Image-guided variable spot stimulation-based electrophysiological evaluation device for determining changes in disease progression and functional health of biological samples during treatment

Abstract

To provide an image-guided variable spot light stimulation electrophysiology device applicable to structural / functional imaging and evaluation.SOLUTION: To provide an application of a device for performing image-guided functional assessment based on variable spot light stimulation of a light-activated biological sample to determine changes in functional health during disease progression and treatment. More specifically, the invention relates to the application of the device in the diagnosis of vision and neurological disorders.SELECTED DRAWING: Figure 1

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a joint venture of U.S. Provisional Application No. 63 / 383,979 (filed November 16, 2022, entitled "Disease Progression and Image-guided variable spot for determining changes in functional health of biological samples during surgery and treatment Electrophysiological assessment device based on spot stimulation (IMAGE GUIDED VARIABLE SPOT STIMULA TION-BASED ELECTROPHYSIOLOGY ASSESSMENT DEVICE TO DETERMINE CHANGES IN THE FUNCT IONAL HEALTH OF BIOLOGICAL SAMPLES DURING DISEASE PROGRESSION AND THERAPY) The benefit of priority is claimed, and the entirety of that application is incorporated herein by reference. The references (which may include publications, patents, and patent applications) are cited and incorporated herein by reference. Citation and / or discussion of these references does not constitute an explanation of the present invention. It is provided for clarity of explanation and is not intended to be a substitute for the inventions described herein. All information cited and discussed herein is not an admission that it is "prior art." All references are incorporated by reference in their entirety, unless each reference is individually incorporated by reference. shall be incorporated to the same extent as if in conjunction with the

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with funding from Nanoscope Instruments, Inc. The government has no rights in this invention. [Technical Field]

[0003] The present invention generally relates to imaging-guided variable spot stimulation for a variety of biomedical applications. Specifically, the present invention relates to an electrophysiological evaluation device based on a photoactivatable Image-guided functional assessment based on variable spot light stimulation of biological specimens is performed to assess disease progression and treatment More specifically, the present invention relates to the application of the device to determine changes in functional health status during treatment. The disclosure relates to the application of the device in the diagnosis of visual and neurological disorders. [Background technology]

[0004] Retinopathy occurs at different rates and locations of dysfunction / degeneration of various retinal layers and photoreceptors. The development of novel therapeutic interventions such as gene therapy and cell replacement therapy is However, the clinical significance of geographic atrophy or treatment is unclear. The functional characterization of a spatially targeted region of the retina alone, such as a treatment site, is not a spatial target. This is often complicated by the lack of cellular reactivity and the secondary hemorrhagic response of nearby cells. Therefore, the development of high-resolution ERG based on multicolor variable spot stimulation to identify cellular changes is needed. There is a clear need for critical assessment of disease progression and therapeutic interventions in the retina. It becomes possible. Summary of the Invention

[0005] To address these challenges, the present invention facilitates targeted stimulation and provides structural / functional imaging. We provide an image-guided variable spot light stimulation electrophysiology device that can be applied to imaging and evaluation. do.

[0006] Specifically, the present invention combines 3D imaging and electrophysiological functions to It includes a T-guided variable spot ERG platform and is used for the diagnosis and management of retinal diseases. High-resolution biosensors for structural and functional assessment of the retina are emerging as new tools in neuroscience. Real-time imaging guide for intravital microscopy imaging and electrophysiological analysis Prepare to dance.

[0007] In one embodiment, the device of the present invention generates a light signal that is transmitted by the retina and visual nervous system. It measures the electrical signals generated by the brain, such as digitized ERG, visual evoked potential (VEP), and EP) signal, power spectrum and topographic map. Spatial representation of the stimulus field. The size is predetermined by the user under guidance from OCT imaging. Flash / flicker, monochromatic or white light, or onset control A last light stimulus is presented to the desired location on the retina of the subject's eye. The elicitation signal is generated by the software. Algorithmically analyzed by temporal filtering and artifact removal Data are presented in numerical and graphical form.

[0008] In one embodiment, the present invention provides a method for detecting optical stimulation of a sample using optical coherence tomography (OCT) and / or a holographic imaging system. In the case of OCT, three-dimensional imaging is performed using a fundus microscope. Use of a low-coherence broadband light source and a spectroscopic camera detector, or photodiode detection This can be achieved by either a wavelength-swept source combined with a laser.

[0009] In yet another embodiment, the present invention integrates stimulation beams of different wavelengths and modes of operation. It includes OCT integration to control the spot size of the target stimulation, and the spot size is determined by diffraction. It can be varied from a marginal spot to a spot that covers the entire field of view.

[0010] In yet another embodiment, the present invention provides an image-guided variable spot stimulating light having different wavelengths. and electroretinogram (ERG), visual evoked potentials (VEP), and other light-activatable potential changes. The present invention contemplates a device that includes an integrated electrical signal detection system for functional evaluation.

[0011] In yet another embodiment, the present invention provides an OCT-guided tunable spot electrophysiology device. and / or adjusting the focal plane at the depth of interest and the wavelength for the cell type of interest. , layer-specific photoreceptor cells by photobleaching other cell types with wavelength-tuned background light. Or it can stimulate the neural retina or other light-sensitive neurons.

[0012] In another embodiment, the present invention is directed to monitoring the activity of specific cell types. Localized and patterned stimulation with light beams of different wavelengths and spot sizes The present invention encompasses an image-guided variable spot device for

[0013] In yet another embodiment, the present invention provides a method for detecting light of different colors (wavelengths) and different pulse rates (periods). A unique method for multiplexed measurements of different photoreceptors using simultaneous stimulation with multiple light beams of different wavelengths. The present invention provides a method for:

[0014] In yet another embodiment, the present invention provides a method for reducing measurement time during long-term measurements at multiple spots. We assume space-time multiplexing for

[0015] In yet another embodiment, the present invention provides a method for detecting cerebral palsy using patterned stimulation with OCT-vs. ERG. A time-efficient method for functional mapping of the visual field is included.

[0016] In yet another embodiment, the present invention provides a method for fast and precise movement of deflection mirrors and liquid lenses. Scanning control and positioning of the image-guided light stimulus spot to the selected area of ​​the retina This includes methods for making the image scalable.

[0017] In another embodiment, the present invention provides image-guided electrophysiology with different stimulation wavelengths and modes of operation. Physiological systems include dry AMD, retinitis pigmentosa, cone-rod dystrophy, and diabetes The present invention provides a method for generating functional assessments of retinal abnormalities, including diabetic macular edema and diabetic retinopathy.

[0018] In yet another embodiment, the present invention provides indirect fundus imaging using an OCT-guided variable spot. Includes configurations that integrate with electrophysiology systems to improve image guidance for targeted stimulation .

[0019] In a broader embodiment, the present invention provides an image-guided variable spot electric field having multiple stimulation wavelengths. Provides a method for evaluating local therapeutic effects using physiological systems: gene replacement, opioid therapy, Transgenetics gene therapy, regenerative cell (transplant) therapy.

[0020] Any embodiment of the methods, devices or compositions described herein may be used in combination with any of the methods, devices or compositions described herein. It is contemplated that the invention may be practiced with respect to any other method, device, or composition described. can be.

[0021] Further details relating to these and other embodiments are provided below.

[0022] Other objects, features and advantages of the present invention will become apparent from the following detailed description. However, the detailed description and specific examples are intended to illustrate particular embodiments of the invention. It should be understood that the present invention is by way of example only, as the detailed description herein Therefore, it will be apparent to those skilled in the art that various changes and modifications can be made within the spirit and scope of the present invention. This is because it becomes clear.

[0023] The following drawings are for illustrative purposes and not for limiting purposes. Therefore, all features of a given structure are always labeled The following drawings form part of this specification and illustrate certain aspects of the present invention. The present invention is provided to further illustrate one or more of these drawings and the principles of this specification. The present invention will be more fully understood by reference to the accompanying drawings in conjunction with the detailed description of the embodiments of the present invention. can be well understood. [Brief explanation of the drawings]

