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

Abstract

The present invention generally relates to an image-guided variable spot stimulation-based electrophysiological assessment device for various biomedical applications. Specifically, the present invention relates to the application of the device to perform image-guided functional assessment based on variable spot light stimulation of photoactivated biological samples to determine changes in functional health during disease progression and treatment. More specifically, the present invention relates to the application of the device in the diagnosis of visual and neurological disorders.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is related to U.S. Provisional Application No. 63 / 383,979 (filed November 16, 2022, entitled "IMAGE GUIDED VARIABLE SPOT STIMULATION-BASED ELECTROPHYSIOLOGY ASSESSMENT DEVICE TO DETERMINE CHANGES IN THE FUNCTIONAL HEALTH OF BIOLOGICAL SAMPLES DURING DISEASE PROGRESSION AND TREATMENT"). This application claims the priority benefit of "Patent Citation and Discussion of the Invention," filed on October 1, 2007, entitled ...

[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 an image-guided variable spot stimulation-based electrophysiological assessment device for various biomedical applications. Specifically, the present invention relates to the application of the device to perform image-guided functional assessment based on variable spot light stimulation of photoactivatable biological samples to determine changes in functional health during disease progression and treatment. More specifically, the present invention relates to the application of the device in the diagnosis of visual and neurological disorders. [Background technology]

[0004] Retinal disorders are characterized by dysfunction / degeneration of various retinal layers and photoreceptors occurring at different rates and locations. The progress of novel therapeutic interventions, such as gene therapy and cell replacement therapy, relies on functional measurements at disease and treatment sites. However, the independent functional characterization of spatially targeted areas of the retina, such as geographic atrophy or treatment sites, is often complicated by the lack of spatial targetability and collateral hemorrhagic responses of neighboring cells. Therefore, there is a clear need for the development of high-resolution ERGs based on multicolor variable spot stimuli to identify cellular changes, which will enable critical assessment of disease progression and therapeutic interventions in the retina. Summary of the Invention

[0005] To address these challenges, the present invention provides an image-guided, variable-spot light stimulation electrophysiology device that facilitates targeted stimulation and is applicable to structural / functional imaging and assessment.

[0006] Specifically, the present invention combines 3D imaging and electrophysiology capabilities and includes an OCT-guided tunable spot-ERG platform, providing real-time imaging guidance for high-resolution biomicroscopic imaging and electrophysiological analysis for structural and functional assessment of the retina as a novel tool in the diagnosis and management of retinal diseases.

[0007] In one embodiment, the device generates optical signals and measures electrical signals generated by the retina and visual nervous system. It displays digitized ERG, visual evoked potential (VEP) signals, power spectra, and topographic maps. The spatial size of the stimulus field is predetermined by the user under guidance from OCT imaging. Flash / flicker, monochromatic or white light, or onset contrast light stimuli are presented to the desired location on the retina of the subject's eye. The evoked signals are analyzed by software algorithms through temporal filtering and artifact removal. Data is presented in numerical and graphical formats.

[0008] In certain embodiments, the present invention contemplates devices in which spatiotemporal guidance of optical stimuli to a sample is achieved by OCT and / or fundus scopy. In the case of OCT, three-dimensional imaging is achieved either by using a low-coherence broadband light source and a spectroscopic camera detector, or by using a wavelength-swept light source in combination with a photodiode detector.

[0009] In yet another embodiment, the invention encompasses OCT integration that combines stimulation beams of different wavelengths and modes of operation to control the spot size of the target stimulation, which can be varied from a diffraction-limited spot to a spot that covers the entire field of view.

[0010] In yet another embodiment, the present invention contemplates a device that integrates image-guided variable spot stimulation light with different wavelengths and an electrical signal detection system for functional assessment, including electroretinogram (ERG), visual evoked potential (VEP) and other light-activatable electrical potential changes.

[0011] In yet another embodiment, the present invention provides an OCT-guided tunable spot electrophysiology device that allows stimulation of layer-specific photoreceptors or neural retina or other light-sensitive neurons by adjusting the focal plane at the depth of interest and tuning the wavelength to the cell type of interest, and / or photobleaching other cell types with wavelength-tuned background light.

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

[0013] In yet another embodiment, the present invention provides a unique method for multiplexed measurements of different photoreceptor cells using simultaneous stimulation with multiple light beams of different colors (wavelengths) and different pulse rates (frequencies).

[0014] In yet another embodiment, the present invention contemplates space-time multiplexing for shortening measurement times during long-term measurements at multiple spots.

[0015] In yet another embodiment, the invention encompasses a time-efficient method for functional mapping of the visual field using patterned stimulation with OCT-vsERG.

[0016] In yet another embodiment, the invention encompasses a method for enabling precise magnification / reduction of an image-guided light stimulation spot through rapid and precise scanning control of deflecting mirrors and liquid lenses and positioning at selected sites on the retina.

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

[0018] In yet another embodiment, the present invention encompasses a configuration that integrates indirect fundus imaging with an OCT-guided variable spot electrophysiology system to enhance image guidance for targeted stimulation.

[0019] In broader embodiments, the present invention provides a method for assessing the efficacy of localized treatments using an image-guided tunable spot electrophysiology system with multiple stimulation wavelengths: gene replacement, optogenetic gene therapy, regenerative cell (transplant) therapy.

[0020] It is contemplated that any embodiment of a method, device, or composition described herein can be implemented with respect to any other method, device, or composition described herein.

[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, it should be understood that the detailed description and specific examples are intended to illustrate particular embodiments of the present invention and are given by way of example only. This is because, from the detailed description herein, it will become apparent to those skilled in the art that various changes and modifications can be made within the spirit and scope of the present invention.

[0023] The following drawings are for purposes of illustration and not limitation. For purposes of brevity and clarity, every feature of a given structure is not always labeled in every figure in which that structure appears. The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of the embodiments of the specification set forth herein. [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 is associated with loss of photoreceptors or the retinal pigment epithelium (RPE), which are involved in visual transmission. 2,3,4. is becoming the leading cause of blindness among working-age people in developed countries. A significant proportion of this population suffers from retinitis pigmentosa (RP). 5 and then Stargardt disease 6,7These degenerative diseases are characterized by the degeneration of different types of photoreceptor cells, resulting in retinal dysfunction. The dysfunction or death of photoreceptor cells leads to the loss of signals that initiate visual perception. 8 In cone-rod dystrophies, rod loss precedes cone loss, whereas in cone-rod dystrophies, cone loss precedes rod loss. With the advent of preventative treatments, early diagnosis of these diseases can help slow progression. These diseases have highly variable spatial patterns of degeneration (e.g., central vision in AMD, peripheral vision in RP), and the degree of visual loss varies with age. 9 Because retinal disease progresses at different rates with different stages of progression, measuring the structural and functional integrity of the retina with spatial resolution can better guide the various therapeutic interventions that will become available in the future.

