an image stimulus focusing module. The visual stimulus control module is used to output a specified visual stimulus type or batch type and set control signals for various visual stimuli according to the user's experimental needs. The control center is used to generate visual stimulus control signals, including parameters for visual stimulus initiation, type marking, key node marking, and continuous marking, and sends them to the image output module, the spectrum switching module, and the optical imaging system, and feeds them back to the visual stimulus control module to store various visual stimulus parameters in a timely manner. The image output module is used to generate a specific visual stimulus without a green spectral emission source and control the corresponding light intensity according to the received visual stimulus control signal; the specific spectral range emitted by the specific green spectral emission source is the range that avoids the green fluorescence signal spectrum generated by neurons and the detection spectrum of the two-photon calcium imaging system. The spectral switching module is used to select a specified spectral range module, perform double filtering, and generate a first specific spectral visual stimulus based on the visual stimulus control signal, a specific spectral visual stimulus without a green emission source, and light intensity control. The infrared light source module is used to output infrared spectral visual stimulation to the spectral processing module in parallel, thereby realizing the infrared imaging function of retinal neurons. The infrared light source control module is used to control the switch of the infrared light source module output and the intensity of its infrared light, so as to ensure clear infrared imaging of retinal neurons. The spectral processing module is used to perform triple filtering on the specific green spectral signal remaining from the first specific spectral visual stimulus output by the spectral switching module, then integrate the infrared spectral visual stimulus, and turn it 90 degrees to synchronously and vertically output the second specific spectral visual stimulus without green spectrum. The image stimulus focusing module is used to select a specified projection range and resolution for the second specific spectral visual stimulus output by the spectral processing module according to the needs of scientific research, and to clearly focus the visual stimulus without green spectrum on the visual tissue area, thereby inducing the effective light response characteristics of neurons.

2. The intelligent visual projection system for optical imaging according to claim 1, characterized in that: The visual stimulation control module includes a stimulation control interface and a stimulation workstation. The stimulation control interface offers various visual stimulation options, including WhiteLight stimulation, ColorFlash stimulation, Bar stimulation, Chirp stimulation, WhiteNoise stimulation, LuminanceGradient stimulation, NaturalScenes stimulation, NaturalMovie stimulation, and ipRGCs stimulation, along with control over their visual parameters. The stimulation workstation is a computer workstation used to regulate the signal output of the visual control center based on the visual parameter information input by the user through the stimulation control interface.

3. The intelligent visual projection system for optical imaging according to claim 1, characterized in that: The image output module includes an image stimulator without a green emission source; the image stimulator is used to generate various flashing stimuli, image stimuli and movie stimuli based on visual stimulus control signals. The image stimulator has a spectral output without a green emission source, thereby minimizing noise interference to the neuronal signals acquired by the optical imaging system and damage to the optical recording system at the stimulation source.

4. The intelligent visual projection system for optical imaging according to claim 3, characterized in that: For image stimulators without green emission sources, gamma values ​​are calibrated according to the specific spectral range of the output to ensure that the intensity of the visual stimulus output light is linearly related to the analog control value; the number of cycles, duration, interval between cycles, and dark adaptation time of the image stimulus parameters are adjustable. The flashing stimuli include WhiteLight stimulation, ColorFlash stimulation, LuminanceGradient stimulation, and ipRGCs stimulation. The image stimuli include Bar stimulation, Chirp stimulation, White Noise stimulation, and NaturalScenes stimulation; The movie stimuli include NaturalMovie stimuli; Among them, the brightness value distribution of each grid of the WhiteNoise stimulus is Gaussian, while the neuron signal acquired by the optical imaging system is optical information with a large time delay. Both ensure the effective analysis of the spatiotemporal characteristics of the neuron. For the assessment of neuronal orientation selectivity and directional selection, Bar stimulation was used for analysis; for the assessment of neuronal frequency and contrast, Chirp stimulation was used for analysis, wherein Chirp stimulation presented stimulation patterns of different frequencies with uniform brightness and the same frequency with gradually increasing brightness; for the assessment of neuronal light sensitivity, LuminanceGradient stimulation was used for analysis; for the analysis of the group encoding of neurons to natural visual images and movie stimuli, NaturalScenes stimulation and NaturalMovie stimulation were used for analysis. In addition to selecting a single narrow-spectrum stimulus that deviates from the green detection band of the optical imaging system for the ColorFlash stimulus, the DarkAdaptation time for each visual stimulus is set to ensure that it is incubated with extremely low background light before operation, thereby enabling further analysis of the changes in neuronal signals acquired by the optical imaging system.

5. The intelligent visual projection system for optical imaging according to claim 1, characterized in that: The spectral switching module includes an optical wheel module, an optical wheel switching control center, an optical wheel power supply, and a neutral density filter group. The optical wheel module includes a targeted narrow-band filter for ColorFlash stimulation and dual-band spectral filters for other visual stimuli. The former aims to target and activate the spectral stimulation corresponding to the S-, M-, and L-type cone cells in the retina while acquiring the green fluorescence signal of neurons, thereby activating the color-related circuitry in the retina. The latter is a dual-band spectral filter that selects a more comprehensive spectral stimulation output while avoiding optical noise interference. In addition, the optical wheel module can optionally add more filter modules as needed. The light wheel switching control center receives the visual stimulus spectrum switching signal sent by the visual stimulus control module through the control center, and then automatically switches the light wheel module to the specified filter group according to the visual stimulus type. Then, it further processes the input specific spectrum visual stimulus without green emission source and outputs the first specific spectrum visual stimulus. The neutral density filter group is used to control the light intensity of the first specific spectral visual stimulus output. The light intensities filtered by the various targeted narrow-band filters of the ColorFlash stimulus are not equal to those filtered by the dual-band spectral filters of the other visual stimuli. By adding different neutral density filter groups, the intensity of all visual stimulus spectra is ensured to be equal, and the optimal light intensity range for retinal neuron visual response is guaranteed. The stimulus control interface in the visual stimulus control module can also optionally be configured to further set the light intensity of various visual stimuli output by inputting analog control values ​​from the image stimulator. The light wheel switching control center, the neutral density filter group, and the stimulus control interface work together to ensure that the final image output intensity is within the optimal sensitive light intensity range of the visual neurons.