[0024] [Figure 1]Figure 1A shows the first configuration of the OCT-induced variable spot stimulation and electrophysiology system. 1001: Blue light source; 1002: Green light source; 1003: Red light source; 1004: Beam combiner 1; 1005: Imaging light source; 1006: Beam combiner 2; 1007: Laser isolator; 1008: Beam splitter; 1009: Mirror 1; 1010: Mirror 2; 1011: Dynamic focusing element; 1012: Focusing lens; 1013: Reference arm; 1014: Detector; 1015: Microcontroller; 1016: Computer; 1017: Display; 1018: Electrode. Figure 1B shows the second configuration of the OCT-induced variable spot stimulation and electrophysiology system. 1001: Blue light source; 1002: Green light source; 1003: Red light source; 1004: Beam combiner 1; 1005: Beam steering optics; 1006: Beam combiner 2; 1007: Imaging light source; 1008: Laser isolator; 1009: Beam splitter; 1010: Mirror 1; 1011: Mirror 2; 1012: Dynamic focusing element; 1013: Focusing lens; 1014: Reference arm; 1015: Detector; 1016: Microcontroller; 1017: Computer; 1018: Display; 1019: Electrode. [Figure 2]Figure 2A shows configuration 3 of the OCT and fundus image-guided variable spot stimulation and electrophysiology system. 1001: Blue light source; 1002: Green light source; 1003: Red light source; 1004: Beam combiner 1; 1005: Imaging light source; 1006: Beam combiner 2; 1007: Laser isolator; 1008: Beam splitter; 1009: Mirror 1; 1010: Mirror 2; 1011: Dynamic focusing element; 1012: Condenser lens; 1013: Reference arm; 1014: Detector; 1015: Microcontroller; 1016: Computer; 1017: Display; 1018: Electrode; 1019: Imaging light source 2; 1020: Collimating optics; 1021: Beam combiner for imaging optics 2; 1022: Condenser lens; 1023: Detector for imaging optics 2; 1024: Beam combiner for imaging modality 2. Figure 2B shows configuration 4 of the fundus image-guided variable spot stimulation and electrophysiology system. 1001: Blue light source; 1002: Green light source; 1003: Red light source; 1004: Beam combiner 1; 1005: Dynamic focusing element 1; 1006: Mirror 1; 1007: Mirror 2; 1008: Imaging light source; 1009: Collimating optics; 1010: Dichroic mirror for imaging light source; 1011: Focusing lens; 1012: Focusing lens; 1013: Detector; 1014: Microcontroller; 1015: Computer; 1016: Display; 1017: Electrode. Figure 2C shows the configuration 5 of the OCT and fundus image-guided variable spot stimulation and electrophysiology system. 1001: Blue light source; 1002: Green light source; 1003: Red light source; 1004: Beam combiner 1; 1005: Imaging light source; 1006: Beam combiner 2; 1007: Laser isolator; 1008: Beam splitter; 1009: Mirror 1; 1010: Mirror 2; 1011: Dynamic focusing element; 1012: Focusing lens; 1013: Reference arm; 1014: Detector; 1015: Microcontroller; 1016: Computer; 1017: Display; 1018: Electrode; 1019: Imaging light source 2 with focusing element; 1020: Focusing lens; 1021: Detector for imaging optics 2; 1022: Beam splitter for imaging modality 2. [Figure 3] Figure 3A shows the OCT-guided ERG system housing with the scanner head. Figure 3B shows a live fundus image of a rodent retina (rat). Figure 3C shows an OCT en-face (i.e., en face) reconstruction from 3D OCT imaging. The marked circle indicates the diagnostic ERG stimulation site. This modality allows for localized and variable-sized stimulation. Figure 3D shows a multicolor ERG profile demonstrating differential responses to blue, green, and red stimuli for functional evaluation of different cones (S, M, L). Figure 3E shows the OCT-guided ERG system scanner head with a fundus imaging light source imaging a model eye (white sphere on the right). Figure 3F shows a fundus image of the model eye. Black curved features are shown to represent blood vessels. The white patch represents the optic nerve. Figure 3G shows an OCT en-face image of the model eye. Black curved features are shown to represent blood vessels. The white patch represents the optic nerve. [Figure 4] Figure 1 shows variable control of stimulation spot size with a focusing element: The steering optics provide precise localization of the stimulus, while the focusing element allows for varying the stimulation spot size from a focused spot to the entire field of view. [Figure 5] Figure 5A shows spatial resolution determination of vsERG. En face OCT images after OCT-guided targeted laser microirradiation (peripheral retinal degeneration model). Figure 5B shows ERG responses at the non-damaged site. Figure 5C shows ERG responses at the damaged site, demonstrating reduced ERG amplitude at the damaged site. [Figure 6]Figure 6A shows OCT-guided variable spot ERG measurements using a scaled ISCEV standard protocol to assess regional function. A standard cone ERG protocol was used with a 2 mm stimulus spot (3 cd·s / m² stimulation under light adaptation). Figure 6B shows a standard rod and cone ERG protocol with a 2 mm stimulus spot (3 cd·s / m² stimulation under dark adaptation). Figure 6C shows a scotopic rod ERG protocol with a 2 mm stimulus spot (0.01 cd·s / m² under dark adaptation). Figure 6D shows the same scotopic rod ERG protocol with a 4 mm stimulus spot, demonstrating the stimulus size-dependent response. [Figure 7] Figure 7A shows OCT-induced variable spot visual evoked potential (VEP) measurements. Two different stimulation locations are shown in the OCT en face. Figure 7B shows VEP measurements in the visual cortex with white light stimulation at location 1. Figure 7C shows VEP measurements in the visual cortex with white light stimulation at location 2. The VEP response from the optic nerve area (location 1) is shown to be low. Figure 7D shows that stimulation with green light at location 2 results in a higher VEP response in the visual cortex compared to stimulation with white light. [Figure 8]Figure 8A illustrates a multiplexed stimulation scheme using multiple wavelengths and frequencies to simultaneously detect the responses of different photoreceptors. The timestamps of the light pulses for Color 1 (Frequency 1) are denoted by t1, t2, ..., and the timestamps of the light pulses for Color 2 (Frequency 2) are denoted by τ1, τ2, .... Figure 8B illustrates the use of frequency filtering in the Fourier domain to separate the responses of different cones based on their frequency responses. 1001: Signal acquisition; 1002: Frequency bandpass filter; 1003: Averaging based on Color 1 timestamps (t1, t2, t3, ...); 1004: Frequency 1 signal extraction; 1005: Photoreceptor type 1 response; 1006: Averaging based on Color 2 timestamps (τ1, τ2, τ3, ...); 1007: Frequency 2 signal extraction; 1008: Photoreceptor type 2 response. Figure 8C illustrates flicker ERG responses using a 14 Hz red stimulus. Figure 8D illustrates flicker ERG responses using a 31 Hz blue stimulus at the same location. Figures 8E and 8F show flicker ERG responses to a linear combination of 14 Hz red and 31 Hz blue light, averaged based on the red light stimulus timestamp. [Figure 9] Schematic of spatial and temporal stimulation to increase the signal-to-noise ratio (SNR). For multi-location stimulation, approximately half of the locations are randomly stimulated at a given time point, and the local response at each individual stimulation location is extracted from a linear combination of the stimuli, resulting in an increased SNR due to per-stimulus averaging compared to single-location stimulation. [Figure 10] Figure 10A illustrates an integrated OCT-guided ERG scanner for simultaneous evaluation, Figure 10B illustrates the experimental setup for an OCT-guided variable spot ERG system for clinical research, Figure 10C illustrates an integrated OCT-guided wearable ERG system, and Figure 10D illustrates the experimental setup for an OCT-guided variable spot size ERG goggle system. [Figure 11]Figure 11A shows an OCT-guided variable spot ERG system for clinical research. Figure 11B shows en face OCT images with a 2 mm spot size stimulus. Figure 11C shows a standard cone ERG measurement. Figure 11D shows a 30 Hz flicker ERG measurement. Figure 11E shows an OCT-guided wearable variable spot ERG system for clinical research. Figure 11F shows a standard rod-cone ERG measurement using OCT-guided variable spot size ERG. [Figure 12] Figure 1 shows the software interface of the OCT-guided variable spot ERG system for clinical trials. The left panel shows individual measurements, and the center panel shows the average signal. The image in the upper right corner shows the OCT en face and tomographic image of the stimulated area (dark circle with white crosshairs). [Figure 13] Figure 13A shows peripheral measurements using a ring pattern, illustrating multiple stimulation pattern schemes generated by OCT-guided variable spot ERG. Figure 13B shows a linear square pattern for OCT-guided variable spot ERG measurements. Figure 13C shows a concentric circle pattern for OCT-guided variable spot ERG measurements. Figure 13D shows an overlay of ERG response measurements using a peripheral ring pattern (NHP). Figure 13E shows an overlay of ERG response measurements using a concentric circle pattern (human). DETAILED DESCRIPTION OF THE INVENTION

[0025] Chorioretinal dystrophies and outer retinal dystrophies, e.g., dry age-related macular degeneration 1 Vision loss due to diseases such as dry AMD occurs when the photoreceptor cells or retinal pigments involved in visual transmission are damaged. Associated with loss of the RPE. Inherited retinal disease 2,3,4. is the number of working years in developed countries A significant proportion of this population suffers from retinitis pigmentosa (RP). ) 5 and then Stargardt disease6,7 and other diseases affecting photoreceptor cells These degenerative diseases cause retinal dysfunction due to the degeneration of different types of photoreceptor cells. Dysfunction or death of photoreceptor cells leads to loss of signals that initiate visual perception. RP 8 In dystrophies, rod loss precedes cone loss, whereas in cone-rod dystrophies Conversely, loss of cones leads to loss of rods. With the advent of preventative treatments, the treatment of these diseases has become more common. Early diagnosis helps slow progression. The spatial patterns of degeneration vary widely in these diseases. (e.g., central vision in AMD, peripheral vision in RP), and the degree of visual loss also varies with age. 9 and Both travel at different speeds, allowing for spatial resolution of the structural and functional integrity of the retina. Measurement in a way that will better guide the various therapeutic interventions that will become available in the future. It is possible.

[0026] Evoked potentials are used to measure electrical activity at specific sites in a biological sample. To generate electrical activity in a living specimen, stimulation of specific sensory nerve pathways is required. The functional response of stimulated tissue to electrophysiological signals has been measured. Although fixed, variable spots can be spatially targeted to localized areas with high resolution. This has not been achieved prior to the present invention. Stimulation requires image guidance and a stimulation device aligned with the image-guided modality. It is difficult.