[0026] Evoked potentials are used to measure electrical activity at specific locations in a biological specimen. Stimulation of specific sensory nerve pathways is required to generate electrical activity in the biological specimen of interest. While functional responses of stimulated biological tissue to electrophysiological signals have been measured before, spatially targeted, variable spot stimulation of localized locations with high resolution has not been achieved prior to the present invention. Spatially targeted stimulation is difficult without image guidance and a stimulation device aligned with the image-guided modality.

[0027] The most widely accepted method for assessing retinal function is electroretinography (ERG). ERG measures the electrical responses of various cell types within the retina, including photoreceptors, inner retinal cells, and retinal ganglion cells. ERG measurements can be performed using a variety of stimulation modes, including global stimulation, multifocal stimulation, flicker stimulation, and pattern stimulation, to access selective functional information of the retina.

[0028] Full-field flash electroretinogram (ERG) allows for measurement of the function of the entire retina. 10However, this technique may not be able to detect retinal abnormalities in the early stages of the disease. 11,12 However, the multifocal ERG (mfERG) technique, which measures retinal activity topographically, does not provide functional information with the resolution necessary to assess local atrophy or treatment effects. 13 Even in mfERG, it is not possible to separate localized regions of function with a sufficient signal-to-noise ratio to distinguish between the health states of different photoreceptors. Therefore, the use of different ERG protocols for rods and cones has not been achieved with mfERG. Furthermore, conventional mfERG illumination uses white light, which makes it impossible to distinguish between different cone functions.

[0029] Furthermore, accurate diagnosis requires co-registered structural and functional measurements. To address this challenge, we developed an ERG system based on multicolor variable spot light stimulation guided by optical coherence tomography (OCT) and / or fundus imaging to discern cellular function with high resolution. This allows for spatially resolved, isolated measurements of retinal function while minimizing responses from non-target areas, enabling accurate assessment of disease progression and the effectiveness of therapeutic interventions.

[0030] In one embodiment, the present invention provides a device that integrates an OCT-guided stimulation light beam with an electrophysiology system that can change wavelength and operation mode to control the target stimulation spot size, where the spot size can be tunable from a diffraction-limited spot size to a spot that covers the entire field of view.

[0031] In yet another embodiment, the present invention contemplates a biomicroscopic imaging and electrodiagnostic device incorporating a fundoscope and bioelectrical signal detection hardware for structural and functional assessment of the retina as an aid in the diagnosis and management of retinal diseases.

[0032] In another embodiment, the invention encompasses an apparatus in which the spatiotemporal guidance of optical stimulation of a sample is provided by OCT and / or a fundoscope. In the case of OCT, the three-dimensional image is generated using a low-coherence broadband light source in combination with a spectroscopic camera detector, or a wavelength-swept light source in combination with a photodiode detector.

[0033] In yet another embodiment, the invention encompasses a method for precisely scaling an image-guided light stimulation spot by controlling the high-speed and precise scanning of deflection mirrors and liquid lenses and positioning them at selected locations on the retina. Image-guided variable-spot ERG (vsERG) systems can measure signals in a controlled manner (position, size, wavelength, intensity, duration) from small, diffraction-limited spot sizes to large spot sizes covering the entire visual field.

[0034] In yet another embodiment, the present invention provides an OCT-guided variable spot ERG system with multiple stimulation wavelengths, allowing for assessment of different photoreceptor functions. The OCT-guided variable spot ERG system is capable of multicolor stimulation and can acquire OCT B-scans and en face images to select the region of interest and position the stimulation spot.

[0035] In yet another embodiment, the present invention provides a time-efficient method for functional mapping of the visual field using patterned stimulation with OCT-vsERG, allowing real-time structural and functional assessment to facilitate spatially controlled retinal therapeutic decision-making.

[0036] In yet another embodiment, the present invention encompasses a unique method for multiplexed measurements of different photoreceptor cells using simultaneous stimulation with multiple light beams of different colors (wavelengths) and different pulse rates (frequencies). For fast electrophysiological responses, such as cone responses to flicker stimuli, multiple stimuli targeting different wavelength-dependent photosensitive cells are achieved through frequency multiplexing. Because various photosensitive cells have distinct and separate absorption peaks, multiple stimuli of different wavelengths can be combined with unique stimulation frequencies to extract the photoresponses of different types of photoactivated cells within a common stimulation region in a single measurement. Stimulated cell types respond only to light flicker that matches their own absorption peaks, and the combined responses from individual cells can be deconvolved by averaging the acquired electrophysiological signals against the corresponding color stimulus timestamps. In addition to temporal averaging, frequency filtering can be used to apply notch and bandpass filters to further reduce contributions from other frequency responses.

[0037] In yet another embodiment, the present invention contemplates spatiotemporal multiplexing to reduce measurement time for long-term measurements at multiple spots. When electrophysiological responses are orders of magnitude longer than the stimulus duration and require a buffer period between subsequent stimuli, the present invention encompasses a method for stimulating multiple non-overlapping sites using short bursts of stimulation to improve the signal-to-noise ratio of the averaged signal. Rather than stimulating one location and waiting for full recovery at the stimulated site, the method immediately stimulates the next region. By stimulating a significant percentage of the total number of targeted spots in each burst stimulation window, the measured electrophysiological signal resulting from a single burst of stimulation comprises a linear combination of response signals from multiple regions. While there is an inherent time delay between individual stimuli, the stimulation order among multiple targeted spots can be varied because the locations and times of stimulation are known and synchronized. The average signal from each spot location can be obtained by solving a linear equation based on the locations and times of stimulation.

[0038] In yet another embodiment, the device: i) an image-guided photostimulation beam that provides a variable spot size at a sample for electrophysiological measurements of said sample; ii) an image-guided photostimulation beam assembly; The image-guided photostimulating beam assembly includes: an imaging subassembly for illuminating and collecting back-reflected light from the sample for imaging, the imaging subassembly comprising near-infrared (NIR) light from a low-coherence light source, the near-infrared (NIR) light being capable of being split into a sample beam and a reference beam for interference detection to obtain a depth-resolved image; a light stimulation subassembly comprising light beams of different wavelengths, the light stimulation subassembly having controllable intensity and / or pulse rate; iii) For each optical stimulation beam wavelength, the power of the stimulation beam at the sample plane is 0.01 to 50 cd.s / m 2 is in the range of iv) the photostimulation beam can be combined with the sample beam and directed towards the sample; v) the sample is selected from in vitro or in vivo neurons or photosensitive cells; vi) a region of interest for electrophysiological measurements on the sample in response to a variable spot stimulus is preselected and controlled by a scanning mirror that deflects the stimulus beam; vii) the variable spot size is generated by a dynamic focusing element (such as a liquid lens), e.g., from a diffraction-limited spot to the full field size; viii) the light stimulus is capable of being targeted to a preselected region of interest on the sample; ix) the optical stimulation beam can be switched off when preselecting a region of interest for optical stimulation, and the preselection of the region of interest is based on morphological / tomographic imaging; x) for morphological / tomographic imaging, the sample beam can be transmitted through an optical fiber or free space, collimated, and deflected onto the sample by the scanning mirror and optical components; xi) the optical component is coated with an anti-reflective material to avoid scattering and multiple reflections; xii) a back-reflected sample beam for morphological / tomographic imaging from said sample can be routed back to a detector; xiii) a tomographic image can be reconstructed by recording and analyzing the interference of the back-reflected sample beam and the reference beam; xiv) the morphological / tomographic images can be marked with selected areas of variable size to correspond to light stimulation spots for electrophysiological measurements; xv) Electrophysiological measurements can be performed using electrodes connected to biosensing hardware.

