Photometric system for neurophysiological measurements

Abstract

A photometric system for measuring one or more neurophysiological parameters of a target, the photometric system comprising an optical arrangement. The optical arrangement includes a first optical input for receiving input light from a light source, a first optical output for providing output light to a target, a first light path from the first optical input to the first optical output, a second optical input for receiving light from the target, a second optical output, a second light path from the second optical input to the second optical output, a single-photon avalanche detector configured to receive light from the second optical output, and a processor element configured to receive data from the detector and to carry out single-photon counting.

Description

[0001] Photometric System for Neurophysiological Measurements

[0002] FIELD OF INVENTION

[0003] The present disclosure is in the field of photometric systems for measuring one or more neurophysiological parameters of a target. The present disclosure also relates, particularly but not exclusively, to photometric systems including a single-photon avalanche detector that can measure at least one neurophysiological parameter. The present disclosure also relates generally to imaging systems.

[0004] BACKGROUND TO INVENTION

[0005] Tools for observing the activity of neuronal populations are vital for fundamental neuroscience, translational research into disease mechanisms and for drug discovery. They are also of increasing importance for assessment of epileptic seizures and for brain machine interfaces. Electrophysiology has been the gold standard for monitoring neuronal population activity due to its high temporal resolution. However, it suffers a major limitation in that it is challenging to attribute observed electrophysiological signals to particular neuronal populations.

[0006] Fibre photometry has emerged as a valuable alternative to electrophysiology for monitoring the activity of neuronal populations in vivo. This method relies on the use of optical fibres to deliver excitation light to target neurons and to collect emitted fluorescence signals. By utilising fluorescent biosensors, for example genetically encoded calcium indicators (GECIs), it allows measurements of neural activity. A key advantage of fibre photometry over electrophysiology is its ability to target specific neuronal populations. However, current fibre photometry methods are unable to match electrophysiology in their temporal resolution, substantially limiting their range of applications.

[0007] Two obstacles limit the application of fibre photometry. First, fibre photometry typically relies on and is optimised for fluorescent indicators that have slow kinetics relative to the activity they are reporting. For example, GECIs have time constants on the order of 10s of milliseconds or longer, whereas single action potentials have a duration of approximately 1 millisecond. GECIs also don’t report sub-threshold membrane potential activity. Second, the sampling rates of hardware used for fibre photometry are typically limited, resulting in a loss of temporal precision when trying to observe fast neuronal events, such as action potentials and high frequency brain oscillations. This restricts the ability to capture rapid and dynamic changes in neuronal activity.

[0008] The first limitation has recently been addressed through development of genetically encoded voltage indicators (GEVIs) that can reveal subthreshold activity and resolve spike timing with millisecond resolution from identified neuronal populations. GEVIs are suitable for monitoring both subthreshold activity and action potentials from specific cell populations. Recently developed GEVIs, such as Voltron (Abdelfattah et al., 2019), ASAP3 (Villette et al., 2019), ASAP4 (Evans et al., 2021), and Jedi-2p (Liu et al., 2022), can be targeted to specific types defined by their molecular identity or projection targets. Other recently developed fluorescent biosensors for neurotransmitters and neuromodulators such as genetically encoded glutamate sensor iGluSnFR3 (Aggarwal et al., 2023) and GABA sensor iGABASnFR (Marvin et al., 2019), also have potential applications for resolving neural dynamics with millisecond precision.

[0009] Maximally capitalising on these indicators will require advances in hardware to improve sensitivity and speed. This is challenging given that fast biosensors impose stringent requirements due to their millisecond dynamics, limited photon budget and rapid photobleaching. Current imaging techniques that can exploit the fast kinetics of GEVIs and allow for deep brain imaging require animals to be head-fixed, involve complex hardware and are difficult to apply to extended recording sessions for behavioural tasks or sleep studies. Fibre photometry approaches have been demonstrated for imaging GEVIs, but their current utility is limited to physiological events with frequencies below 100 Hz. These approaches used amplitude modulated laser beams for illumination and photodiodes for detection of the fluorescent signals, with blind source separation to disambiguate physiology from movement related signals.

[0010] It is therefore an aim of at least one embodiment of at least one aspect of the present disclosure to obviate or at least mitigate at least one of the above identified shortcomings of the prior art. SUMMARY OF INVENTION

[0011] According to a first aspect of the disclosure, there is provided a photometric system for measuring one or more neurophysiological parameters of a target, the photometric system comprising: an optical arrangement comprising: a first optical input for receiving input light from a light source; a first optical output for providing output light to a target; a first light path from the first optical input to the first optical output; a second optical input for receiving light from the target; a second optical output; a second light path from the second optical input to the second optical output; and a single-photon avalanche detector configured to receive light from the second optical output; and a processor element configured to receive data from the single-photon avalanche detector and to carry out single-photon counting.

[0012] The optical arrangement may include one or more optical conduits, which may be optical fibres.

[0013] The target may be an in vivo target or an in vitro target. The target may be a region, or internal region of tissue of a living organism, such as a human or animal, or a cultured sample. The target may be or may include a portion of tissue comprising one or more or a plurality of neurons, or population of at least 100, or at least 1 ,000, or at least 10,000 neurons. The target may be a tissue sample suitable for measuring neurophysiological activity.

[0014] The system may be configured for use with a free moving organism, such as a free moving human or animal. The system may be configured for measuring the one or more neurophysiological parameters when the target, or the organism, is freely movable or moving.

[0015] The first optical output may be configured to be engageable with the target. The first optical output may be configured to be implantable at least partially within the target. The first optical output may be configured for long-term implantation at least partially within the target. “Long-term” in this context may be at least 24 hours, optionally at least 1 week, optionally at least 4 weeks. The system may comprise a first output element. The first output element may comprise a first end and a second end. The first output element may include the first optical output, which may be at the first end thereof. The first output element may be configured to be at least partially implantable in the target. The first output element may be configured for transcutaneous implantation in the target.

[0016] The first output element may comprise a cannula. The first output element may be adapted to engage with a cannula. The cannula may be an optical fibre cannula. The cannula may be located or locatable at the first end of the first output element.

[0017] The first output element may include one or more, or a single optical conduit, which may be optical fibre element(s), to optically connect the second end thereof to the first end thereof. An optical conduit of the first output element may be connected or connectable to the cannula.