[0027] The most widely accepted method for assessing retinal function is the electroretinogram (ERG). ERG is expressed by various cells in the retina, including photoreceptors, inner retinal cells, and retinal ganglion cells. ERG measurement involves measuring the electrical response of the retina. To access the There are various stimulation modes.

[0028] Full-field flash electroretinogram (ERG) allows for measurement of the function of the entire retina. There are 10 However, this technique does not detect retinal abnormalities in the early stages of the disease. On the other hand, it may not be possible to achieve large spot size stimulation in existing ERGs. 11, 12 In this study, it was found that functional information could be obtained with the resolution required to assess regional atrophy or therapeutic effects. Similarly, multifocal ERG (mfE), which measures retinal activity topographically, RG) method 13 Even in the presence of a signal-to-noise ratio sufficient to distinguish between different photoreceptor health states, It is not possible to separate the functions of local regions. Therefore, different ERG prototypes are generated in rods and cones. The use of different cols has not been realized with mfERG. Because white light is used for illumination, it is not possible to distinguish between different cone functions.

[0029] Furthermore, accurate diagnosis requires structural measurements and functional registration at the same location. To address this challenge, we are using coherent optical imaging to identify cellular functions with high resolution. Multicolor variable spotting guided by optical coherence tomography (OCT) and / or fundus imaging We have developed an ERG system based on fast light stimulation, which allows us to obtain spatially resolved information on retinal function. This allows for isolated measurements and minimizes responses from non-target areas. This makes it possible to accurately assess disease progression and the effects of therapeutic intervention.

[0030] In one embodiment, the present invention provides an OCT-guided stimulating light beam and a target Electrophysiology system with variable wavelength and operating mode to control stimulation spot size. and a stem, wherein the spot size is diffraction limited. The spot size can be varied from a spot of 1000 nm to a spot covering the entire field of view.

[0031] In yet another embodiment, the present invention provides a method for detecting retinal diseases by administering a retinal immunoglobulin (RI) to the retina as an aid in the diagnosis and management of retinal diseases. Fundoscopes and bioelectrical signal detection hardware for structural and functional assessment The present invention envisions a biomicroscopic imaging and electrodiagnostic device incorporating the technology.

[0032] In another embodiment, the present invention provides a method for providing spatiotemporal guidance of optical stimulation of a sample using OCT and / or In the case of OCT, it includes a device that is composed of a three-dimensional image. The images are captured using a combination of a low-coherence broadband light source and a spectroscopic camera detector, or a photo It is produced by a wavelength swept source combined with a diode detector.

[0033] In yet another embodiment, the present invention provides a method for fast and precise movement of deflection mirrors and liquid lenses. Scanning control and positioning of the image-guided light stimulus spot to the selected area of ​​the retina allows precise spreading. Image-guided variable spot ERG (vsERG) system. The system ranges from diffraction-limited small spot sizes to large spot sizes that cover the entire field of view. The signal can be measured in a controlled (position, size, wavelength, intensity, duration) manner.

[0034] In yet another embodiment, the present invention provides an OCT-guided variable spot with multiple stimulation wavelengths. We provide an ERG system that allows evaluation of different photoreceptor functions. OCT-guided variable spot The ERG system is capable of multicolor stimulation and provides OCT B-scan and en face imaging. can be obtained to select the area of ​​interest and position the stimulation spot.

[0035] In yet another embodiment, the present invention provides a method for detecting cerebral palsy using patterned stimulation with OCT-vs. ERG. A time-efficient method for functional mapping of the visual field is provided. Functional assessment facilitates decision-making for retinal spatial control treatments.

[0036] In yet another embodiment, the present invention provides a method for detecting light of different colors (wavelengths) and different pulse rates (periods). A unique method for multiplexed measurements of different photoreceptors using simultaneous stimulation with multiple light beams of different wavelengths. In the case of fast electrophysiological responses such as cone responses to flicker stimuli, Multiple stimulations targeting different wavelength-dependent photosensitive cells are realized by frequency multiplexing. The various photosensitive cells have different and separated absorption peaks, so they are sensitive to different wavelengths. Multiple stimuli of different lengths combined with unique stimulation frequencies were used to measure different types of photoactivities within a common stimulation area. The light response of stimulated cells can be extracted in a single measurement. The stimulated cell type reacts to its own absorption The mixed responses from individual cells were measured using the acquired voltage. The physiological signals are deconvolved by averaging them against the corresponding color stimulus timestamps. In addition to time averaging, frequency filtering allows for notch and bandpass filtering. By applying a pass filter, contributions from other frequency responses can be further reduced.

[0037] In yet another embodiment, the present invention provides a method for measuring a measurement time in a case where a measurement is performed over a long period of time at multiple spots. We assume spatiotemporal multiplexing for shortening. The electrophysiological response is an order of magnitude longer than the stimulus duration. If a buffer time is required for subsequent stimulation, the present invention uses short bursts of stimulation. It includes methods for stimulating multiple non-overlapping sites to improve the signal-to-noise ratio of the averaged signal. In this method, instead of stimulating one location and waiting for the stimulation site to fully recover, the next area is stimulated. Stimulate immediately. In each burst stimulation window, a significant proportion of the total number of spots of interest The measured electrophysiology resulting from a single stimulus burst group by stimulating the ratio of The target signal contains a linear combination of response signals from multiple regions. There is a specific time delay between the individual stimuli. Although there is a delay, the location and time of stimulation are known and synchronized, so there is no delay between multiple target spots. The stimulus order can be changed. Individual stimulus sequences can be calculated by solving a linear equation based on the stimulus position and time. The average signal from the pot positions can be taken.

[0038] In yet another embodiment, the device: i) performing variable spot sizes on a sample for electrophysiological measurements of said sample; an image-guided light stimulation beam that produces an image; ii) an image-guided photostimulation beam assembly; The image-guided photostimulating beam assembly includes: Illumination and collection of back-reflected light from the sample for imaging, providing low-cost The imaging subassembly was equipped with near-infrared (NIR) light from a Hearing Light Source. The near-infrared (NIR) light is then sampled for interference detection to obtain depth-resolved images. an imaging subassembly capable of splitting a pull beam and a reference beam; , - a light stimulator with light beams of different wavelengths and controllable intensity and / or pulse rate; a super subassembly; iii) For each optical stimulation beam wavelength, the power of the stimulation beam at the sample plane is 0.01 ~50cd.s / m 2 is in the range of iv) the optical stimulus beam can be combined with the sample beam and directed to the sample; and the light can be guided by the v) the sample is derived from neurons or photosensitive cells in vitro or in vivo; Selected, vi) a region of interest for electrophysiological measurements in said sample in response to variable spot stimulation; a preselected area is controlled by a scanning mirror that deflects the stimulation beam; vii) The variable spot size is achieved by a dynamic focusing element (e.g., a liquid lens). , for example, from diffraction-limited spots to the full field of view size, viii) the light stimulus may be targeted to a preselected region of interest on the sample. It is possible, ix) the photostimulation beam is switched off when preselecting a region of interest for photostimulation; and the preselection of the region of interest is based on morphological / tomographic imaging. This is done, x) For morphological / tomographic imaging, the sample beam is delivered via optical fiber or free space. and collimated by the scanning mirror and optical components. It is possible to deflect the beam to a simple xi) the optical component is provided with anti-reflective materials to avoid scattering and multiple reflections It is coated with xii) A back-reflected sample beam for morphological / tomographic imaging from said sample is detected. can be routed back to the source, xiii) tomographic imaging is performed by recording the interference of the back-reflected sample beam with the reference beam. It can be reconstructed by analyzing and xiv) The morphological / tomographic images are matched to photostimulation spots for electrophysiological measurements. It is possible to mark with a selection area of ​​variable size, like xv) Electrophysiological measurements are performed using electrodes connected to biosensing hardware. It is possible to implement.

[0039] Optionally, image guidance for electrophysiological measurements, realizing a variable spot size on the sample. The guided light stimulation beam can be a tabletop system or a wearable system, and optionally, can be electrically The wearable system for physiological measurements may comprise goggles or glasses. A wearable system for physiological measurements may consist of goggles or glasses.

[0040] According to yet another embodiment, temporally and spatially multiplexed variable spot light stimulation A method for performing electrophysiological measurements in a short time is provided, the method being averaged by: Equalization improves signal-to-noise ratio and reduces measurement time: i) stimulating multiple non-overlapping sites using short stimulation bursts; ii) shortening the time interval between stimulations at multiple spots; iii) changing the order of synchronized stimulation between various spots of interest; iv) Electrical stimulation resulting from single bursts of stimulation to various spots at different locations measuring physiological signals; iv) Deconvolving the signal responses from multiple regions to obtain a signal for each spot location To obtain the average signal.

[0041] In yet another embodiment, multiple light-responsive cell types with different absorption spectra can be simultaneously Stimulus frequency multiplexing, which is used to stimulate and record simultaneously at the same stimulation location, is described below. It is envisaged that the method will be carried out by: i) combining multiple wavelength stimulation beams modulated at different frequencies; ii) stimulating different photosensitive cells with different absorption peaks and separated; iii) A single measurement of the light responses of different types of light-activated cells within a common stimulation site. Extraction at a fixed rate, iv) Average the acquired electrophysiological signals based on the timestamp of the corresponding color stimuli. deconvolving the mixed responses from individual cells by combining / or v) Notch frequency filtering to eliminate the contribution of frequency / wavelength-dependent responses of other cell types To reduce.