[0039] Optionally, the image-guided optical stimulation beam for electrophysiological measurements that achieves a variable spot size on the sample can be a tabletop system or a wearable system, and optionally, the wearable system for electrophysiological measurements can include goggles or glasses. Optionally, the wearable system for electrophysiological measurements can consist of goggles or glasses.

[0040] According to yet another embodiment, there is provided a method for performing rapid temporally and spatially multiplexed variable spot light stimulation electrophysiological measurements, which allows for improved signal to noise ratio and reduced measurement time through averaging by: 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) measuring the electrophysiological signals resulting from single stimulus bursts to various spots at different locations; iv) Deconvolving the signal responses from multiple regions to obtain an average signal for each spot location.

[0041] In yet another embodiment, a method is contemplated in which stimulation frequency multiplexing measurements, utilized to simultaneously stimulate and record multiple photoresponsive cell types with different absorption spectra at the same stimulation location, are performed by: i) combining multiple wavelength stimulation beams modulated at different frequencies; ii) stimulating different photosensitive cells with different absorption peaks and separated; iii) extracting the light responses of different types of light-activated cells within a common stimulation site in a single measurement; iv) deconvolving mixed responses from individual cells by averaging the acquired electrophysiological signals based on the timestamps of the corresponding color stimuli; and / or v) Reducing the contribution of frequency / wavelength dependent responses of other cell types by notch frequency filtering.

[0042] According to yet another embodiment, the present invention provides that local disease progression or treatment effect is measured using an image-guided tunable spot electrophysiology system with multiple stimulation wavelengths and identified by: i) comparing baseline measurements of healthy / normal and abnormal regions with the same stimulation parameters; ii) comparing spatially localized electrophysiological measurements with measurements at the same location at a previous time point; iii) Identifying functional changes in various light-sensitive cell types by matching stimulation wavelength and / or stimulation intensity; iv) mapping the electrophysiological function of the visual field with different stimulation patterns, such as peripheral and array stimulation patterns; v) To determine local dystrophic progression or therapeutic improvement by administering a series of concentrically expanding stimuli to assess gradients of electrophysiological changes in local disease areas.

[0043] In another embodiment, the present invention contemplates a device that integrates OCT-induced stimulation light with an electrophysiology system, the electrophysiology system consisting of graphical user interface (GUI) software that provides a platform for user interaction, allowing the user to control the desired stimulation location within the field of view of the en-face image.

[0044] In yet another embodiment, the invention encompasses a device comprising an OCT-guided variable spot electrophysiology system comprising graphical user interface (GUI) software that allows a user to initiate an electroretinogram (ERG) upon selection of any stimulation location on an en face image and presents the user with a list of stimulation and recording parameters.

[0045] In another embodiment, the present invention contemplates a device comprising an OCT-guided variable spot electrophysiology system, which includes graphical user interface (GUI) software that guides a user through the initiation of the image / signal recording process after certain parameters have been selected, such as stimulation power, duration, interstimulus interval, number of stimuli, etc. The image / signal recording process is automatic after the initiation phase.

[0046] In another embodiment, the invention encompasses a device for use in a method, wherein the OCT-guided stimulation light integrated electrophysiology system with different wavelengths and modes of operation is capable of selectively stimulating rod or cone photoreceptors to generate functional assessments from dry AMD, retinitis pigmentosa, or cone-rod dystrophy models.

[0047] In another embodiment, the present invention contemplates a wearable or portable benchtop OCT-guided tunable spot electrophysiology system with different wavelengths and modes of operation capable of selectively stimulating rod or cone photoreceptors to perform model-based functional assessment of dry AMD, retinitis pigmentosa, or cone-rod dystrophy.

[0048] In another embodiment, the present invention contemplates a wearable OCT-guided variable spot electrophysiology system, where the wearable portion comprises, but is not limited to, goggles, glasses, and the like.

[0049] In another embodiment, the present invention provides for the evaluation of localized therapeutic efficacy using an OCT-guided tunable spot ERG system with multiple stimulating wavelengths. 14 , optogenetics and gene therapy 15 , and regenerative cell (transplant) therapy 16 Optogenetics-treated atrophic retinal regions show promise for restoring vision loss. Importantly, accurate measurement of visual function allows for quantitative assessment of retinal function recovery after treatment. This provides further insight into the integration efficiency of gene transfer or transplanted cells that enable vision restoration. The use of an OCT-guided variable-spot ERG system has enabled measurement of improved visual responses in optogenetically treated atrophic retinal regions lacking the outer retina.

[0050] The present disclosure will now be described in detail with reference to the accompanying drawings, in which several exemplary embodiments of the present invention are shown. However, the present invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the present invention to those skilled in the art.

[0051] In the claims and / or specification, when used in conjunction with the word "comprising," the words "a" or "an" may mean "one," but are also consistent with the meanings of "one or more," "at least one," and "one or more."

[0052] Use of the term "or" in the claims is intended to mean "and / or" unless expressly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the present disclosure supports both definitions of alternatives only and "and / or."

[0053] The terms "a" and "an" are defined to mean "one or more," unless the present disclosure expressly requires otherwise. The term "substantially," as one of ordinary skill in the art will understand, refers to approximately, but not necessarily entirely, the specified content (but includes the specified content; e.g., "substantially 90 degrees" includes 90 degrees, and "substantially parallel" includes parallel). In any disclosed embodiment, the terms "substantially," "approximately," and "about" can be substituted for "within [a percentage]" of the specified content, including 0.1%, 1%, 5%, and 10%.

[0054] Throughout this application, the term "about" is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0055] Furthermore, a device or method configured in a particular manner may be configured in at least that manner, but may also be configured in a manner other than that specifically described, even if the device is capable of or configured to perform the functions described herein, and such implications are also encompassed by the disclosure herein.