6. The intelligent visual projection system for optical imaging according to claim 5, characterized in that: The final output visual stimulus control spectrum range deviates from the excitation and emission spectrum of the neuronal fluorescence signal, thus avoiding the overlap and misalignment caused by direct induction of visual stimulus and the autonomous induction of the excitation spectrum of the neuronal fluorescence signal. In other words, the visual stimulus spectrum cannot directly induce the artifact response of abnormal visual excitation produced by the neuronal excitation spectrum, otherwise the study of neuronal visual function encoding will fail.

7. The intelligent visual projection system for optical imaging according to claim 1, characterized in that: The spectral processing module includes a light filtering system and a projection integration system, namely a green light filtering system for triple filtering of residual green spectral signals and a projection integration system for integrating the filtered spectral visual stimuli and infrared spectral visual stimuli. Among them, the green light filtering system has a wider spectral range than that detected by the optical imaging system. By selecting a dichroic mirror, the residual green spectral signal in the first specific spectral visual stimulus can be triple-filtered, thereby further reducing the contamination of the neuronal optical signal. The projection integration system can integrate the triple-filtered visual stimulus signal with the infrared spectral visual stimulus signal in parallel and turn it 90 degrees to form a vertical output of visual stimulus, thus generating a second specific spectral visual stimulus.

8. The intelligent visual projection system for optical imaging according to claim 1, characterized in that: The image stimulation focusing module includes a focusing system and an outer ring clutter filtering and waterproofing system; The focusing system is used to control the size of the projection area of ​​the image stimulus output. Due to the inconsistent needs of neural tissue research, some require a larger projection area while others require higher resolution and a smaller projection area. The focusing system can controllably change the projection area to the required size by adjusting the lens group while ensuring that the image stimulus is clearly projected onto the neural tissue area. The output port of the image stimulation focusing module is also equipped with an outer ring clutter filtering and waterproofing system, including a black light shield with a transparent aperture glass slide and a water flow system; the former ensures clutter filtering of the outer ring of the image stimulation and shields the system from damage caused by the overflow of the solution from the vertical top; the latter further ensures that the overflowing liquid flows out in time to avoid affecting the accuracy of the visual stimulus projection.

9. The intelligent visual projection system for optical imaging according to claim 4, characterized in that: The control center includes a visual control center and a visual feedback center; the former is mainly used to accurately identify the start time of various visual stimuli and the time of their internal key nodes; the latter is responsible for saving all visual stimulus parameters according to the recording time and feeding them back to the control center and the visual stimulus control module in a timely manner. The visual control center implements four different coded control strategies by synchronously outputting visual stimulus control signals through a digital output channel, as follows: Low-latency, high-speed signal markers are used to mark the key nodes responsible for the start time of each frame of WhiteNoise stimulus, the start time of each random direction of Bar stimulus, the start and end time of flicker stimulus, frequency stimulus and contrast stimulus in Chirp stimulus, the time of light stimulation and light removal stimulus in flicker stimulus, the stimulus initiation time after dark adaptation, and the end time of each type of stimulus. Precision type signal labeling is used to label each type of visual stimulus. That is, the same number of waves with the same number as the visual stimulus type are automatically input for automatic identification of stimulus type during subsequent data analysis. The output of this stimulus type matches and offsets the spectral range acquired by the optical imaging system to avoid signal contamination caused by visual stimuli. Color signal markers, used for markers of flashing stimulus types, have an inconsistent number of repeats; The light wheel switching signal is used for targeted switching of the light wheel switching control center in the spectrum switching module. It switches to the specified light wheel sequence by switching digital signals, and at the same time identifies the current sequence number of the light wheel module in real time to ensure the accuracy of light wheel switching. The control center also includes a visual feedback center, which is used to promptly feed back and retain the visual stimulus type and parameters output by the image stimulus focusing module each time to the stimulus workstation; Specifically, this includes various visual stimulus parameters, the brightness of each square in each frame of WhiteNoise, the random direction sequence of Bar stimulation, and the random sequence marker information of colors. These visual information are then stored in the stimulation workstation in real time according to the stimulation time.

10. A smart visual projection method suitable for optical imaging, characterized in that, The system applicable to any one of claims 1 to 9 includes the following specific steps: Set visual stimulation parameters to induce fluorescence signal responses in several neurons within the target visual tissue region; Based on the aforementioned visual stimulus parameters, specific visual stimuli without green spectral emission sources are generated, and the corresponding light intensity is controlled. Based on the visual stimulus parameters, the specific visual stimulus without green spectral emission source and the light intensity, the specified spectral range module is selected to double filter the residual green spectral signal in the first specific spectral visual stimulus and generate the first specific spectral visual stimulus. Set up infrared spectral visual stimulation; The system uses a triple-filtering technique to extract residual green spectral information from the first specific spectral visual stimulus and integrates infrared spectral visual stimulus. It then redirects and directs the signal perpendicular to the visual tissue direction to output the second specific spectral visual stimulus. By focusing a second specific spectral visual stimulus onto the visual tissue area with a specific projection area, neurons can be visualized in real time, inducing the light response characteristics of neurons without visual signal contamination.

Images