[0042] According to yet another embodiment, the present invention provides a method for determining whether local disease progression or therapeutic effect is associated with multiple The measurements were performed using an image-guided variable spot electrophysiology system with a stimulation wavelength of 1000 nm. Provided that it is identified as: i) At baseline in healthy / normal and abnormal regions with identical stimulation parameters comparing the measurements, ii) spatially localized electrophysiological measurements from the same location at a previous time point Compare with the measurements, iii) By matching the stimulation wavelength and / or stimulation intensity, various photosensitive cells Identifying functional changes in types; iv) Different stimulation patterns, such as peripheral and array stimulation patterns Mapping the electrophysiological function of the visual field; v) Concentric electrophysiological measurements to assess gradients of electrophysiological changes in localized disease areas By administering an expanding series of stimuli, local dystrophic progression or therapeutic improvement can be achieved. To judge.

[0043] In another embodiment, the present invention provides an integrated OCT-guided stimulation light and electrophysiology system. The present invention contemplates a device, wherein the electrophysiology system includes: Graphical user interface (GUI) software that provides the platform This allows the user to control the desired stimulus position within the field of view of the en face image. This makes it possible.

[0044] In yet another embodiment, the present invention comprises an OCT-guided variable spot electrophysiology system. The OCT-guided variable spot electrophysiology system includes a device that can It consists of GUI software that allows users to view the face image. It is possible to start an electroretinogram (ERG) when any stimulation position on the A list of stimulation and recording parameters is presented to the user.

[0045] In another embodiment, the present invention provides a device with an OCT-guided variable spot electrophysiology system. The OCT-guided variable spot electrophysiology system is designed for a graphical user interface. The software includes a GUI software that controls the stimulation power, Image / signal recording is performed after certain parameters such as duration, interstimulus interval, and number of stimuli have been selected. The image / signal recording process continues after the start phase. It's done automatically.

[0046] In another embodiment, the invention encompasses a device for use in the method. 2. OCT-Guided Stimulation Optical Integrated Electrophysiology System with Different Wavelengths and Operation Modes By selectively stimulating rod or cone photoreceptors, it is possible to treat dry AMD, retinal pigment epithelium, and other conditions. Functional assessments can be generated from degenerative or cone-rod dystrophy models.

[0047] In another embodiment, the present invention provides a method for detecting rod or other photoreceptor cells with different wavelengths and modes of operation. By selectively stimulating cone photoreceptors, it can treat dry AMD, retinitis pigmentosa, or cone Wearable or portable devices capable of performing model-based functional assessment of rod dystrophy A versatile benchtop OCT-guided variable spot electrophysiology system is envisioned.

[0048] In another embodiment, the present invention provides a wearable OCT-guided variable spot electrophysiology system. The wearable part is assumed to be a goggle, eyeglasses, etc., but is not limited to these.

[0049] In another embodiment, the present invention provides OCT-guided variable spot ERG with multiple stimulation wavelengths. The system provides an evaluation of local therapeutic efficacy. 14 , Optoje Netics gene therapy 15 , and regenerative cell (transplant) therapy 16 in degenerative areas of the retina It is hoped that this will lead to the recovery of vision loss. This allows for quantitative evaluation of the recovery of retinal function after surgery. This will provide further insight into the integration efficiency of transfected or transplanted cells. Using the Odd Spot ERG System, optogenetic treatment was performed on a patient with a defect in the outer retina. It became possible to measure improvements in visual response in the atrophic retinal areas.

[0050] The present disclosure will now be described in detail with reference to the accompanying drawings, in which some of the inventions are shown. Although only a few exemplary embodiments are shown, the invention may be embodied in many different forms. The present invention is not limited to the embodiments described herein. These embodiments will fully and completely understand the present disclosure and will allow those skilled in the art to easily understand the scope of the present invention. It is provided to fully convey the scope of

[0051] In the claims and / or specification, the term "comprising" When used in conjunction with "a" or "an," the term "one" or "one" means "one It can also mean "one or more," "at least one," or "one This is also consistent with the meaning of "or more than."

[0052] Use of the term "or" in the claims refers only to alternatives or to alternatives. means "and / or" unless expressly stated to be mutually exclusive. However, the present disclosure is not intended to be construed as limiting the scope of the invention, and should not be construed as limiting the scope of the invention. The definition is also supported.

[0053] The terms "a" and "an" are used to expressly indicate that this disclosure is not intended to be limiting. "Substantially" is defined to mean "one or more" unless otherwise required. The term "subject matter" generally refers to the subject matter specified, but not necessarily to the full specification, as will be understood by those skilled in the art. (However, it does include the specified content. For example, "effectively 90 degrees" is 9 0 degrees, and "substantially parallel" includes parallel.) In any disclosed embodiment, The terms "substantially," "approximately," and "about" mean "to be" or "to be" in relation to the specified subject matter. "within a percentage" can be replaced with "within a percentage" and this percentage includes 0.1% , 1%, 5%, and 10%.

[0054] Throughout this application, the term "about" refers to the device by which a value is determined. or used to indicate that the standard deviation error of the method is included.

[0055] Furthermore, a device or method configured in a particular form may be configured in at least that form. Although the device is configured in a particular manner, it may be configured in other ways than those specifically described. may be capable of or configured to perform the functions described herein. Such implications are also encompassed in the disclosure of this specification.

[0056] "Prepare" (and all forms of "prepare", e.g., "prepare" and "prepared") , "have" (and all forms of "have", e.g., "have", "have", etc.) , "enclose" (and all forms of "enclose", e.g., "enclose", "enclosed", etc.) and "Contains" (and all forms of "contains", e.g., "contains" and "containing" ", etc.) are open-ended linking verbs. As a result, they can be used to "contain" one or more elements. An apparatus that includes, has, contains, or contains one or more elements in question. However, it is not limited to having only one or more of these elements. A method that "comprises," "has," "involves," or "contains" more than one step is or multiple steps, but only one or more of those steps Not determined.

[0057] As used in this specification and claims, "comprises" (and "comprising" any form of "comprise," "comprises," etc.), "having" "and all forms of "have", e.g. "have" and "have" "has"), "contains" (and all forms of "contains", e.g., "includes" "includes," "include," etc.) or "containing" (and " Any form of "contains," e.g., "contains," "contain ) etc.) may be generic or open-ended and may include additional, unlisted elements or does not exclude method steps.

[0058] Any embodiment of any of the apparatus, devices, systems and methods described The phrase "comprises / includes / contains" any of the steps, elements and / or features included in the Instead of " / having" you can use "consisting of" or "consisting essentially of" Therefore, in any claim, the scope of the claim should be limited to the above open-ended These open-ended linking verbs are used to differentiate from the use of the Use the words "consisting of" or "consisting essentially of" in place of any of the terms You can be there.

[0059] Features of an embodiment may be used unless expressly prohibited by the nature of the disclosure or the embodiment. Unless otherwise described or illustrated, the invention may be applied to other embodiments.

[0060] Any specific disclosure in the above cited documents or other documents is not intended to be limiting of any of the general aspects of the present invention. To the extent any of these is contemplated, the present disclosure excludes such previously disclosed species. It should be understood to include any proviso to exclude or disallow such. Aspects of the present invention not anticipated by the disclosure of the relevant literature are not disclosed or addressed herein. The disclosures in these documents are at least in part due to the unexpectedly superior results achieved. It's not self-evident.

[0061] The presently disclosed invention is further illustrated by the following examples, which are not intended to be limiting. These are provided for illustrative purposes only and therefore should not be construed as limiting the scope of the invention. should not be interpreted. [Example]

[0062] We are developing a novel method based on image-guided variable spot stimulation for various biomedical applications. We have developed an electrophysiological evaluation device that can measure the electrical activity of a photoactivated living specimen under image guidance. Functional assessment based on variable spot light stimulation of the material can be performed.

[0063] Example 1: FIG. 1A shows configuration 1 of an OCT-guided variable spot stimulation and electrophysiology system. The fiber-coupled visible light source (1001, 1002, 1003) is a beam combiner. The light emitted from these sources was combined using a 1004 laser. A near-infrared imaging light source with a central wavelength of 860 nm and a bandwidth of approximately 100 nm ( The low-coherence light from the second beam combiner (1005) is then combined with the second beam combiner (1006). The laser isolator (1007) is used as an imaging light source. Used to block back-reflected visible laser beams. Fiber optic 50 / 50 Beam splitter (1008) splits the imaging light beam into two arms The beam in the sample arm was focused by mirror 1 (1009) and XY M The light was guided and scanned using EMS mirror 2 (1010). 1011, liquid lens) in combination with the condenser lens assembly (1012) A fiber optic cable was used to focus the optics and visible laser beam onto the sample. The second beam from the optical beam splitter (1008) passes through a collimating lens and a mirror. The reference arm (1013) has an adjustable optical path length and is used to guide the reference beam. The backscattered light from the sample was reflected back by a splitter (1008). Assembly (1012), dynamic focusing element (1012), XY MEMS mirror 2 (1010), mirror 1 (1009) and a fiber optic beam splitter (100 8), where the backscattered light from the sample is split and routed to a detector (spectrophotometer ) (1014) and split by a fiber optic beam splitter (1008) The back-reflected reference beam after being reflected is also received by a detector (1014) equipped with a spectroscopic camera. The interference signal between the back-reflected signal from the reference mirror and the back-scattered light signal from the sample The signal was detected by a spectroscopic CCD detector as a function of wavelength. Detected signal (function of wavelength) is plotted as a function of wavenumber and Fourier transformed to obtain optical coherence tomography ( Optical Coherence Tomography (OCT) information, i.e., the intensity profile as a function of depth, was obtained. and 3D imaging to allow real-time adjustment of OCT imaging position and acquisition. Rendering was performed on a software platform.