[0018] The first output element may comprise a coupling mechanism for coupling an optical conduit to the cannula. The coupling mechanism may be a sleeve, a casing, a tube member, or the like. The coupling mechanism may be engageable with and / or connectable to an optical conduit and to the cannula.

[0019] The first output element may include at least a portion of the first light path. The first output element may include at least a portion of the second light path.

[0020] The second optical input may be configured to be engageable with the target. The second optical input may be configured to be implantable at least partially within the target. The second optical input may be configured for long-term implantation at least partially within the target. “Long-term” in this context may be at least 24 hours, optionally at least 1 week, optionally at least 4 weeks.

[0021] The first output element may include the second optical input, which may be at the first end thereof.

[0022] The first optical output and the second optical input may be a common optical input / output. The first optical output and the second optical input may be integrally formed.

[0023] The first output element may comprise a single optical conduit linking the first end to the second end. The single optical conduit may be substantially devoid of any further optical components.

[0024] At least a portion of the first and second light paths may be common. The portion of the first and second light paths within the output element may be common. The first output element may be configurable for being fixed relative to the target. The first end of the first output element may be configurable in a fixed position on or at the target.

[0025] The optical arrangement may comprise a stationary part and a movable part. The movable part may be a flexible part. The movable part may include one or more flexible optical conduits.

[0026] The movable part may include the first output element. The first output element may be connected to the stationary part. The system may comprise a coupling element connecting the stationary part to the movable part.

[0027] The coupling element may be a rotary coupling, or the like. The coupling element may be connected between one or more stationary optical conduits and the second end of the first output element to permit at least partial rotation of the first output element relative to the one or more stationary optical conduits. The coupling element may be configured to permit at least partial rotation of the first output element about at least one axis of rotation.

[0028] The system may comprise, in terms of optical components, only one or more optical conduits between the coupling element and the first optical output. The system may comprise, in terms of optical components, only one or more optical conduits between the coupling element and the second optical input. The system may comprise, in terms of optical components, only one or more optical conduits between the coupling element and the cannula. The first output element may comprise, in terms of optical components, only one or more optical conduits between the first end and the second end thereof.

[0029] The single-photon avalanche detector may comprise one or more or a plurality of detector elements. The, or each detector element may be a single-photon avalanche diode (SPAD). The SPAD(s) may be configured to operate in Geiger mode.

[0030] The detector element or elements may be implemented in an integrated circuit.

[0031] The detector may comprise an array of detector elements. The array may include between 1 and n row detector elements and 1 and n’ column detector elements, n and n’ may each be between 2 and 1 ,000, optionally between 10 and 500, optionally 200 and 400. The array may be a SPAD array.

[0032] The system may be configured to operate the detector with a sampling rate of up to 20 kHz, optionally up to 15 kHz, optionally up to 12 kHz, optionally up to 11 kHz, optionally up to 10 kHz, optionally up to 5 kHz, optionally up to 3 kHz, optionally up to 2.2 kHz, optionally between 200 Hz and 20 kHz, optionally between 200 Hz and 12 kHz, optionally between 500 Hz, and 11 kHz, optionally between 800 Hz and 11 kHz, optionally between 800 Hz and 5 kHz, optionally between 200 Hz and 2.2 kHz, optionally between 400 Hz and 2.2 kHz, optionally between 500 Hz and 10 kHz, optionally between 500 Hz and 2 kHz optionally at least 100 Hz, optionally at least 200 Hz, optionally at least 500 Hz, optionally at least 1 kHz, optionally at least 2kHz, optionally at least 5 kHz.

[0033] The sampling rate may be a frame rate that includes substantially all of the detector elements.

[0034] The one or more neurophysiological parameters may include individual or population neuronal activity, which may include at least one of action potentials, subthreshold membrane potential activity, neural oscillations such as theta oscillations, sharp wave ripples (SWRs), high frequency gamma oscillations, high frequency brain oscillations, or any suitable neurophysiological parameter.

[0035] The input light may be provided from a light source. The system may comprise the light source. The light source may be or may comprise at least one light emitting diode (LED) and / or laser light source.

[0036] The light source may be configured to emit visible light. The light source may be configured to emit light at one or more wavelengths between 380 nm to 740nm, optionally between 380 nm to 435 nm, optionally between 435 nm to 500 nm, optionally between 500 nm to 600 nm, optionally between 550 nm and 740 nm.

[0037] The first light path may be a visible light path. The second light path may be a visible light path. The second light path may be configured for transmission of fluorescent light from the second optical input to the second optical output.

[0038] The first light path and / or the second light path may be configured for the transmission of light at one or more wavelengths between 380 nm to 740nm, optionally between 380 nm to 435 nm, optionally between 435 nm to 500 nm, optionally between 500 nm to 600 nm, optionally between 550 nm and 740 nm.

[0039] The system may comprise one or more first optical inputs. The system may comprise a plurality of first optical inputs each for receiving input light from a light source. The system may comprise one or more light sources. The system may comprise a plurality of light sources. The light sources may be independent light sources.

[0040] The system may comprise two or more different light sources for providing light at substantially different wavelengths to at least two of the first optical inputs.

[0041] The first light path may be configured to provide the input light from each of the plurality of first optical inputs to the first optical output. The system may be configured to provide the output light as excitation light configured to induce fluorescence in the target. The excitation light may be configured to induce fluorescence in a genetically encoded voltage indicator when present in the target.

[0042] The measurement of the one or more neurophysiological parameters may be based, at least in part, on the received light, or received fluorescence from the second optical input to the second optical output.

[0043] The system may comprise a driver element for controlling the, or each light source. The driver element may include an LED driver. The driver element may include a waveform generator, which may be operable to provide at least one of: sinusoidal, square, or any suitable waveform for driving the or each light source.

[0044] The system may be operable to perform continuous sampling of the detector. The system may be operable to perform the continuous sampling at the sampling rate.

[0045] The system may comprise a first light source for providing input light to a first optical input, and a second light source for providing input light to another first optical input. The system may be operable to modulate the first and second light sources. The modulation may be time division modulation, or frequency modulation, or any suitable modulation.

[0046] The driver element may be configured to modulate the first and second light sources.