[0056] "Comprises" (and all forms of "comprises", e.g., "comprises", "comprising", etc.), "has" (and all forms of "have", e.g., "have", "having", etc.), "encompasses" (and all forms of "encompasses", e.g., "encompass", "encompassing", etc.), and "contains" (and all forms of "contains", e.g., "contains", "containing", etc.) are open-ended linking verbs. Consequently, an apparatus that "includes", "has", "includes", or "contains" one or more elements has those one or more elements, but is not limited to having only those one or more elements. Similarly, a method that "comprises", "has", "encompasses", or "contains" one or more steps has those one or more steps, but is not limited to having only those one or more steps.

[0057] As used in this specification and claims, the words "comprising" (and all forms of "comprising", e.g., "comprise", "comprises", etc.), "having" (and all forms of "having", e.g., "have", "has", etc.), "including" (and all forms of "comprising", e.g., "includes", "include", etc.) or "containing" (and all forms of "containing", e.g., "contains", "contain", etc.) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0058] Any embodiment of any of the apparatus, devices, systems, and methods may "consist of" or "consist essentially of" any of the recited steps, elements, and / or features, rather than "comprising / including / containing / having." Thus, in any claim, the terms "consisting of" or "consisting essentially of" may be used in place of any of these open-ended linking verbs to define the scope of the claim differently than would be the case if the open-ended linking verbs above were used.

[0059] Features of one embodiment may be applied to other embodiments even if not described or illustrated, unless expressly prohibited by the nature of this disclosure or the embodiment.

[0060] To the extent that any specific disclosure in the above-cited or other documents may anticipate any of the general aspects of the present invention, the present disclosure should be understood as including provisos to exclude or disclaim such previously disclosed species, and aspects of the present invention not anticipated by such document disclosures are not obvious from the disclosure of those documents due, at least in part, to the unexpected and superior results disclosed or claimed herein.

[0061] The presently disclosed invention is further described below by way of examples, which are provided for illustrative purposes only and therefore should not be construed as limiting the scope of the invention. [Example]

[0062] We have developed an image-guided variable spot stimulation-based electrophysiological evaluation device for various biomedical applications. Under image guidance, this device can perform variable spot light stimulation-based functional evaluation of photoactivated biological specimens.

[0063] Example 1: Figure 1A shows the configuration 1 of the OCT-guided variable spot stimulation and electrophysiology system. Fiber-coupled visible light sources (1001, 1002, 1003) were combined using Beam Combiner 1 (1004). The light emitted from these sources was used for targeted stimulation of biological tissue. Low-coherence light from a near-infrared imaging light source (1005) with a central wavelength of 860 nm and a bandwidth of approximately 100 nm was combined with the visible light beam using a second Beam Combiner 2 (1006). A laser isolator (1007) was used to block back-reflected visible laser beams returning to the imaging light source. A fiber-optic 50 / 50 beam splitter (1008) was used to split the imaging light beam into two arms. The beam in the sample arm was guided and scanned using Mirror 1 (1009) and XY MEMS Mirror 2 (1010). A dynamic focusing element (1011, liquid lens) was used in combination with a collecting lens assembly (1012) for imaging and focusing the visible laser beam onto the sample. A second beam from a fiber optic beam splitter (1008) served as an adjustable reference arm (1013) consisting of a collimating lens and mirrors, back-reflecting the reference beam back to the beam splitter (1008). Back-scattered light from the sample was routed through the collecting lens assembly (1012), the dynamic focusing element (1012), XY MEMS mirror 2 (1010), and mirror 1 (1009) to the fiber optic beam splitter (1008), where the back-scattered light from the sample was split and received by a detector (spectrometer) (1014). The back-reflected reference beam after splitting by the fiber optic beam splitter (1008) was also received by the 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 was detected by a spectroscopic CCD detector as a function of wavelength. The detected signal (as a function of wavelength) was plotted as a function of wavenumber and Fourier transformed to obtain optical coherence tomography (OCT) information, i.e., the intensity profile as a function of depth.To simplify operation and allow real-time adjustment of OCT imaging position and acquisition, 3D rendering was performed on a software platform.

[0064] To perform variable-spot target stimulation and electrophysiological measurements, user-friendly GUI software was used to control the visible light stimulation beam, NIR imaging beam, and OCT sensor, acquire imaging / electrophysiological measurements, and then process and display the results. Once the measurement spot was identified on the image, a dynamic focusing element (1011, liquid lens) was used to control the laser stimulation spot size at each measurement location. The software control panel was used to change the power and exposure time (scan speed) of the visible laser beam and OCT imaging beam. A computer (1016) with a microcontroller (1015) and display (1017) was used to control and automate the visible light source (1001, 1002, 1003), low-coherence imaging light source (1005), XY MEMS mirrors 1 and 2 (1009, 1010), adjustable path length reference arm (1013), and detector (1014). Electrodes (1018) were used on the samples (eye, brain, retina explants) to measure the electrophysiological activity of the tissue while performing 3D image-guided laser stimulation.

[0065] This integrated device enabled a simple, multimodal workflow for electroretinogram (ERG) and visual evoked potential (VEP) measurements based on OCT-guided variable spot laser stimulation, using electrodes placed in the brain or on the visual cortex. OCT offers good deep penetration and excellent depth resolution, and the integration of OCT and ERG enabled spatially targeted local stimulation with depth resolution. ERG measurements provided electrical responses of various cell types within the retina, including photoreceptors, inner retinal cells, and retinal ganglion cells. Different ERG measurements included various laser mode stimulations, including global, focal, flicker, and pattern stimulation, to obtain selective functional information of the retina. The phase of the interference signal was measured to measure label-free photoelectrophysiology. Easily interchangeable imaging lenses enabled imaging and illumination of a wide range of species, from small animals (mice and rats) to large animals (rabbits and pigs / NHPs) and humans. Meanwhile, by irradiating the retina with a focused polychromatic laser beam, responses evoked in different retinal layers in multiple regions of the retina were measured, allowing rapid identification of which areas of the retina exhibited abnormal functional responses and precise guidance of focal stimulation to specific points.