[0064] A user-friendly G-sensor for performing variable spot-target stimulation and electrophysiological measurements UI software is used to measure the visible light stimulation beam, NIR imaging beam, and OCT sensor. The sensors are controlled, imaging / electrophysiological measurements are acquired, and further processing and results are Once the measurement spot was identified on the image, the dynamic focusing element (1 011, liquid lens) was used to control the laser stimulation spot size at each measurement position. The software control panel controls the visible laser beam and the OCT image. The microcontroller was used to change the power of the scanning beam and the exposure time (scanning speed). A computer (1016) with a laser (1015) and a display (1017) Light source (1001, 1002, 1003), low coherence imaging light source (1005) , XY MEMS mirrors 1 and 2 (1009, 1010), and a reference axis with adjustable optical path length. It was used to control and automate the beam (1013) and the detector (1014). 018) measured the electrophysiological activity of tissue while performing 3D image-guided laser stimulation. It was used on samples (eye, brain, retina explants) to

[0065] This integrated device is based on OCT-guided variable spot laser stimulation for electroretinography ( ERG) and visual evoked potentials (VEP: when electrodes are placed in the brain or over the visual cortex) Convenient measurement workflows on one multimodal platform OCT offers good deep penetration and excellent depth resolution, making it ideal for integrating OCT and ERG. This allows for spatially targeted local stimulation with depth resolution. The determination involves the electrical activity of various cell types within the retina, including photoreceptors, inner retinal cells, and retinal ganglion cells. Different ERG measurements were performed to obtain selective functional information of the retina. Various lasers including global, local, flicker, and pattern stimuli Mode stimulation was performed. To measure label-free photoelectrophysiology, the phase of the interference signal was measured. The easily interchangeable imaging lenses allow for imaging of small animals (mice, rats) Imaging and testing of a wide range of species from small to large animals (rabbits, pigs / NHPs) and humans On the other hand, irradiation of multiple regions of the retina by focused polychromatic laser beams has become possible. The responses evoked in different retinal layers in the region are measured to determine which parts of the retina exhibit abnormal functional responses. The focal stimulus was precisely guided to a specific point while rapidly identifying which stimulus indicated the presence of the stimulus.

[0066] Example 2: Figure 1B shows configuration 2 of the OCT-guided variable spot stimulation and electrophysiology system. The visible light sources (1001, 1002, and 1003) are connected to the beam combiner 1 ( 1004) in free space, and the beams are combined using a beam steering optical system (1005, XY The beam is guided by a MEMS mirror and shares the same optical path as the imaging beam. The light emitted from these sources was used for targeted stimulation of biological tissue. The low-coherence light from the near-infrared imaging light source (1007) with a width of about 100 nm is The back-reflected light passes through the laser isolator (1008) and is blocked from passing through the laser isolator (1008) toward the imaging light source. After that, a fiber optic 50 / 50 beam splitter (1009) was used to separate the sample beam. The imaging beam in the sample arm was split into a reference beam and a reference beam. 1010) and XY MEMS mirror 2 (1011) guide and scan the light. After that, it was combined with the visible stimulus light by a dichroic beam combiner (1006). A focusing element (1012, liquid lens) and a focusing lens assembly (101 3) is used in combination to direct the imaging beam and the visible laser beam to the sample. The second beam from the fiber optic beam splitter (1009) was focused onto the The reference arm (1014) is made up of a realignment lens and a mirror, and has an adjustable optical path length. The reference beam was reflected back to the beam splitter (1009). The scattered light is reflected by the focusing lens assembly (1013), the dynamic focusing element (1 012), Dichroic Beam Combiner (1006), XY MEMS Mirror 2 (1 011), and through mirror 1 (1010) to fiber optic beam splitter (1 009), where the backscattered light from the sample is split and routed to a detector ( The light was received by a fiber optic beam splitter (1009). The split back-reflected reference beam is also detected by a detector (1014) equipped with an optical camera. The back-reflected signal from the reference mirror and the back-scattered light signal from the sample are The interference signal was detected by a spectroscopic CCD detector as a function of wavelength. The signal (a function of wavelength) is plotted as a function of wavenumber and then Fourier transformed to obtain the optical Optical Coherence Tomography (OCT) information, i.e., intensity profile as a function of depth Simplify operation and adjust OCT imaging position and acquisition in real time To enable this, 3D rendering was performed on a software platform. .

[0067] A user-friendly G-sensor for performing variable spot-target stimulation and electrophysiological measurements The UI software is used to measure the visible light stimulation beam, NIR imaging beam, and OCT. The sensors are controlled and imaging / electrophysiological measurements are acquired, processed, and analyzed. The dynamic focusing lens allows for imaging at multiple depths. After the 3D rendering was performed, the user could Change the position and size of the stimulation spot in the software's graphical interface The scanning mirror and dynamic focus lens allow for 3D rendering. Based on the imaging image and the selected stimulation area, the exact location can be pinpointed. The software control panel controls the visible laser beam and the OCT imaging beam. The stimulus intensity and repetition rate of the stimulus were changed. To synchronize the signal processing and acquisition hardware, a microcontroller (1016 ) was used. A computer (1017) equipped with a display (1018) was used. It communicates with duino and outputs visible light sources (1001, 1002, 1003), low coherence images, XY MEMS for oscillating light source (1007), visible light beam and imaging beam Mirrors (1005, 1011), a reference arm (1014) with an adjustable optical path length, and The detector (1015) is controlled and automated. The electrode (1019) is connected to the sample (eye, brain, It is used in retinal explants to measure the electrical conductivity of tissue during 3D image-guided electrophysiological stimulation. was used to measure physiological activity.

[0068] This integrated device is a simple-to-use electrophysiological device based on OCT-guided tunable spot laser stimulation. Electroretinogram (ERG, when electrodes are placed on the cornea) and visual evoked potentials (VEP, VEP is The measurement workflow (electrodes placed in the brain and visual cortex) is integrated into one multimodal platform. OCT has good deep penetration and excellent depth imaging. provides 3D resolution, and OCT and ERG integration allows for spatially targeted imaging with depth resolution. ERG measurements are performed on photoreceptors, inner retinal cells, and retinal nerve cells. The different ERG measurements provided electrical responses of various cell types within the retina, including ganglion cells. To obtain selective functional information of the retina, global, local, and flicker stimuli were used. Various laser mode stimulations, such as , and pattern stimulation, were performed. To measure electrophysiology, the phase of the interference signal was measured. Glens (1013) has demonstrated that the genotypes of small animals (mice, rats) and large animals (rabbits, pigs, etc.) Imaging and irradiation of a wide range of species, from NHPs to humans, has become possible. Irradiation with a focused polychromatic laser beam induces changes in different retinal layers in multiple regions of the retina. The resulting responses are measured to rapidly identify which areas of the retina show abnormal functional responses. In both cases, the focal stimulus was precisely guided to a specific point.

[0069] Example 3: Figure 2A shows OCT and fundus image-guided variable spot stimulation and electrophysiology simulation. The third configuration of the system is shown. Most of the elements of this configuration are the same as those of the first configuration. , an additional device to realize fundus imaging in addition to the OCT image-guided variable spot electrophysiology system. The components 1001 to 1000 described in the first embodiment are added. In addition to the 18, a second imaging light source (1019) is placed. The illumination light is then focused The beam is focused (1020), passes through a semi-reflecting mirror (1020), and then passes through a multi-wavelength optical bandpass filter. The light is reflected by the filter (1024) towards the sample, illuminating the sample. The light reflected from the fundus illumination source then passes through a bandpass filter and is then reflected by a semi-reflecting mirror (1 The fundus passes through the microscope (1020) and is then focused onto the camera (1023). Although the fundus does not offer the depth penetration that OCT offers, this configuration allows for live fundus imaging, allowing for the observation of OC Enhance image guidance without interfering with the imaging and electrophysiological function of the system. This system allows for the use of a second imaging light source (1019) instead of a second imaging light source (1020). By using a visible laser as the illumination source (1001-1003), the sample The fluorescent signal from the XY scanning mirrors (1009, 1010) can be detected. This allows point-by-point scanning and excitation at selective wavelengths (blue, green, red) and redshifted or blueshifted in a wider bandwidth than the stimulation source (narrowband 1001-1003). The shifted signal is reflected by a multi-wavelength bandpass filter and transmitted to a fundus camera (1023 )

[0070] Example 4: Figure 2B shows the configuration of a fundus image-guided variable spot stimulation and electrophysiology system. 4. This configuration abandons the depth-resolved image guidance, but instead relies on the fundus image. ing samples (eye, brain, retinal explants, and other photoactivatable tissue specimens) ) to simplify variable spot electrophysiology by inducing local stimulation of a visible light source (10 1001, 1002, 1003) were combined using beam combiner-1 (1004). The light emitted from these sources was used for targeted stimulation of living tissue. The liquid lens (1005) is a steering mirror (1006) that is a MEMS mirror. Used in conjunction with A visible laser beam was then used to target the dichromic microparticles. The fundus image is directed towards the imaging patch by the lens (1007). The light source (1008) is collimated by a collimation optical system (1009), The light is reflected by a semi-reflecting mirror (1010) towards the sample and then focused by a focusing optical system (1011). The light reflected from the sample is then focused into a focusing optics (101 1), semi-reflecting mirror (1010), dichromic mirror (1017), focusing optical system (101 After passing through 2), it is detected by the fundus camera (1013).