[0047] The time division modulation may include applying a square wave from the waveform generator to each first and second light source. This may include modifying the duty cycle of the square wave drive of each light source. The duty cycle of each light source may be less than 50%, optionally less than 40%, optionally less than 35 %, optionally 30% or less. The time division modulation may activate the first light source and deactivate the second light source for a first period of time. The time division modulation may activate the second light source and deactivate the first light source for a second period of time. The time division modulation may include one or more third periods of time in which the first and second light sources are deactivated. The third time period may be between the first and second time periods. The frequency of the drive waveform of the first and second light sources may be substantially identical. The frequency may be between 100 Hz and 2 kHz, optionally between 200 Hz and 1 kHz, optionally between 300 Hz and 700 Hz, or any suitable frequency.

[0048] The frequency modulation may include applying a sinusoidal drive to the first and second light sources. The frequency modulation may include applying a sinusoidal drive having a first frequency to the first light source, and a second frequency to the second light source. The first frequency may be different to the second frequency. The first frequency and / or the second frequency may be a prime number. The first frequency may be between about 100 Hz and 2 kHz, optionally between 200 Hz and 1 kHz, optionally between 300 Hz and 700 Hz. The second frequency may be between about 100 Hz and 2 kHz, optionally between 500 Hz and 1.5 kHz, optionally between 700 Hz and 1.1 kHz.

[0049] Modulated input light may be provided to the first optical output, such that the detector receives modulated light from the target. The processor may be configured to demodulate the detector data.

[0050] The system may comprise a housing for at least some of the components thereof.

[0051] The optical arrangement may be configured to combine input sub-paths from each of the plurality of first optical inputs into a common part of the first light path. The optical arrangement may comprise one or more first optical components configured to combine the input sub-paths of the first optical inputs. The first optical components may include or may be one or more mirrors, or dichroic mirrors.

[0052] The optical arrangement may be configured to divide received light from the second optical input into output sub-paths. The division of the output-sub paths may be wavelength dependent. The optical arrangement may comprise one or more second optical components configured to divide the received light into the output sub-paths. The second optical components may include one or more mirrors, or dichroic mirrors configured to divide the received light into the output sub-paths.

[0053] The optical arrangement may comprise inputs / outputs for coupling optical conduits to the first and second optical components.

[0054] The system may comprise one or more second optical outputs or a plurality of second optical outputs, optionally two or more, three or more, four or more, eight or more, or any suitable number of second optical outputs. The optical arrangement may be configured to provide received light from the second optical input to the plurality of second optical outputs. The optical arrangement may be configured to provide the output sub-paths to the plurality of second optical outputs. The optical arrangement may be configured to provide each of the output sub-paths to at least one second optical output.

[0055] The optical arrangement may be configured to provide a different output sub-path to each second optical output. The optical arrangement may be configured to provide each output sub-path to a sub-region of the detector, or to a sub-region of detector elements, or to a sub-region of SPADs. The sub-regions may be non-overlapping or partially overlapping. The sub-regions may include one or more discrete pixels of the detector, or two or more, or three or more, or four or more, or ten or more discrete pixels, optionally between 1 and 1 ,000 pixels, optionally between 1 and 100 pixels, between 1 and 10 pixels, optionally between 1 and 5 pixels, optionally between 1 and 4 pixels. The, or each sub-regions may be less than or equal to 50% of the detector pixels of the detector, optionally less than or equal to 40%, optionally less than or equal to 30 %, optionally less than or equal to 20%, optionally less than or equal to 10%, optionally less than or equal to 5%, optionally less than or equal to 1 %.

[0056] The optical arrangement may comprise a second output element. The second output element may include the, or each, or some of the second optical output(s). The second output element may include a first end and a second end. The first end of the second output element may include the second optical output(s).

[0057] The second end of the second output element may be optically connected to the second optical input, which may be an indirect connection. The second end of the second output element may be connected to the one or more second optical components. The second output element may include one or more optical conduits, which may be optical fibres, connecting the second optical output to the second optical component(s) or to intermediate optical conduits to the second optical component(s).

[0058] The system may be configured to maintain a fixed distance between the second optical output and the detector. The system may comprise an adapter for positioning the second optical output relative to the detector. The adapter may be configured to maintain the second optical output a fixed distance to the detector or to the detector element(s) thereof. The adapter may comprise an adjustment mechanism configured to allow the distance between the second optical output and the detector to be adjusted prior to or during use.

[0059] The optical arrangement may comprise one or more light focussing elements configured to focus light to the detector or detecting elements thereof. The light focussing element may be a collimator. The light focussing element may be located adjacent to, at, in, or integral with the adapter.

[0060] The adapter may be configured to provide a linear distance from each detector element to the second optical output or the light focussing element.

[0061] The adapter may be configured to receive an end of an optical conduit therein, optionally a fibre optic conduit therein.

[0062] The adapter may comprise a securing mechanism configured to secure the optical conduit therein. The adapter may be mounted to a support member, which may be a detector support member. The adapter may comprise a mounting mechanism for mounting to the support member.

[0063] The system may comprise a total light path from the, or each light source, to one or more first optical components, to the coupling element, to the first output element, to the first optical output, to the target, to the second optical input, to the first output element, to the coupling element, to one or more second optical components, to the second output element, to the one or more second optical outputs.

[0064] The system, in terms of optical components, and in the second light path between the coupling element and the second output element may comprise only one or more optical conduits and the second optical component(s) and optionally one or more first optical components. The system, in terms of optical components, may comprise only one or more optical conduits and the light focussing element between the first and second ends of the second output element.

[0065] The system, in terms of optical components between the second optical input and the second optical output, and in the second light path, may comprise only one or more optical conduits, the one or more second optical components, and the light focussing element, and optionally the one or more first optical components.

[0066] The system may be an optical fibre-based photometry system.

[0067] The system may comprise a user interface. The user interface may include or may be a graphical user interface. The user interface may include a display element. The processor element may be operable to display image data from the single-photon detector. The processor element may be operable to permit a user to select one or more regions of interest of the detector. The processor element may be operable to permit a user to move the region of interest to a new region of interest.

[0068] The processor element may be operable to display data from the detector, such as intensity data (image data) or temporal photon count data, in real time. The intensity data is based on photon counting. The temporal data may be photon counts over time.

[0069] The processor element may be configured to permit the user to select and / or move the region of interest in real time.

[0070] The processor element may be operable to carry out single-photon counting for the, or each detector element, or pixel of the detector. The processor element may be operable to display pixel intensity data dependent on the photon counting for the, or each pixel. The processor element may be operable to display temporal data of photon count for a pixel or for a region of interest of pixels by concatenating pixel counts for the region of interest of pixels.