[0066] Example 2: Figure 1B shows Configuration 2 of the OCT-guided variable spot stimulation and electrophysiology system. Visible light sources (1001, 1002, and 1003) were combined in free space using Beam Combiner 1 (1004) and directed by beam steering optics (1005, XY MEMS mirror) to share the same optical path as the imaging beam. The light emitted from these sources was used for targeted stimulation of biological tissue. Low-coherence light from a near-infrared imaging source (1007) with a central wavelength of 860 nm and a bandwidth of approximately 100 nm passed through a laser isolator (1008) to block back-reflected light toward the imaging source, and then split into a sample beam and a reference beam by a fiber-optic 50 / 50 beam splitter (1009). The imaging beam in the sample arm was guided and scanned by mirror 1 (1010) and XY MEMS mirror 2 (1011), and then combined with the visible stimulus beam in a dichroic beam combiner (1006). A dynamic focusing element (1012, liquid lens) and a focusing lens assembly (1013) were used in combination to focus the imaging beam and the visible laser beam onto the sample. A second beam from a fiber optic beam splitter (1009) served as an adjustable reference arm (1014), consisting of a collimating lens and mirrors, which reflected the reference beam back to the beam splitter (1009). The backscattered light from the sample was routed through a focusing lens assembly (1013), a dynamic focusing element (1012), a dichroic beam combiner (1006), an XY MEMS mirror 2 (1011), and a mirror 1 (1010) to a fiber optic beam splitter (1009), where the backscattered light from the sample was split and received by a detector (spectrometer) (1014). The back-reflected reference beam after being split by the fiber optic beam splitter (1009) was also received by a detector (1014) equipped with an optical camera. The interference signal between the back-reflected signal from the reference mirror and the backscattered light signal from the sample was detected by a spectroscopic CCD detector as a function of wavelength.The detected signal (as a function of wavelength) was plotted as a function of wavenumber and then Fourier transformed to obtain optical coherence tomography (OCT) information, i.e., the intensity profile as a function of depth. To simplify operation and allow real-time adjustment of OCT imaging position and acquisition, 3D rendering was performed on a software platform.

[0067] To perform variable-spot target stimulation and electrophysiological measurements, user-friendly GUI software was used to control the visible light stimulation beam, NIR imaging beam, and OCT sensor, acquire imaging / electrophysiological measurements, and process and display the results. A dynamic focusing lens was used to maintain focus at multiple depths during imaging. After 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 allowed for precise location based on the 3D-rendered image and the selected stimulation area. The software control panel was used to change the stimulation intensity and stimulation repetition rate of the visible laser beam and OCT imaging beam. A microcontroller (1016) was used to synchronize the high-speed scanning, dynamic focusing, and acquisition hardware. A computer (1017) with a display (1018) communicates with the Arduino to control and automate the visible light sources (1001, 1002, 1003), the low-coherence imaging light source (1007), the XY MEMS mirrors for the visible light beam and the imaging beam (1005, 1011), the reference arm with adjustable optical path length (1014), and the detector (1015). Electrodes (1019) were used in samples (eye, brain, and retinal explants) to measure the electrophysiological activity of the tissue during 3D image-guided electrophysiological stimulation.

[0068] This integrated device allows for a convenient measurement workflow of electroretinogram (ERG, with electrodes placed on the cornea) and visual evoked potential (VEP, with electrodes placed on the brain or visual cortex) based on OCT-guided variable spot laser stimulation, all in one multimodal platform setup. OCT provides good deep penetration and excellent depth resolution, and the integration of OCT and ERG enables spatially targeted local stimulation with depth resolution. ERG measurements provided electrical responses of various cell types within the retina, including photoreceptors, inner retinal cells, and retinal ganglion cells. Different ERG measurements were performed using various laser modes, including global, focal, flicker, and pattern stimulation, to obtain selective functional information of the retina. The phase of the interference signal was measured to measure label-free photoelectrophysiology. Easily interchangeable imaging lenses (1013) enable imaging and illumination of a wide range of species, from small animals (mice and rats) to large animals (rabbits and pigs / NHPs) and humans. Meanwhile, by irradiating the retina with a focused polychromatic laser beam, responses evoked in different retinal layers in multiple regions of the retina were measured, allowing rapid identification of which areas of the retina exhibited abnormal functional responses and precise guidance of focal stimulation to specific points.

[0069] Example 3: Figure 2A shows Configuration 3 of an OCT and fundus image-guided variable spot stimulation and electrophysiology system. Most elements of this configuration are identical to Configuration 1 of Example 1, except for the addition of additional components to achieve fundus imaging in addition to the OCT image-guided variable spot electrophysiology system. In addition to components 1001-1018 described in Example 1, a second imaging light source (1019) is also included. The illumination light is then focused (1020), passes through a semi-reflecting mirror (1020), and is then reflected by a multi-wavelength optical bandpass filter (1024) toward the sample, illuminating it. The light reflected from the fundus illumination light source then passes through a bandpass filter, passes through the semi-reflecting mirror (1020), and is then focused (1022) onto a camera (1023). While fundus imaging does not offer the depth penetration capabilities of OCT, this configuration allows for live fundus imaging to enhance image guidance without interfering with the OCT imaging and electrophysiology functions of the system. This system can detect fluorescent signals from a sample by using a visible laser as the illumination light source (1001-1003) instead of the second imaging light source (1019). Point-by-point scanning and excitation at selective wavelengths (blue, green, red) are realized by controlling the XY scanning mirrors (1009, 1010). Red-shifted or blue-shifted signals with a bandwidth wider than that of the excitation source (narrow band 1001-1003) are reflected by a multi-wavelength bandpass filter and acquired by a fundus camera (1023).

[0070] Example 4: Figure 2B shows Configuration 4 of the fundus image-guided variable-spot stimulation and electrophysiology system. This configuration simplifies variable-spot electrophysiology by abandoning depth-resolved image guidance and instead guiding localized stimulation of samples (eye, brain, retinal explant, or other light-activatable tissue specimens) through funduscopic imaging. Visible light sources (1001, 1002, 1003) were combined using a beam combiner-1 (1004). The light emitted from these sources was used for targeted stimulation of biological tissue. A dynamic focusing element (1005, liquid lens) was used in combination with a steering mirror (1006, MEMS mirror) to change the size of the stimulation spot within the imaging field of view and enable precise location targeting. The visible laser beam was then directed toward the imaging patch by a dichromic mirror (1007). The fundus imaging light source (1008) is collimated by the collimation optics (1009), reflected by the semi-reflecting mirror (1010) towards the sample, and then focused onto the sample by the focusing optics (1011). The light reflected from the sample then passes through the focusing optics (1011), the semi-reflecting mirror (1010), the dichromic mirror (1017), and the focusing optics (1012) before being detected by the fundus camera (1013).

[0071] Example 5: Figure 2C shows Configuration 5 of the OCT and fundus image-guided variable spot stimulation and electrophysiology system. Most elements of this configuration are identical to Configuration 3 of Example 3, except that a second imaging light source is combined with a focusing element (1019) in a ring-shaped configuration and placed in front of the focusing lens 1012. Light reflected from the fundus is then routed by a beam splitter (1022) for imaging modality 2 before finally being focused (1020) onto a camera (1021). This configuration simplifies live funduscopic imaging while enabling OCT imaging and electrophysiological measurements.

[0072] To perform variable-spot target stimulation and electrophysiological measurements, user-friendly GUI software was used to control the intensity of the visible light stimulation beam, the fundus imaging beam, and the fundus camera exposure time, acquire imaging / electrophysiological measurements, and process and display the results. Once the measurement spot was identified on the image, a dynamic focusing element (1005, liquid lens) was used to control the size of the laser stimulation spot at each measurement location. A computer (1015) with a microcontroller (1014) and display (1016) was used to control and automate the visible light sources (1001, 1002, 1003), MEMS mirror (1006), and fundus detector (1013). Electrodes (1017) were used in samples (eye, brain, and retinal explants) to measure tissue electrophysiological activity while performing image-guided laser stimulation.