[0071] Example 5: Figure 2C shows OCT and fundus image-guided variable spot stimulation and electrophysiology simulation. The system configuration 5 is shown. Most of the elements of this configuration are the same as those of the configuration 3 in Example 3. , a second imaging light source is combined with a focusing element (1019) to form a ring-shaped The difference is that it is configured as a 1012 and is placed in front of the focusing lens. The emitted light is then filtered by a beam splitter (1022) for imaging modality 2. After being routed, it is finally focused (1020) by the camera (1021). This configuration simplifies live funduscopic imaging while providing OCT imaging and electrical Allows physiological measurements.

[0072] A user-friendly G-sensor for performing variable spot-target stimulation and electrophysiological measurements The UI software is used to measure the intensity of the visible light stimulation beam, the fundus imaging beam, and the fundus The camera exposure time is controlled and imaging / electrophysiological measurements are acquired and processed. Once the measurement spot was identified on the image, the dynamic focus was A lens element (1005, liquid lens) is used to measure the size of the laser stimulation spot at each measurement position. The size was controlled by the microcontroller (1014) and the display (1016). A computer (1015) with a visible light source (1001, 1002, 1003), a MEM Used to control and automate the S mirror (1006) and fundus detector (1013). The electrode (1017) is used in the sample (eye, brain, retina explant) and the image Electrophysiological activity of the tissue was measured while induced laser stimulation was performed.

[0073] This integrated device allows funduscopic image guidance in one multimodal platform setting. Electroretinography (ERG) and visual evoked potential measurements based on variable spot laser stimulation We have achieved a simplified workflow for VEP (when electrodes are placed in the brain or visual cortex). ERG measurements are performed on various cells within the retina, including photoreceptors, inner retinal cells, and retinal ganglion cells. The ERG measurements provide specific electrical responses to the retina. To achieve this, various stimuli such as global stimuli, local stimuli, flicker stimuli, and pattern stimuli are used. Laser mode stimulation was performed. Easily interchangeable imaging lenses allow for easy imaging of small animals. A wide range of species, from mice and rats to large animals (rabbits, pigs / NHPs) and humans On the other hand, imaging and irradiation with a focused polychromatic laser beam have become possible. This allows measurements of responses evoked in different retinal layers in multiple regions of the retina, allowing researchers to determine which part of the retina is affected. Rapidly identify whether a location shows an abnormal functional response and ensure that focal stimulation is focused on that specific point. was closely guided.

[0074] Example 5: FIG. 3A shows a schematic diagram of an OCT-guided variable ERG system with a scanner head. Figure 3B shows a live fundus image of a rodent (rat) retina. Figure 3C shows an OCT en face image reconstructed from 3DOCT imaging. The marked circle indicates the diagnostic ERG stimulation site. Localized stimulation of various sizes was achieved. Figure 3D shows the stimulation of a wild-type mouse (C57 The differences in response to blue, green, and red stimuli for different cones (S and M) in the BL / 6J Figure 1 depicts the multicolor ERG functional assessment profiles showing different responses. The ERG profiles show distinct and complex responses from individual photoreceptors and higher-order neurons. Mice lack L cones, so ERG responses to red stimuli are minimal, whereas S cones The body shows a more spread out response (blue light stimulus) compared to the M cone (green light stimulus).

[0075] To simplify fundus imaging that integrates OCT and electrophysiological measurements, The LEDs (LEDs) are arranged in a ring shape and illuminate the eye. Figure 3E shows the fundus imaging light source. The scanner head of the OCT-guided ERG system is shown, and a model eye (white sphere on the right) is shown. Figure 3F shows the fundus image of the model eye, and the black curve indicates the blood flow. The white area in the fundus image of the model eye mimics the optic nerve. G shows an OCT en face image of the model eye, with the black curves mimicking blood vessels. The white part mimics the optic nerve.

[0076] Example 6: Figure 4 shows how a dynamic focusing element can change the size of the stimulation spot. The steering optics are used to change the OCT and / or Accurate imaging of the stimulation spot aligned with the image generated by fundoscopic imaging. The focusing element allows precise positioning from a microfocused spot. The size of the stimulation spot can be varied from a small spot to a large spot that covers the entire visual field. The imaging system's field of view ranges from small rodent systems (mice, rats) to large animals. Dynamic focusing lenses are used in a wide range of imaging systems (rabbit, pig, NHP) and clinical settings. Fine focusing in the sample plane is required to obtain high spatial resolution for gauging. You can also change the lens to fill the aperture of the focusing objective, It is also possible to stimulate the entire imaging field.

[0077] Example 7: Experimental determination of the spatial resolution of a variable spot OCT-guided ERG system Instead of a stimulation laser, a laser with the same specifications as the stimulation laser but with a higher output was used. A laser with a variable spot OCT-guided ERG system was then coupled to the dynamic flash. By changing the focusing element, different spot sizes were irradiated onto the mouse retina, and localized The accuracy and positioning of the laser stimulation were visualized by inducing significant tissue damage. To measure functional deficits in a mouse model of dry AMD created by laser injury, To achieve this, an OCT-guided laser microirradiation integrated electrophysiology system was used. Figure 5A shows the en face ( OCT image showing a damaged spot in the peripheral retina. OCT-guided visual illumination. were presented separately at various intensities to measure activity in the control and laser-damaged areas. Figure 5B shows a typical ERG response obtained from an undamaged retinal region. Scotopic variable spot electroretinogram (vsERG) responses are shown. As shown, the scotopic vs. ERG responses obtained from the laser-damaged area showed a decreased ERG response. The spatial resolution of the vsERG measurements was determined to be approximately 0.05 mm.

[0078] To further determine the accuracy of the OCT-guided variable spot ERG system, A pig model of geographic atrophy was created using a laser. B-scan and immunostaining images were obtained. -Indicates focal outer nuclear layer damage due to injury, where the photoreceptor and RPE layers are lost However, there was no damage to the inner retina. OCT-guided vsERG signals were measured in healthy pig retina outside the laser-damaged area. The sERG measurement showed a blue light vs. ERG response, but the ERG signal was significantly increased when red light stimulation was used. There was no signal response (because pigs do not have L cones). However, When the retinal area was selected with the same stimulation parameters (stimulation spot size, wavelength, intensity), blue No response was detected using color or red light stimulation. These experiments were OCT-induced vsERG establishes structure-function relationships with high spatial resolution in the partially atrophied retina Not only does it detect minute changes occurring in the retina in almost real time (several days after laser damage), It was shown that they can be distinguished by the time (minutes).

[0079] Example 8: To assess disease progression or evaluate treatment outcome, specific It is often necessary to measure function from multiple retinal cell types, e.g., photoreceptors. and glaucoma for inherited retinal degenerative diseases and dry AMD in which RPE damage occurs. is associated with RGC damage. Evaluate the pathological progression of GA or the development of new atrophy. Therefore, spatiotemporal control of electrophysiological assessments using OCT guidance offers a unique opportunity. Furthermore, in retinitis pigmentosa, the loss of rod cells precedes the loss of cone cells, whereas In cone-rod dystrophies, loss of cone cells leads to loss of rod cells. Controllable By varying the wavelength of the OCT-guided visible laser stimulation beam with a suitable spot size, rods This makes it possible to selectively evaluate the function of specific (S, M, L) pyramidal cells. Figure 6 A is an OCT-guided variable spot E based on a scaled ISCEV standard protocol. This shows the assessment of regional function using RG measurements. , 2 mm stimulus spot (3 cd·s / m under light adaptation 2 color-dependent responses were observed in Figure 6B shows the results of the stimulation of a 2 mm stimulus spot (3 cd·s / m under dark adaptation). 2 ) was used Figure 6C shows a standard rod and cone ERG protocol. (0.01 cd·s / m under dark adaptation 2 ) and a scotopic rod ERG protocol using Figure 6D shows the same scotopic rod ERG protocol using a 4 mm stimulus spot. The stimulus size dependent responses are shown.

[0080] Example 9: OCT-guided variable spot electrophysiology system detects localized electrical networks within the retina It is not limited to membrane measurements, but also includes visual evoked potentials (VEPs) generated by local stimulation, It is also used to measure OCT-induced OCT images showing two different stimulation locations in the rodent retina for variable spot VEP measurements. Figure 7B shows the image of the visual cortex measured using a white light stimulus at position 1. Figure 7C shows the VEP measured in the visual cortex using white light stimulation at position 2. P. The VEP response in the optic nerve area (position 1) shows a low response. Figure 7D shows the VEP response in the position 1. In 2, VEPs measured in the visual cortex using green light stimulation were compared with white light stimulation of the same intensity. This indicates that the α-glucan-containing β-glucan solution exhibits a higher response than the α-glucan-containing β-glucan solution.