[0071] The processor may be operable to display both pixel intensity data and temporal photon count data, which may be in the same pane of the user interface.

[0072] The processor may be operable to downsample the received data from the detector. This may be carried out when modulation is used or not. In this example, the downsampling may be by a factor of at least 2, or at least 5, or at least 10, or at least 20. The processor may be operable to downsample and display the photon count data from the detector in real time.

[0073] The processor element may be configured to identify defective pixel(s), which may be based, at least in part, on the average values of some or all of the pixels, or on the average values of adjacent or neighbouring pixels. The defective pixel(s) may be “hot pixels”. The processor element may be configured to remove or dynamically adjust the defective pixel(s) in the temporal pixel data and / or the intensity plots of the detector or the region(s) of interest.

[0074] According to a second aspect of the disclosure, there is provided an imaging system comprising: a first optical input for receiving input light from a light source; a first optical output for providing output light to a target; a first light path from the first optical input to the first optical output; a second optical input for receiving light from the target; a second optical output; a second light path from the second optical input to the second optical output; and a detector configured to receive light from the second optical output.

[0075] The detector may comprise one or more single-photon detectors, single-photon avalanche detectors, or any suitable detector element.

[0076] According to a third aspect of the disclosure, there is provided a method of carrying out photometry on a target, the method comprising: providing light to at least a part of the target; receiving light from at least a part of the target; and performing single-photon counting on at least a portion of the received light from the target. The above summary is intended to be merely an example and non-limiting. The disclosure includes one or more corresponding aspects, embodiments or features in isolation or in various combinations whether or not specifically stated (including claimed) in that combination or in isolation. It should be understood that features defined above in accordance with any aspect of the present disclosure or below relating to any specific embodiment of the disclosure may be utilized, either alone or in combination with any other defined feature, in any other aspect or embodiment or to form a further aspect or embodiment of the disclosure.

[0077] BRIEF DESCRIPTION OF DRAWINGS

[0078] These and other aspects of the present disclosure will now be described, by way of example only, with reference to the accompanying drawings, wherein:

[0079] Figure 1 Shows a photometric system in accordance with embodiments of the invention;

[0080] Figure 2 Is a schematic of a single-photon avalanche diode (SPAD) pixel of the system of Figure 1 ;

[0081] Figure 3 Depicts a SPAD array of the system of Figure 1 ;

[0082] Figure 4 Shows two configurations of the light path design;

[0083] Figure 5 Illustrates the detector of Figure 1 in more detail;

[0084] Figure 6 Shows the user interface of the embodiment of Figure 1 ;

[0085] Figure 7 Illustrates modulation of the input light sources of Figure 1 ;

[0086] Figure 8 Is a simulation of time-division modulated transmission and reception of light in accordance with embodiments of the invention;

[0087] Figure 9 Is a simulation of frequency modulated transmission and reception of light;

[0088] Figure 10 Shows the creation of temporal data from the SPADS, for a region of interest;

[0089] Figure 11 Shows an example of animal recording data;

[0090] Figure 12 Is a comparison of signal to noise ratios for three types of detector;

[0091] Figure 13 Shows recording data from a freely moving animal;

[0092] Figure 14 Shows another set of recording data from a freely moving animal;

[0093] Figure 15 Shows simulated sinusoidal waves tested on different detectors;

[0094] Figure 16 Shows a comparison of temporal resolution of detectors using square wave signals; and Figure 17 Shows a comparison of temporal resolution of detectors using sinusoidal wave signals.

[0095] DETAILED DESCRIPTION OF DRAWINGS

[0096] With reference to Figures 1 to 17, embodiments of a photometric system 1 for measuring one or more neurophysiological parameters of a target 2 are illustrated and described. The one or more neurophysiological parameters may include individual or population neuronal activity, which may include action potentials, sub-threshold membrane potential activity, neural oscillations such as theta oscillations, sharp wave ripples (SWRs), or high frequency gamma oscillations, high frequency brain oscillations, or any suitable neurophysiological parameter.

[0097] Turning to Figure 1 , the photometric system 1 comprises an optical arrangement 4 comprising a plurality of first optical inputs 6 for receiving input light from light sources 38a, 38b, a first optical output 10 for providing output light to a target 2, a first light path 12 from the first optical input 6 to the first optical output 10, a second optical input 14 for receiving light from the target 2, two second optical outputs 16, a second light path 18 from the second optical input 14 to the second optical output 16, a single-photon avalanche detector 20 configured to receive light from the second optical output 16, a processor element 22 configured to receive data from the detector 20 and to carry out single-photon counting.

[0098] The optical arrangement 4 includes a plurality of optical fibre conduits 24 for defining portions of the first and second light paths 12, 18. The system 1 is a fibre-optic based photometric system 1.

[0099] The system 1 can be used with in vivo or in vitro targets 2, and the target 2 may be a region, or internal region of tissue of a living organism, such as a human or animal, or a cultured sample. The target 2 will typically be a portion of tissue comprising one or more or a plurality of neurons, or population of at least 100, or at least 1 ,000, or at least 10,000 neurons. In some examples, the target 2 can be a tissue sample suitable for measuring neurophysiological activity. Other uses of the system 1 will be readily apparent to those of skill in the art.

[0100] In the embodiments of Figures. 1 to 14, the system 1 is configured for use with a free moving organism, such as a free moving human or animal. The system 1 is configured for measuring the one or more neurophysiological parameters when the target 2 is freely movable or moving. This is advantageous over photometric systems that require the target to be fixed in place, such as those that require the animal’s head to be held in a fixed position, particularly as it allows for the measurement of neurophysiological activity when the animal is carrying out various activities, such as travelling through a maze or the like.

[0101] In the embodiment shown in Figure. 1 , the system 1 is used with an animal target 2, and the first optical output 10 is configured to be engageable and implantable within the target 2, typically for a number of weeks or longer.

[0102] The system 1 comprises a first output element 26 having a first end 26a and a second end 26b. The first optical output 10 is at the first end 26a. The first output element 26 is configured for transcutaneous implantation in the target 2.

[0103] The first output element 26 is adapted to engage with an optical fibre cannula 28 at the first end 26a. Whilst it is depicted in Fig. 1 that the cannula is implanted in the brain of the animal target 2, it will be appreciated that other implantation locations are possible, or the system could be used with samples taken from the target 2 for in vitro analysis.