[0073] This integrated device enabled a simple workflow for electroretinography (ERG) and visual evoked potential (VEP) measurements (when electrodes are placed in the brain or visual cortex) using fundus-scope image-guided variable-spot laser stimulation in a single multimodal platform setup. ERG measurements provided electrical responses of various cell types within the retina, including photoreceptors, inner retinal cells, and retinal ganglion cells. Different laser modes of stimulation, such as global, focal, flicker, and pattern stimulation, were implemented for different ERG measurements to obtain selective functional information of the retina. Easily interchangeable imaging lenses enabled imaging and illumination of a wide range of species, from small animals (mice and rats) to large animals (rabbits and pigs / NHPs) and humans. Meanwhile, illumination with a focused polychromatic laser beam measured responses evoked in different retinal layers in multiple regions of the retina, rapidly identifying which areas of the retina exhibited abnormal functional responses and precisely guiding focal stimulation to specific points.

[0074] Example 5: Figure 3A shows the chassis of the OCT-guided variable ERG system with the scanner head. Figure 3B shows a live fundus image of a rodent (rat) retina. Figure 3C shows an en face OCT image reconstructed from 3D-OCT imaging, with the marked circle indicating the diagnostic ERG stimulation site. The modality allows for localized stimulation of various sizes. Figure 3D depicts multicolor ERG functional assessment profiles showing different responses of different cones (S and M) to blue, green, and red stimuli in a wild-type mouse (C57BL / 6J). The different ERG profiles using multicolor stimuli indicate distinct composite responses from individual photoreceptors and higher-order neurons. Because mice lack L cones, the ERG response to red stimuli is minimal, and S cones show a more diffuse response (blue light stimuli) compared to M cones (green light stimuli).

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

[0076] Example 6: Figure 4 shows how a dynamic focusing element can be used to vary the size of the stimulation spot. The steering optics enable precise positioning of the stimulation spot in alignment with the image generated by OCT and / or fundus imaging. The focusing element can vary the size of the stimulation spot, from a microfocused spot to a large spot that covers the entire field of view. The imaging system's field of view ranges from small rodent systems (mice, rats) to large animal systems (rabbits, pigs, NHPs) and clinical systems. The dynamic focusing lens can achieve a fine focus at the sample plane to obtain high spatial resolution for imaging, and it can also be varied to fill the aperture of the focusing objective and stimulate the entire imaging field of view.

[0077] Example 7: The spatial resolution of the variable-spot OCT-guided ERG system was experimentally determined. Instead of the stimulating laser, a laser with the same specifications but higher power output was coupled to the variable-spot OCT-guided ERG system. The dynamic focusing element was then varied to illuminate the mouse retina with different spot sizes, causing localized tissue damage to visualize the precision and positioning of the laser stimuli. To measure functional deficits in a mouse model of dry AMD created by OCT-guided laser damage, an OCT-guided laser microirradiation integrated electrophysiology system was used, followed by integrated variable-spot ERG. Figure 5A shows an en face (OCT) image of a laser-treated retina, revealing a damaged spot in the peripheral retina. OCT-guided visual illumination was presented at various intensities separately, and activity in control and laser-damaged regions was measured. Figure 5B shows a scotopic variable-spot electroretinogram (vsERG) response, showing a typical ERG response obtained from an undamaged retinal region. Scotopic vsERG responses obtained from the laser-damaged region show a reduced ERG response, as shown in Figure 5 C. 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, we created a porcine model of geographic atrophy using laser injury. B-scan and immunostaining images showed localized outer nuclear layer damage due to laser injury, with loss of the photoreceptor and RPE layers, but no damage to the inner retina. We also performed vsERG signal analysis between normal and abnormal (laser-damaged) areas within the same retina. OCT-guided vsERG measurements in healthy porcine retinas outside the laser-damaged area showed blue-light vsERG responses, but no ERG signal response using red light stimulation (due to the absence of L-cones in pigs). However, when the retinal portion within the laser-damaged area was selected with the same stimulation parameters (stimulus spot size, wavelength, and intensity), no response was detected using either blue or red light stimulation. These experiments demonstrated that OCT-guided vsERG can not only establish structure-function relationships in partially atrophied retinas with high spatial resolution, but can also distinguish subtle changes occurring within the retina in near real time (minutes after laser injury).

[0079] Example 8: To assess disease progression or evaluate treatment outcomes, it is often necessary to measure function from specific or multiple retinal cell types. For example, in contrast to inherited retinal degenerative diseases and dry AMD, which result in photoreceptor and RPE damage, glaucoma is associated with RGC damage. Spatiotemporal control of electrophysiological assessments using OCT guidance offers a unique opportunity to assess the pathological progression of glaucoma or the development of new atrophy. Furthermore, in retinitis pigmentosa, rod loss precedes cone loss, whereas in cone-rod dystrophies, cone loss leads to rod loss. Varying the wavelength of the OCT-guided visible laser stimulation beam with a controllable spot size enabled selective functional assessment of rods and specific (S, M, L) cones. Figure 6A shows the assessment of regional function using OCT-guided variable-spot ERG measurements based on a scaled ISCEV standard protocol. The standard cone ERG protocol uses a 2 mm stimulation spot (3 cd·s / m under light adaptation). 2Figure 6B shows the color-dependent response for a 2 mm stimulus spot (3 cd·s / m under dark adaptation). 2 Figure 6C shows a standard rod and cone ERG protocol using a 2 mm stimulus spot (0.01 cd·s / m under dark adaptation). 2 Figure 6D shows the same scotopic rod ERG protocol using a 4 mm stimulus spot, demonstrating stimulus size-dependent responses.

[0080] Example 9: The OCT-guided variable spot electrophysiology system is not limited to measuring localized electroretinograms within the retina, but can also be used to measure visual evoked potentials (VEPs, when electrodes are placed in the brain / visual cortex) arising from localized stimulation. Figure 7A shows an OCT en face image showing two different stimulation locations in a rodent retina for OCT-guided variable spot VEP measurements. Figure 7B shows a VEP measured in the visual cortex using white light stimulation at location 1. Figure 7C shows a VEP measured in the visual cortex using white light stimulation at location 2. The VEP response in the optic nerve area (location 1) shows a low response. Figure 7D shows a VEP measured in the visual cortex using green light stimulation at location 2 shows a higher response compared to white light stimulation at the same stimulation intensity.