[0081] Example 10: Flicker stimulation using pulse train stimulation of a specific frequency within a given time window In this paper, we combine multiple color stimuli and deliver individual colors from the same area with a single stimulus pulse train. The response can be measured by frequency multiplexing. Different types of photoreceptors respond differently to different wavelengths. The combination of multicolor stimuli with different frequencies due to their different absorption profiles When different types of photoreceptors are stimulated, each photoreceptor responds only to the color stimulus of the corresponding stimulus frequency. Example 8A shows a frequency-multiplexed stimulus for simultaneously detecting the responses of different cone cells. The upper pulse train shows the blue light flicker as a function of time. -The intensity profile of the stimulation is shown. The central pulse train has a slightly faster repetition rate. The figure shows the red light flicker stimulus at different speeds. t1, t2, and t3 are the stimulus pulse trains for color 1. represent the timestamps of the color 1 stimuli, and τ1, τ2, and τ3 represent the timestamps of the color 2 stimuli. The lower pulse train consists of a train of blue flicker stimuli and a train of red stimuli superimposed on each other. This mixed single pulse train sequence shows a linear combination of blue and This can be used to simultaneously detect the responses of photoreceptors from white and red light stimuli. 8B shows a method for detecting different visual fields based on frequency response using frequency filtering in the Fourier domain. The method for separating cellular responses is shown. Based on the timestamps (t1, t2, t3…) in color 1, Synchronous averaging (1003) of the filtered acquired signal generates the frequency 1 signal (1004) ) and reduce other frequency signal components. This extracted signal is Similarly, the time-series response of color 2 is shown. Synchronous averaging (100 s) of the acquired signals filtered based on the amplitudes (τ1, τ2, τ3, ...) 6) extracts the frequency 2 signal (1007) and reduces other frequency signal components. The extracted signal represents the response of photoreceptor cell type 2 (1008).

[0082] Figure 8C shows the flicker ERG response using red light and 14 Hz stimulation, and Figure 8D shows Figure 8 shows the flicker ERG response using blue light and 31 Hz stimulation at the same location. E is the timestamp averaged over the red light stimulus timestamp for 14 Hz red and 31 Hz blue light. Figure 8F shows the flicker ERG response using a linear combination of colored lights, and Figure 8F shows the time series of blue light stimuli. A linear combination of 14Hz red light and 31Hz blue light averaged over a time stamp. Shown is a Licker ERG response.

[0083] Example 11: Electrophysiological signals with time-delayed / long-lasting profiles By utilizing spatial and temporal stimulus multiplexing, multiple locations can be stimulated simultaneously. By stimulating one location at a time, the signal pair of the average signal is increased compared to stimulating one location at a time sequentially. This method can increase the signal-to-noise ratio (SNR). It is widely used to shorten acquisition time and increase the signal-to-noise ratio per stimulus. Figure 9 shows a schematic diagram of spatial and temporal stimulation to increase the signal-to-noise ratio. For stimulation at multiple locations, approximately half of the random locations were stimulated at a given time, and no stimulation was observed from each location. These local responses can be extracted from linear combinations of stimuli. Compared to , averaging increases the SNR per stimulus.

[0084] Example 12: Figure 10A shows integrated OCT-guided variable spot electrophysiology for clinical studies. The platform shown is a PC and all hardware in a compact system. It is housed in a muscular chassis, making it easy to use in a clinic or office environment. An adjustable LED light mounted on the stand serves as a fixed target during testing. 10B shows the experimental setup of the OCT-guided tunable spot-ERG system, with electrodes connected. (The signal electrode is placed on the cornea, the reference electrode is placed under the eye, and the ground electrode is placed on the forehead.) The chin rest provides stability for the patient, and the scanner is attached to a chin rest with translational and rotational adjustment. It has a maintenance function.

[0085] Figure 10C shows an integrated wearable OCT-guided variable spot electrophysiology system for clinical research. The goggles are shown. The separated binocular configuration allows for independent electrophysiological measurements for each eye. Finally, Figure 10D shows the OCT-guided wearable variable sensor. This shows the setup of the wearable ERG goggles. The form further reduces motion artifacts in local ERG stimulation. This is because body and head movements are canceled out by the wearable goggles.

[0086] Example 13: Figure 11A shows the clinical setup during OCT-guided variable spot ERG measurements. Here is an example. First, we acquire OCT images and evaluate the structural information with depth resolution of the retina. Based on the structural assessment, the user graphically overlays the stimulation location and visualizes it in an en face image. The size of the stimulation area can be adjusted along with other stimulation parameters. An example of a face OCT image is shown. A standard cone E was obtained using a 2 mm spot size stimulus. RG protocol (Figure 11C) and 30 Hz flicker ERG protocol (Figure 11D). Variable spot ERG profiles were measured using

[0087] Figure 11E shows clinical results during OCT-guided variable spot ERG measurements using wearable goggles. An example of a floor setup is shown. Similar to the benchtop system, retinal structural evaluation is performed. The ERG stimulation area is then defined by the user. The image-guided ERG system uses goggles that completely block out ambient light, allowing the user to see clearly in the room. For photometric measurements, the background light is controlled within the goggles. The goggles block out ambient light and nearby activity, ensuring the safety of the goggles. This setup significantly reduces noise and helps ensure consistent measurements.

[0088] Example 14: Figure 12 shows the OCT-guided variable spot ERG system software for clinical trials. The left panel shows the software interface. The left panel uses a standard cone ERG protocol. The individual measurements are shown, and the middle panel shows the average signal. The image is shown at the top, with the marked black circle indicating the stimulation area. The white crosshairs indicate the xy-section. The OCT B-scan images on the right and bottom show the Within the dataset, the stimulus color, the stimulus mode (different ISCEV protocols), the number of stimuli to be averaged, etc. Measurement parameters can be digitally controlled, and data processing and display of results are also possible. The software provides different filtering and averaging methods, and all data is saved. , can be read and reprocessed.

[0089] Example 15: Precise electrophysiological stimulation anywhere within the field of view of the imaging modality The flexibility of placement allows for functional responses of multiple regions of interest to be measured using different stimulation patterns. Figure 13A shows a peripheral MRI scan using a ring pattern surrounding the macular region. Figure 13B shows a linear square pattern for OCT-guided variable spot ERG measurements. Figure 13C shows the same stimulation spot size without changing the stimulation position. 1 shows a concentric circle pattern measurement scheme that realizes changing the size of different concentric circles. Measure electrophysiological responses with spot sizes of various sizes and compare responses from small areas with those from large areas. By subtracting it from the response from the surrounding area, the response from the center can be calculated. Figure 13D shows the ERG response measurements superimposed using a peripheral ring pattern. Figure 13E shows the results of the analysis using the concentric circle pattern in the software. Figure 1 shows ERG response measurements overlaid with the sham-like response (human).

[0090] Acquire a 3D volumetric OCT scan and navigate within the acquired volume to locate regions of interest The stimulation spot can be selected while the cross-sectional OCT scan is also displayed. When this is done, the software will prompt you to select two depths for the phase difference measurement. Once the depth of interest is determined, the stationary OCT signal over time (M-scans) is calculated. It will start generating phase transitions and display them in a separate window at the default B-scan frame rate. Finally, once the stimulation options have been initiated with the set pulse width and repetition rate, the interactive The active software provides information on the stimulation conditions: (i) bulk loading; potential damage due to thermal effects; and (ii) power adjustment suggestions based on overall phase change calculations. User-friendly GUI software provides a platform for user interaction. It provides a platform that allows users to perform multiple scan modes: A-scan for functional probing; Structural imaging for stimulation location by B&C scan, and OCT imaging The software allows you to control the acquisition with adjustable scan range and focal depth. This allows you to navigate the region of interest within the OCT scan. Within the software platform, users can adjust power, pulse duration, stimulation frequency, and Controls for customizing the stimulation laser, including selecting specific ROIs within the OCT scan Once the stimulation option is initiated, the user is presented with a list of parameters. The integrated The device and software provide 3D OCT image-guided microfocus laser stimulation control and temperature The software also provides on-line or off-line phase OCT signal measurements. raw M-scan data to examine functional (neuronal) changes by measuring A simple analysis is also provided.

[0091] The specification and examples provide a complete description of the structure and use of exemplary embodiments. The following description may be omitted to refer to a particular embodiment in some detail or to one or more individual embodiments. Although the invention has been described with reference to the accompanying drawings, those skilled in the art will be able to adapt the disclosed embodiments without departing from the scope of the invention. Many variations on the embodiment may be made. Thus, various examples of the device The illustrative embodiments are not intended to be limited to the particular forms disclosed. , including all modifications and alternatives that fall within the scope of the claims, and The embodiments may include some or all of the features of the depicted embodiments. Components may be omitted or combined into a single structure and / or Furthermore, where appropriate, any of the examples described above may be used. Any embodiment may be combined with other example embodiments described, having similar or different characteristics, Further examples can be created that address the same or different problems. The benefits and advantages disclosed may relate to one embodiment or may be applicable to several embodiments. It will be appreciated that the terms "condition" and "conditions" may also be relevant.

[0092] Although the devices, compositions and methods of the present invention have been described in terms of preferred embodiments, Those skilled in the art will readily appreciate the devices, compositions and / or methods described herein. No change may be made to the steps or sequence of the method without departing from the concept, spirit and scope of the present invention. It will be apparent that the following can be applied to the present invention. More specifically, chemical and physiological Certain agents related to the present invention may be substituted for, and are similar or It will be apparent that similar results can be obtained. All such similar substitutes and modifications are deemed to be within the spirit, scope and concept of the present invention. It is considered.