[0104] The first output element 26 includes a single fibre optical conduit 24 optically connecting the second end 26b (and the cannula 28) to the first end 26a. The single fibre optic conduit is substantially devoid of any further optical components, which is advantageous as the light path within this part of the system 1 can be kept as short as possible, which mitigates optical losses and is particularly important when imaging low- light producing samples. For example, when using genetically encoded voltage indicators, the sample will be excited by the light from the first optical output 10, and may fluoresce at low light levels. It is therefore advantageous to keep the optical path length short, at least of the second light path 18 to maximise the signal to noise ratio at the detector 20.

[0105] The first output element 26 comprises a coupling mechanism 30 for coupling the fibre optical conduit 24 to the cannula 28. In the embodiments illustrated here, the coupling mechanism 30 is a sleeve, but it will be understood that any suitable coupling mechanism may be used, such as a casing, a tube member, connector or the like. The coupling mechanism 30 is engageable with and connectable to the optical conduit 24 and to the cannula 28.

[0106] The first output element 26 includes portions of the first light path 12 and the second light path 18.

[0107] In the embodiments illustrated in Figs. 1 to 17, the first optical output 10 and the second optical input 14 are formed as a common optical input / output, and are integrally formed. It will be appreciated that in other embodiments, the first optical output 10 and the second optical input 14 may be separate, and it may be that there is less or no overlap between the first and second light paths 12, 18.

[0108] The first end 26a of the first output element 26 is configurable for being fixed relative to the target 2, for example by straps, headgear, or the like.

[0109] The optical arrangement 4 comprises a stationary part 32 and a flexible, movable part 34 including the flexible optical conduit 24a and the first output element 26. The first output element 26 is connected to the stationary part 32 by a rotary coupling element 36. The coupling element 36 connects a stationary optical conduit 24b and the second end 26b of the first output element 26 to permit rotation of the first output element 26 relative to the stationary optical conduits 24b about at least one axis of rotation to permit movement of the animal.

[0110] In the embodiments shown in Figure. 1 , the system 1 comprises, in terms of optical components, only a single fibre optical conduit 24a between the coupling element 36 and the first optical output 10 and the second optical input 14. The system 1 comprises, in terms of optical components, only a single fibre optical conduit 24a between the coupling element 36 and the cannula 28.

[0111] As shown in Figure 3, the single-photon detector 20 comprises a 320 x 240 array of detector elements, which in this embodiment are single-photon avalanche diodes (SPAD) configured to operate in Geiger mode. The SPADS are implemented in an integrated circuit. For brevity, a detailed description of the operating principle of SPADs will not be provided, however, Figure 2 shows a schematic of a SPAD pixel and accompanying waveform diagrams illustrating the known photon-counting principle of operation.

[0112] The advantage of SPADs is that they detect the exact time of arrival of photons on the detector 20, enabling high frame rates, while having exceptionally low background noise levels, enabling excellent signal to noise ratios to be achieved. SPADs can be used to image individual neurons in ex vivo preparations.

[0113] The system 1 is configured to operate the detector 20 at a sampling rate of up to 10kHz when continuous sampling is used. The sampling rate is the rate of sampling one frame, and is the number of times the system 1 obtains a photon-count of all of the pixels in the detector 20, per second. In other examples, such as when modulation of the input light is used, the sampling rate may be different to the continuous mode, which will be described later.

[0114] As shown in Figures 1 and 4, the system 1 comprises a first light source 38a and a second light source 38b for providing input light to the first optical inputs 6, which in this embodiment are light emitting diodes (LEDs) configured to emit visible light. The light sources 38a, 38b are independent and different.

[0115] In the example of Figure 1 and Figure 4 (a), the first light source 38a has a peak wavelength transmission at 465 nm and the second light source 38b has a peak wavelength transmission at 560nm. In the example of Figure 4 (b), the first light source 38a emits at 465nm and the second light source at 405 nm (peak wavelengths). Any suitable light sources for providing input light can used, and each light source may be configured to emit light at one or more wavelengths between 380 nm to 740nm, optionally between 380 nm to 435 nm, optionally between 435 nm to 500 nm, optionally between 500 nm to 600 nm, optionally between 550 nm and 740 nm. It will also be understood that in some embodiments, only one or more than two light sources may be employed.

[0116] In a typical use of the system, the first light path 12 is for transmitting visible light to the target 2 and the second light path 18 is for the transmission of fluorescence from the target 2 to the second optical output 16.

[0117] The first light path 12 and the second light path 18 are configured for the transmission of light at least at wavelengths between 380 nm to 740nm, but this is purely illustrative and in other embodiments, the first and second light paths 12, 18 could be configured for the transmission of other wavelength ranges or values. Likewise, whilst the embodiments use visible light and fluorescence, in other embodiments other regions of the electromagnetic spectrum may be employed.

[0118] Figures 1 and 4 (a) depict emission from the target 2 at 525nm and 600 nm from the excitation of the 465nm and 560nm light sources respectively, and Figure 4 (b) depicts emission at 525 nm from the light sources. This is purely an example, and the embodiments are compatible with other emission wavelengths, or received wavelengths of light.

[0119] In the embodiments illustrated and described here, the system 1 comprises two different light sources 38a, 38b for providing light at substantially different wavelengths to the two first optical inputs 6. This is useful as one of the first optical inputs 6 can be used to provide a reference excitation light, which does not excite the marker used for neuronal activity (e.g. a genetically induced voltage indicator (GEVI)), and one of the first optical inputs 6 can be used to provide excitation light that will activate the marker (e.g. the GEVI). In a free moving organism, this helps to identify and remove artefacts from the detector data that are attributable to things like movement of the animal. It will be appreciated that in some embodiments the use of a reference channel is unnecessary. Furthermore, it may be desirable to excite different types of markers or indicators, such as GEVIs and genetically encoded calcium indicators (GECI), or other markers, and the system 1 can have any number of light sources and corresponding first optical inputs 6 for this purpose.

[0120] The first light path 12 is configured to provide the input light from each of the plurality of first optical inputs 6 to the first optical output 10.

[0121] Depending on how the system 1 is to be used, the system 1 is configured to provide the output light from the first optical output 10 as excitation light configured to induce fluorescence in the target 2, which will depend on which biomarker / indicator is used.