[0081] Example 10: Flicker stimulation, which uses pulse train stimuli of specific frequencies within a given time frame, allows for the combination of multiple color stimuli to measure individual color responses from the same site using a single stimulation pulse train through frequency multiplexing. Because different types of photoreceptors have different absorption profiles depending on wavelength, when different types of photoreceptors are stimulated with a combination of multicolor stimuli with different frequencies, each photoreceptor responds only to the color stimulus of the corresponding stimulation frequency. Example 8A shows a frequency-multiplexed stimulation scheme for simultaneously detecting the responses of different cone cells. The top pulse train shows the intensity profile of a blue light flicker stimulus as a function of time, while the middle pulse train shows a red light flicker stimulus at a slightly faster frequency repetition rate. t1, t2, and t3 represent the timestamps of the color 1 stimulation pulse train, and τ1, τ2, and τ3 represent the timestamps of the color 2 stimulation pulse train. The bottom pulse train shows a linear combination of the blue flicker stimulus and the red stimulus train superimposed on each other. This mixed single pulse train sequence can be used to simultaneously detect the photoreceptor responses from blue and red light stimuli. FIG. 8B illustrates a method for separating different photoreceptor cell responses based on their frequency responses using Fourier-domain frequency filtering. Synchronous averaging (1003) of the acquired filtered signals based on the timestamps (t1, t2, t3, ...) of color 1 extracts a frequency 1 signal (1004) and reduces other frequency signal components. This extracted signal represents the color- and frequency-dependent response of photoreceptor cell type 1 (1005). Similarly, synchronous averaging (1006) of the acquired filtered signals based on the timestamps (τ1, τ2, τ3, ...) of color 2 extracts a frequency 2 signal (1007) and reduces other frequency signal components. This extracted signal represents the response of photoreceptor cell type 2 (1008).

[0082] Figure 8C shows the flicker ERG response using red light and a 14 Hz stimulus, and Figure 8D shows the flicker ERG response using blue light and a 31 Hz stimulus at the same location. Figure 8E shows the flicker ERG response using a linear combination of 14 Hz red light and 31 Hz blue light when averaged over the timestamp of the red light stimulus. Figure 8F shows the flicker ERG response using a linear combination of 14 Hz red light and 31 Hz blue light when averaged over the timestamp of the blue light stimulus.

[0083] Example 11: For electrophysiological signals with time-delayed / long-lasting profiles, spatial and temporal stimulation multiplexing can be used to simultaneously stimulate multiple locations, thereby increasing the signal-to-noise ratio (SNR) of the average signal compared to sequential stimulation of one location at a time. This method is widely used in multifocal ERG to reduce total 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 multi-location stimulation, approximately half of the random locations are stimulated at a given time, and the local responses from each location can be extracted from a linear combination of the stimuli. This increases the SNR per stimulus by averaging compared to single-location stimulation.

[0084] Example 12: Figure 10A shows an integrated OCT-guided variable spot electrophysiology platform for clinical research. The PC and all hardware are housed in a compact system chassis, facilitating use in a clinic office environment. An adjustable LED light attached to the chin rest serves as a fixation target during testing. Figure 10B shows the experimental setup for the OCT-guided variable spot ERG system, with electrodes connected (signal electrode on the cornea, reference electrode under the eye, and ground electrode on the forehead). The chin rest provides patient stability, and the scanner is attached to the chin rest and features translation and rotation adjustment.

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

[0086] Example 13: Figure 11A shows an example of a clinical setup during OCT-guided variable spot ERG measurements. First, an OCT image is acquired to assess structural information with depth resolution of the retina. Based on the structural assessment, the user can graphically overlay the stimulation location and adjust the size of the stimulation area on the en face image, along with other stimulation parameters. Figure 11B shows an example of an en face OCT image. Using a 2 mm spot size stimulus, variable spot ERG profiles were measured using a standard cone ERG protocol (Figure 11C) and a 30 Hz flicker ERG protocol (Figure 11D).

[0087] Figure 11E shows an example of a clinical setup during OCT-guided variable-spot ERG measurements using wearable goggles. Similar to benchtop systems, a structural assessment of the retina is performed, followed by the user's definition of the ERG stimulation area. Image-guided ERG systems based on wearable goggles allow for dark adaptation even under room lighting, as the goggles completely block ambient light. For photoreceptive measurements, a background light is illuminated in a controlled manner within the goggles. By blocking ambient light and nearby activity, the goggle setup significantly reduces noise and helps ensure consistent measurements.

[0088] Example 14: Figure 12 shows the software interface for an OCT-guided variable spot ERG system for clinical trials. The left panel shows individual measurements using a standard cone ERG protocol, and the center panel shows the average signal. The OCT en face image is displayed at the top, with the marked black circle indicating the stimulation area. White crosshairs indicate the xy-section and are displayed on the OCT B-scan images on the right and bottom. Within the software, users can digitally control measurement parameters such as stimulus color, stimulation mode (different ISCEV protocols), and number of stimuli to average, as well as process the data and display the results. The software offers different filtering and averaging methods, and all data can be saved, loaded, and reprocessed.

[0089] Example 15: With the flexibility to precisely place electrophysiological stimuli anywhere within the imaging modality's field of view, different stimulation patterns can be used to assess the functional responses of multiple regions of interest. Figure 13A shows peripheral stimulation using a ring pattern surrounding the macular region, and Figure 13B shows a rectilinear square pattern for OCT-induced variable spot ERG measurements. Figure 13C shows a concentric circle pattern measurement scheme that allows for varying the stimulation spot size without changing the stimulus location at the same location. By measuring electrophysiological responses with different concentric circle spot sizes and subtracting the responses from the smaller area from the larger area, the peripheral response can be calculated, excluding the central response. Figure 13D shows an overlaid ERG response measurement using a peripheral ring pattern (NHP). Figure 13E shows an overlaid ERG response measurement using a concentric circle pattern (human) after analysis in software.

[0090] A 3D volumetric OCT scan can be acquired and navigated within the acquired volume to target a region of interest, while a cross-sectional OCT scan is also displayed. Once a stimulation spot is selected, the software prompts the user to select two depths for phase difference measurement. Once the two depths of interest are determined, the time-dependent static OCT signal (M-scan) begins to generate calculated phase changes, displayed in a separate window at the default B-scan frame rate. Finally, once the stimulation option is initiated with the set pulse width and repetition rate, the interactive software provides information on the stimulation status, including: (i) potential damage due to bulk heating effects; and (ii) power adjustment suggestions based on the overall phase change calculation. The user-friendly GUI software provides a platform for user interaction and allows the user to control OCT image acquisition in multiple scan modes: functional probing with A-scans, structural imaging for identifying stimulation locations with B&C-scans. The software allows the user to navigate the region of interest within the OCT scan with adjustable scan ranges and focal depths. Within the same OCT software platform, users have access to a control panel for customizing the stimulation laser, including power, pulse duration, stimulation frequency, and specific ROI selection within the OCT scan. Once the stimulation option is initiated, the user is presented with a list of parameters and guided through the delivery process. Thus, this integrated device and software provides 3D OCT image-guided microfocus laser stimulation control and temperature measurement. The software also offers easy analysis of raw M-scan data to examine functional (neuronal activity) changes by measuring the phase OCT signal online or offline.