[0093] Furthermore, the claims may be means-plus-function or step-plus-function. The limitation of the patent uses the phrases "means for" or "steps for," respectively. The use of the present invention should not be construed as including such limitations unless expressly stated in a given claim. It's not that.

[0094] References The following references provide procedural or other details that complement those described above: To the extent applicable, the disclosures herein are specifically incorporated by reference.

[0095] 1. Lim, L.S., Mitchell, P., Seddon, J.M., Holz, F.G. & Wong, T.Y. Age-related ma cular degeneration. The Lancet 379, 1728-1738 (2012). 2. Swain, P.K., et al. Mutations in the cone-rod homeobox gene are associated wi th the cone-rod dystrophy photoreceptor degeneration. Neuron 19, 1329-1336 (1997 ). 3. Perrault, I., et al. Leber congenital amaurosis. Molecular genetics and metab olism 68, 200-208 (1999). 4. Kalatzis, V., Hamel, C.P., MacDonald, I.M. & Symposium, F.I.C.R. Choroideremi a: towards a therapy. American journal of ophthalmology 156, 433-437. e433 (2013 ). 5. Sandberg, M.A., Jacobson, S.G. & Berson, E.L. Foveal cone electroretinograms in retinitis pigmentosa and juvenile macular degeneration. American journal of o phthalmology 88, 702-707 (1979). 6. Han, Z., Conley, S.M. & Naash, M.I. Gene therapy for Stargardt disease associ ated with ABCA4 gene. Adv Exp Med Biol 801, 719-724 (2014). 7. Oh, K.T., et al. Electroretinographic findings in patients with Stargardt dis ease and fundus flavimaculatus. Retina 24, 920-928 (2004). 8. Hartong, D.T., Berson, E.L. & Dryja, T.P. Retinitis pigmentosa. The Lancet 36 8, 1795-1809 (2006). 9. Salvi, S., Akhtar, S. & Currie, Z. Ageing changes in the eye. Postgraduate me dical journal 82, 581-587 (2006). 10. McCulloch, D.L., et al. ISCEV Standard for full-field clinical electroretino graphy (2015 update). Documenta ophthalmologica 130, 1-12 (2015). 11. Seiple, W.H., Siegel, I.M., Carr, R.E. & Mayron, C. Evaluating macular funct ion using the focal ERG. Investigative ophthalmology & visual science 27, 1123-1 130 (1986). 12. Hood, D.C., Seiple, W., Holopigian, K. & Greenstein, V. A comparison of the components of the multifocal and full-field ERGs. Visual neuroscience 14, 533-54 4 (1997). 13. Hood, D.C., et al. Assessment of local retinal function in patients with ret initis pigmentosa using the multi-focal ERG technique. Vision research 38, 163-1 79 (1998). 14. Boye, S.E., Boye, S.L., Lewin, A.S. & Hauswirth, W.W. A comprehensive review of retinal gene therapy. Molecular therapy 21, 509-519 (2013). 15. Batabyal, S., et al. Sensitization of ON-bipolar cells with ambient light ac tivatable multi-characteristic opsin rescues vision in mice. Gene Therapy, 1-15 (2020). 16. Mahato, B., et al. Pharmacologic fibroblast reprogramming into photoreceptor s restores vision. Nature 581, 83-88 (2020).

Claims

1. i) Achieving a variable spot size on a sample for electrophysiological measurements of said sample an image-guided light stimulation beam; ii) an image-guided photostimulation beam assembly, The image-guided photostimulating beam assembly includes: - Illuminating and collecting back-reflected light from the sample for imaging, an imaging subassembly containing near-infrared (NIR) light from a coherent light source; The near-infrared (NIR) light is then sampled for interference detection to obtain depth-resolved images. an imaging subassembly capable of splitting a pull beam and a reference beam; 、 - light, including light beams of different wavelengths, with controllable intensity and / or pulse rate; a stimulation subassembly, iii) For each optical stimulation beam wavelength, the power of the stimulation beam at the sample plane is 0.01 ~50 cd.s / m ２ is in the range of iv) the optical stimulus beam can be combined with the sample beam and directed to the sample; and the light can be guided by the v) the sample is derived from neurons or photosensitive cells in vitro or in vivo; Selected, vi) a region of interest for electrophysiological measurements in said sample in response to variable spot stimulation; a preselected area is controlled by a scanning mirror that deflects the photostimulation beam; vii) the variable spot size is produced by a dynamic focusing element; viii) the light stimulus can be targeted to a preselected region of interest on the sample. It is possible, ix) the photostimulation beam is switched off when preselecting a region of interest for photostimulation; and the preselection of the region of interest is based on morphological / tomographic imaging. This is done, x) For morphological / tomographic imaging, the sample beam is delivered via optical fiber or free space. and collimated by the scanning mirror and optical components. It is possible to deflect the beam to a simple xi) the optical component is provided with anti-reflective materials to avoid scattering and multiple reflections It is coated with xii) The back-reflected sample beam for morphological / tomographic imaging from the sample is detected. can be routed back to the source, xiii) tomographic images are recorded and analyzed by recording the interference of the back-reflected sample beam with the reference beam. It can be reconstructed by analyzing and xiv) The morphological / tomographic images are matched to photostimulation spots for electrophysiological measurements. It is possible to mark with a selection area of ​​variable size, like xv) Electrophysiological measurements are performed using electrodes connected to biosensing hardware. It is possible to implement xvi) For electrophysiological measurements, allowing for variable spot sizes in the sample The image-guided light stimulation beam of claim 1 may be a tabletop system or a wearable system; xvii) The wearable system for electrophysiological measurements includes goggles or glasses. A device that can.

2. 10. The device of claim 1, comprising an electrode, a physical assembly for generating an electroretinogram (ERG) when the electrodes are placed on the cornea of ​​the subject; When the electrodes are placed on the subject's brain or visual cortex, visual evoked potentials (VEPs) are detected. The device is configured to measure.

3. The imaging source is combined with a spectrometer-detector or point detector for tomographic imaging. Low coherence broadband for combined optical coherence tomography (OCT) - Patent Application 20070122997 The device of claim 1 , which is a light source or a wavelength-swept light source.

4. The spatiotemporal guidance of the optical stimulation of the sample is achieved by OCT and / or fundus imaging. The device of claim 1 .

5. If the image-guided light stimulation spot is scanned by a deflection mirror and a dynamic focusing element, and configured to be enlarged or reduced by positioning the image on a selected area of ​​the subject's retina. The device of claim 1 .

6. The multiple stimulation wavelengths of the image-guided variable spot electrophysiology system allow us to investigate the function of different photoreceptor cells. The device of claim 1, wherein the device is capable of examining the function of the device.

7. 10. The device of claim 1, wherein multiple light stimulus beams can be combined to generate mixed color stimuli. Vice.

8. Image-guided variable spot electrophysiological measurement device for visual field stimulation using pattern stimulation The device of claim 1 capable of performing functional mapping.

9. Multiple electrode channels for simultaneous measurement of electroretinogram and visual evoked potentials from a single stimulus The device of claim 1 , comprising:

10. 10. The device of claim 1, Spatial and temporal averaging allows for improved signal-to-noise ratio and shorter measurement times Electrophysiological measurements with systematically multiplexed variable spot light stimulation: i) stimulating multiple non-overlapping sites with short stimulation bursts; ii) shortening the time interval between stimulations at multiple spots; iii) Varying the order of synchronized stimulation between various spots of interest; iv) Electrical stimulation resulting from single bursts of stimulation to various spots at different locations measuring physiological signals; iv) Deconvolving the signal responses from multiple regions to obtain the average for each spot position To obtain an average signal, The device is configured to do this.

11. 10. The device of claim 1, Multiple photoresponsive cell types with different absorption spectra were stimulated simultaneously at the same stimulation position. The stimulus frequency multiplexing measurements utilized to capture and record: i) combining multiple wavelength stimulation beams modulated at different frequencies; ii) stimulating different photosensitive cells with different absorption peaks and separated; iii) A single measurement of the light responses of different types of light-activated cells within a common stimulation site. Extraction at a fixed rate, iv) Average the acquired electrophysiological signals based on the timestamp of the corresponding color stimuli. deconvolving the mixed responses from individual cells by combining / or v) Notch frequency filtering to isolate the contributions of frequency / wavelength-dependent responses of other cell types To reduce The device is configured to do this.

12. 10. The device of claim 1, Measured using an image-guided tunable spot electrophysiology system with multiple stimulation wavelengths Local disease progression or treatment response: i) At baseline in healthy / normal and abnormal regions with identical stimulation parameters comparing the measurements, ii) spatially localized electrophysiological measurements from the same location at a previous time point Compare with the measurements, iii) By matching the stimulus wavelength and / or stimulus intensity, various light sensitivities can be achieved. Identifying functional changes in sex cell types; iv) by different stimulation patterns, such as peripheral and array stimulation patterns; Mapping the electrophysiological function of the visual field; v) Concentric electrophysiological measurements to assess gradients of electrophysiological changes in localized disease areas By administering an expanding series of stimuli, local dystrophic progression or therapeutic improvement can be achieved. To judge, 1. A device configured to be identifiable by

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