[0122] In the embodiments illustrated and described here, the measurement of the one or more neurophysiological parameters is based, at least in part, on the received fluorescence from the second optical input 14 provide to the second optical output 16.

[0123] As shown in Figures 1 and 4, the system 1 comprises a driver element including a waveform generator 40 and an LED driver 42 for controlling each light source 38a, 38b. In the embodiments shown here, the waveform generator 40 is operable to provide a sinusoidal or square waveform to the LED driver 42. These are examples, and it will be understood that any suitable waveform for driving the light sources 38a, 38b can be used.

[0124] The system 1 is operable to perform continuous sampling of the detector 20. Typically, this involves continuous transmission from one of the LEDs 38a or 38b, and continuous sampling of the detector 20 at the sampling rate for continuous mode (up to 10 kHz). In another mode of operation, the system 1 is operable to modulate the first and second light sources 38a, 38b by time division modulation or frequency modulation. These are examples, and any suitable modulation technique can be used.

[0125] The driver element is configured to modulate the first and second light sources 38a, 38b.

[0126] The time division modulation is shown in Figures 7 (a) and 8, and includes applying a square wave from the waveform generator 40 to the first and second light sources 38a, 38b. The square waves each have 30% duty cycle, and are 180 degrees out of phase, so that in a first time period the first light source 38a is active and the second light source is deactivated, in a second period of time the second light source 38b is activated and the first light source 38a is deactivated, and in third periods of time the first and second light sources are deactivated. The third time periods are between the first and second time periods. The frequency of the drive waveform of the first and second light sources 38a, 38b is substantially identical at 500 Hz. The frequency modulation is shown in Figures 7 (b) and 9 and includes applying a sinusoidal drive to the first and second light sources 38a, 38b at different frequencies. The frequency modulation includes applying a sinusoidal drive having a first frequency of 443 Hz to the first light source 38a, and a second frequency of 919 Hz to the second light source 38b. The use of prime numbers mitigates demodulation problems.

[0127] When modulation is used, modulated input light is provided to the first optical output 10, such that the detector 20 receives modulated light from the target 2. The processor element 22 is configured to demodulate the detector data 20.

[0128] Turning back to Figures 1 and 4, the optical arrangement 4 is configured to combine input sub-paths from each of the plurality of first optical inputs 6 into a common part of the first light path 12. The optical arrangement 4 comprises two first optical components 44 configured to combine the input sub-paths of the first optical inputs 6. In the embodiments shown here, the first optical components 44 are dichroic mirrors.

[0129] The optical arrangement 4 is configured to divide received light from the second optical input 14 into output sub-paths. The division of the output-sub paths is wavelength dependent. The optical arrangement 4 comprises a second optical component 46 configured to divide the received light into the output sub-paths. The second optical component 46 is a dichroic mirror.

[0130] As shown in Figure. 1 , one of the output sub-paths passes through the two first optical components 44, and one of the output sub-paths passes through one of the first optical components 44. One of the input sub-paths passes through the second optical component 46.

[0131] Although not shown, the optical arrangement 4 comprises inputs / outputs for coupling the optical fibre conduits 24 to the first and second optical components 44, 46. This may include use of a casing for the optical components and optical input / output ports.

[0132] The optical arrangement 4 is configured to provide received light from the second optical input 14 to the two second optical outputs 16 by providing each of the output subpaths to one of the second optical outputs 16. A different output sub-path is provided to each of the second optical inputs 16.

[0133] As shown in figures 4 (b), (d), Figure 5 (d) and (e), and Figure 10 (b), the optical arrangement 4 is configured to provide each output sub-path to a sub-region 20a, 20b of SPADs of the detector 20. In some cases, the sub-regions 20a, 20b are non-overlapping, and in others there may be an overlap between the sub-regions 20a, 20b. The subregions 20a, 20b are discrete SPAD pixels of the detector 20, which in various embodiments can be or two or more, or three or more, or four or more, or ten or more discrete pixels, optionally between 1 and 1 ,000 pixels, optionally between 1 and 100 pixels, between 1 and 10 pixels, optionally between 1 and 5, optionally between 1 and 4 pixels, optionally less than or equal to 50% of the detector pixels of the detector 20, optionally less than or equal to 40%, optionally less than or equal to 30 %, optionally less than or equal to 20%, optionally less than or equal to 10%, optionally less than or equal to 5%, optionally less than or equal to 1 % of the pixels of the detector 20.

[0134] The optical arrangement 4 comprises a second output element 48, which includes the second optical outputs 16 at a first end 48a thereof. A second end 48b of the second output element 48 is optically and indirectly connected to the second optical input 14. The second end 48b of the second output element 48 is optically connected to the second optical component 46. The second output element 48 includes optical fibre conduits 24c connecting the second optical output 16 to the second optical component 46.

[0135] As best shown in Figure 5, the system 1 comprises an adapter 50 for positioning and maintaining the second optical output 16 a fixed distance apart from the SPAD array of the detector 20. The adapter 50 comprises an adjustment mechanism 50a configured to allow the distance between the second optical output 16 and the detector 20 to be adjusted prior to or during use. In the embodiments shown here, the adjustment mechanism is implemented by two side screws that secure the end-most optical fibre conduit 24c within the adapter. The position of the conduit 24c can be adjusted and then secured in place using the side screws.

[0136] The adapter 50 includes a collimator 52 (an example of a light focussing element), therein for focussing the light from the second optical outputs 16 to the SPAD array.

[0137] The adapter 50 is configured to provide a linear distance from each SPAD to the second optical output 16 (and the collimator 52).

[0138] The adapter 50 is mounted to a detector support member 54, which in the embodiments shown here is a printed circuit board (PCB), by a mounting mechanism 56 including four screws and corresponding apertures on the adapter and in the PCB.

[0139] With reference to Figure 1 , the system 1 comprises a total light path from each light source 38a, 38b, to a first optical component 44, to the coupling element 36, to the first output element 26, to the first optical output 10, to the target 2, to the second optical input 14, to the first output element 26, to the coupling element 36, to the second optical component 46, to the second output element 48, to the second optical outputs 16. The system 1 , in terms of optical components, and in the second light path 18 between the coupling element 36 and the second output element 48 comprises only one or more optical conduits 24b and the second optical component 46. The system 1 , in terms of optical components, comprises only one or more optical conduits 24c and the collimator 52 between the first and second ends 48a, 48b of the second output element 48.