[0091] The specification and examples provide a complete description of the structure and use of exemplary embodiments. While particular embodiments have been described in some detail or with reference to one or more individual embodiments, those skilled in the art will be able to make numerous modifications to the disclosed embodiments without departing from the scope of the present invention. Accordingly, the various exemplary embodiments of the device are not limited to the particular forms disclosed. Rather, they include all modifications and alternatives falling within the scope of the claims, and embodiments other than those shown may include some or all of the features of the depicted embodiments. For example, components may be omitted or combined into a single structure, and / or connections may be substituted. Furthermore, where appropriate, aspects of any of the above-described examples may be combined with aspects of other described examples to form further examples having similar or different characteristics and addressing the same or different problems. Similarly, it will be understood that the benefits and advantages described above may relate to one embodiment or several embodiments.

[0092] While the devices, compositions, and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that variations can be applied to the devices, compositions, and / or methods, and to the steps or sequence of the methods described herein, without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that certain agents which are chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the present invention.

[0093] Furthermore, the claims should not be construed as including means-plus-function or step-plus-function limitations unless such limitations are expressly stated in a given claim using the phrase "means for" or "step for," respectively.

[0094] References The following references, to the extent that they provide procedural or other details supplementary to those set forth above, are specifically incorporated by reference:

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Claims

1. i) an image-guided photostimulation beam that provides a variable spot size at a sample for electrophysiological measurements of said sample; ii) an image-guided photostimulation beam assembly, The image-guided photostimulating beam assembly includes: an imaging subassembly for illuminating and collecting back-reflected light from the sample for imaging, the imaging subassembly including near-infrared (NIR) light from a low-coherence light source, the near-infrared (NIR) light being capable of being split into a sample beam and a reference beam for interference detection to obtain a depth-resolved image; a light stimulation subassembly containing light beams of different wavelengths, the intensity and / or pulse rate of which can be controlled; iii) For each optical stimulation beam wavelength, the power of the stimulation beam at the sample plane is between 0.01 and 50 cd.s / m ２ is in the range of iv) the photostimulation beam can be combined with the sample beam and directed towards the sample; v) the sample is selected from in vitro or in vivo neurons or photosensitive cells; vi) a region of interest for electrophysiological measurements on the sample in response to a variable spot stimulus is preselected and 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 is capable of being targeted to a preselected region of interest on the sample; ix) the photostimulation beam can be switched off when preselecting a region of interest for photostimulation, and the preselection of the region of interest is based on morphological / tomographic imaging; x) the sample beam for morphological / tomographic imaging can be transmitted through an optical fiber or free space, collimated, and deflected onto the sample by the scanning mirror and optical components; xi) the optical component is coated with an anti-reflective material to avoid scattering and multiple reflections; xii) a back-reflected sample beam for morphological / tomographic imaging from the sample can be routed back to a detector; xiii) a tomographic image can be reconstructed by recording and analyzing the interference of the back-reflected sample beam and the reference beam; xiv) the morphological / tomographic images can be marked with selected areas of variable size to match light stimulation spots for electrophysiological measurements; xv) electrophysiological measurements can be performed by electrodes connected to biosensing hardware; xvi) said imaging-guided light stimulation beam for electrophysiological measurements, allowing for a variable spot size at the sample, can be a tabletop or wearable system; xvii) the wearable system for electrophysiological measurements comprises goggles or glasses; xviii) said device for performing spatially and temporally multiplexed variable spot light stimulation electrophysiological measurements, allowing for improved signal-to-noise ratio and reduced measurement times through averaging; (i) the device is configured to stimulate a subset of the total number of spots in the target with short bursts of stimulation; (ii) the device is configured to stimulate a different portion of a plurality of spots to decrease the time interval between stimulations; (iii) the device is configured to vary the order of synchronized stimulation among each portion of the total number of spots of interest within each stimulation burst window; (iv) the device is configured to measure an electrophysiological signal resulting from a single stimulus burst comprising a linear combination of signal responses from a total number of spots of interest; (v) the device is configured to solve a linear equation based on the location and time of stimulation to obtain an average signal from each spot.

2. 10. The device of claim 1, comprising an electrode, wherein the image-guided variable spot stimulation-electrophysiology assembly is configured to measure an electroretinogram (ERG) when the electrode is placed on the cornea of ​​a subject, or a visual evoked potential (VEP) when the electrode is placed on the brain or visual cortex of a subject.

3. 10. The device of claim 1, wherein the imaging light source is a low-coherence broadband or wavelength-swept light source for optical coherence tomography (OCT) used in combination with a spectrometer-detector or point detector for tomographic imaging.

4. The device according to claim 1 , wherein the spatiotemporal guidance of the optical stimulation of the sample is constituted by OCT and / or fundus microscopy.

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

6. The device of claim 1, wherein the multiple stimulation wavelengths of the image-guided variable spot electrophysiology system enable the function of different photoreceptor cells to be investigated.

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

8. 10. The device of claim 1, wherein the image-guided variable spot electrophysiological measurement device allows functional mapping of the visual field using pattern stimulation.

9. The device of claim 1 , comprising multiple electrode channels for simultaneously measuring electroretinograms and visual evoked potentials from a single stimulus.

10. The device of claim 1 for performing stimulus frequency multiplexed measurements and configured to simultaneously stimulate and record multiple photoresponsive cell types having different absorption spectra at the same stimulation location, comprising: i) configured to combine stimulation beams of multiple wavelengths modulated at different frequencies; ii) configured to stimulate different photosensitive cells with different absorption peaks and separated from one another; iii) configured to extract the optical responses of different types of photoactivated cells within a common stimulation site in a single measurement; iv) configured to deconvolve mixed responses from individual cells by averaging acquired electrophysiological signals based on the timestamps of corresponding color stimuli; and / or v) A device configured to reduce the contribution of frequency / wavelength dependent responses of other cell types through notch frequency filtering.

11. The device of claim 1 configured to identify and measure local disease progression or treatment effect using an image-guided tunable spot electrophysiology system with multiple stimulation wavelengths, comprising: i) configured to compare baseline measurements of healthy / normal and abnormal regions with identical stimulation parameters; ii) configured to compare spatially localized electrophysiological measurements with measurements at previous time points in the same locations of the healthy / normal and abnormal regions; iii) configured to identify functional changes in various light-sensitive cell types by matching stimulation wavelengths and / or stimulation intensities; iv) configured to map electrophysiological functions of the visual field with different stimulation patterns, including peripheral stimulation patterns and array stimulation patterns; v) configured to determine local dystrophic progression or therapeutic improvement by administering a series of concentrically expanding stimuli, making it possible to assess the gradient of electrophysiological changes in the local disease area; device.

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