[0140] The system 1 , in terms of optical components between the second optical input 14 and the second optical outputs 16, and in the second light path 18, comprises only optical conduits 24a, 24b, 24c, the first optical components 44, the second optical component 46, and the collimator 52.

[0141] As depicted in Figure 6, the system 1 comprises a user interface 58 including a graphical user interface shown here on a display element (in Fig. 1). The processor element 22 is operable to display image data from the single-photon detector 20 and to permit a user to select and move one or more regions of interest 60 of the detector 20.

[0142] The processor element 22 is operable to display data from the detector 20, including intensity data (image data; left hand side of Figure 6) 62 and temporal photon count data 64 (top right of Figure 6), in real time. The intensity data 62 is from the photon count of each SPAD pixel, and the temporal data 64 represents photon counts over time concatenated for the region of interest 60. That is, the photon counts of the SPAD pixels in the region of interest 60 are concatenated to show a single temporal pixel count trace over time.

[0143] The processor element 22 is configured to permit the user to select and / or move the region of interest 60 in real time.

[0144] The processor element 22 is operable to carry out single-photon counting of each SPAD pixel of the detector 20 and to display pixel intensity data dependent on the photon counting for each SPAD pixel. It will be understood that this example is illustrative for SPADs, but there may be other ways of implementing a single-photon detector 20 and the associated processing thereof.

[0145] In some embodiments, the processor element 22 is operable to downsample the received data from the detector 20, e.g. by processing and / or displaying only 1 / n samples, which may help with the real time display of the data. This may be carried out when modulation of the input light is used or not. For example, the downsampling may be by a factor of at least 2, or at least 5, or at least 10, or at least 20, or any suitable factor. Because SPADs already facilitate a high sampling frequency, downsampling still provides for representative data while easing the computing requirements of displaying the data in real time.

[0146] In the embodiments illustrated and described here, the processor element 22 is configured to identify defective pixel(s) based, at least in part, on the average values of some or all of the pixels, or on the average values of adjacent or neighbouring pixels, as required. The defective pixel(s) may be “hot pixels”. The processor element 22 may be configured to remove or dynamically adjust the defective pixel(s) in the temporal pixel data 64 and / or the intensity plots 62 of the detector 20 or the region(s) of interest 60

[0147] Although not shown, the system 1 has a housing for at least some of the components thereof. Likewise, although not shown, the system may comprise any suitable components for mounting to the target, such as straps, harness(es), securing members, fasteners, garments, sleeves, housing(s), frames, support members, etc.

[0148] The sampling frequencies described in relation to the embodiments are examples. In other embodiments, the sampling frequency could be any suitable value, such as up to 20 kHz, optionally up to 15 kHz, optionally up to 12 kHz, optionally up to

[0149] 11 kHz, optionally up to 10 kHz, optionally up to 5 kHz, optionally up to 3 kHz, optionally up to 2.2 kHz, optionally between 200 Hz and 20 kHz, optionally between 200 Hz and

[0150] 12 kHz, optionally between 500 Hz, and 11 kHz, optionally between 800 Hz and 11 kHz, optionally between 800 Hz and 5 kHz, optionally between 200 Hz and 2.2 kHz, optionally between 400 Hz and 2.2 kHz, optionally between 500 Hz and 10 kHz, optionally between 500 Hz and 2 kHz optionally at least 100 Hz, optionally at least 200 Hz, optionally at least 500 Hz, optionally at least 1 kHz, optionally at least 2kHz, optionally at least 5 kHz.

[0151] Examples of how the system 1 can be used and some further discussion will now be provided.

[0152] With reference to Fig. 1 , the system 1 is used to monitor the activity of specific cell populations within the mouse brain. Photometric measurement of rapid neural signals requires both high sample rates and high sensitivity. Without wishing to be bound by theory, compared with other imaging techniques, the embodiments of the invention provide both an exceptionally high sampling rate of up to 10 kHz, enabling precise capture and analysis of neural activity, and high sensitivity with minimal noise contamination, enabling detection of functionally important signals, which for fast genetically encoded indicators are reported by relatively small changes in fluorescence.

[0153] The use of SPAD sensors for fibre photometry imaging allows for high sensitivity and a continuous sampling rate of up to 10 KHz. The 10 kHz value is not an absolute maximum, and without wishing to be bound by theory, it is possible for the detector 20 or other single photon detectors to achieve higher sampling rates, such as up to 20 kHz or more. However, conventional neuronal imagers are thought to be typically capable only of up to 1 kHz sampling.

[0154] The effective sampling rate with frequency or time-division modulation can reach up to 2 KHz, thought to be higher than all previously reported systems, with much lower noise levels than commonly applied sensors. This feature enables the readout of fast neural population signals reported by GEVIs and other faster fluorescent reporters. The figure of 2 KHz is an example and in other embodiments the effective sampling rate when using modulation may be higher.

[0155] In Figure 1 (a) the fibre cannula 28 is implanted into the target animal’s brain and coupled to a fibre patch cord 24a by the coupling sleeve 30. (b) The fibre patch-cord 24a with the rotary coupling mechanism 36 can be used to avoid twisting of the optical fibre conduits 24 when the animal is moving, (c) A combination of dichroic mirrors 44, 46 are used to guide and filter light of different wavelengths. Minicubes ((RTM) from Doric Lenses) are pre-configured for compatibility with wavelengths that are suitable for neuron imaging, (d) LEDs 38a, 38b for exciting biology fluorophores. (e) a dual fibre patch-cord 24c to direct fluorescent emission light to the detector 20. (f) Collimating lens 52 and customised adaptor 50 to attach the fibre patch cord 24c to the detector 20 and modify the size of the fibre image, (g) PCB board for the detector 20. (h) Imaging software.

[0156] SPADs are electronic devices that, upon activation by a single photon, initiate an avalanche of electrons and generate a substantial electric current (as depicted by the Vc trace in Figure 2). The primary SPAD detector 20 used for demonstrating this invention is a 320 by 240 SPAD array (Figure 3) referred to as SPC (single photon counting) imager. Another SPAD sensor was used for comparison, termed the ATLAS detector. In principle, any SPAD array that allows photon counting at sufficient frame rates can be employed.

[0157] The SPAD detector 20 is attached to a custom designed PCB 54 that includes more components to control the detector 20, and the processor element 22 (Figure 1 (g) and bottom left of Figure 5). The components include the SPAD array 20, the SPAD integrated circuit which supports data transfer and control of the SPAD sensor, and an FPGA (Field Programmable Gate Array) board for controlling the capture of bit-planes and streaming data to a PC through USB 3.0. The FPGA forms part of the processor element 22 of the embodiments, but any suitable processor element 22 for implementing the invention can be adopted. Customised PCB designs with different FPGA boards that are capable of controlling the SPAD sensor can be applied to support different types of SPAD sensors.

[0158] Imaging with multiple excitation wavelengths can be achieved by a design as in Figure 4 (a) and (b). For example, one wavelength can be used to excite a GEVI and the second a reference fluorescence signal. The waveform generator 40 is used to control the LED driver 42, with the possibility of generating square waves or sinusoidal waves to enable time-division or frequency modulation of the LEDs 38a, 38b. Two LEDs 38a, 38b are used as excitation lights to excite two fluorophores. A combination of dichroic mirrors 46, 48 are used to filter and direct excitation light to the target 2 as well as filter and direct emission light from the target 2 to the output fibre patch-cord 24c. To facilitate separate detection of the emission from each fluorophore, lights are fed to a 2-to-1 fanout fibre patch cord 24c with the output fibre-tip focused on the SPAD detector 20 (Figure 4 (b)). Without wishing to be bound by theory, methods to control the size of the fibre image on the SPAD sensor 20 are important for optimising the signal to noise ratio of the detector data. To optimise the signal to noise ratio, the adapter 50 can be used to adjust and then fix the distance between the second optical output 16 and the detector 20. This can be done using test samples, such as coloured cards, or using in vitro neuronal samples to determine an acceptable signal to noise ratio, with both of these methods being illustrative rather than essential.

[0159] The method can be extended to image multiple distinct cell populations by using a different combination of dichroic mirrors and bundling multiple fibre patch-cords together to be collimated and imaged by the detector 20. Consider for example Figure 4 (b) which shows that only two of the possible 7 outputs 16 of the patch cord 24c are in use. It is possible to focus more fibre spots onto the detector, which could involve multiple output elements 48 or fibre patch cords, or the like. The use of one or two sub-regions 20a, 20b, is purely an example.

[0160] Imaging with isosbestic reference can be achieved by the design in Figure 4 (c) and (d), for example to obtain Ca2+-independent reference signals when using a genetically encoded calcium indicator (GECI), such as GCaMP as an indicator. This will also support red-shifted indicators by changing the LED and dichroic mirrors to relevant wavelengths.

[0161] Depending on the size of the SPAD detector 20 and the diameter of the fibre core, the distance from the fibre connector 24c to the detector 20 can be adjusted to allow one or multiple regions of interest to be fully covered by the detector 20. This has been illustrated for two options: Figure 4 (a) and (b) show a dual-colour system with two regions of interest on the detector 20 whereas Figure 4 (c) and (d) show a single-colour, single region of interest system. But in principle additional channels can be added with the primary constraint being the photon counting capacity of the SPADs. For example, the ATLAS SPAD array, which has high bit depth is suited to photometry experiments that use many fibres in parallel.

[0162] To summarise Figure 4 shows a light path design and example for two-colour imaging and single-colour GCaMP imaging. A). A light path design example for two- colour imaging. From left to right, waveform generator 40, LED driver 42, LEDs 38a, 38b, a combination of dichroic mirrors 44, 46. B) An example of a two-colour image on a SPAD sensor obtained with a patch-cord bundle. From right to left, a customised 2-to-1 fan-out fibre patch cord designed by Doric. Inc; an example image of the dual-fibre end on a SPAD sensor. Note: the dark dots in regions of interest 20a, 20b are hot pixels on the SPAD sensor which are removed before analysis. C) A light path design for single-colour imaging. D) An example image of a single fibre tip.

[0163] Figure 5 illustrates the connection between the adaptor 50 and the SPAD imager PCB. The adaptor 50 firmly attaches the fibre patch cord 24c above the SPAD array (Figure 5(b)). The distance between the end of the fibre connector 24c and the SPAD detector 20 is calculated and adjusted according to size of the detector, the diameter of fibre cores and the number of fibres that will be imaged (the number of second optical outputs 16), therefore all regions of interest of the fibre tips are inside the field of view of the detector 20. The collimator 52 is employed to focus multiple fibre tips or modify the fibre image size on the detector 20 if necessary. Optimisation of the fibre image on the SPAD sensor 20 improves the signal to noise ratio of the imaging.

[0164] A collimator 52 (or in some examples, couplers with lenses can be used) is used to focus the light from the fibre tip onto the detector 20 (Figure 5B and C), and an example component is the Thorlabs (RTM) FiberPort Collimator (PAF2P-A10A - Achromatic FiberPort).

[0165] To summarise Figure 5 depicts attaching the fibre patch cord to the SPAD imager with the 3D printed adapter. A). An example design of fibre-to-sensor adapter. The 3D- printed adapter can be fixed on the SPAD imager with screws; the fibre patch-cord can be inserted into the adapter with adjustable distance and fixed with two extra screws. B) The distance between the output ends of the fibre and the sensor can be calculated and adjusted by testing to ensure that all ROIs are fully imaged by the sensor. C) An example fibreport collimator (Thorlab PAF2P-A10A - Achromatic FiberPort). D) Example fibre tip images captured by the SPC imager with a collimator (top) and with a simple 3D printed adapter (bottom). E) Example fibre tip images captured by the ATLAS imager with a collimator (top) and with a simple 3D printed adapter (bottom).

[0166] Figure 6 displays the SPAD imaging software, which is based on the programming language Python. The left panel shows the live image of a fibre tip. The top-right panel displays a trace representing the signal changes over time within the region of interest 60 marked by the circle. The bottom-right panel presents a photon counting histogram of the image.

[0167] Before conducting an imaging experiment, the fibre cannula 28 is implanted into the targeted region of an animal's brain, where specific cells or neurotransmitters are labelled using one or multiple fluorescent biosensors. After 3-4 weeks to allow the viral expression and the animal to recover, neuron population activity can be imaged with a high temporal resolution with the embodiments of the invention shown here. The excitation light excites the fluorescent biosensor(s) and the fluorescence emission light is collected by the detector 20. A different optical arrangement 4, including different combinations of dichroic mirrors 44, 46 can be utilised to support various imaging purposes, such as multi-colour imaging and imaging during optogenetic manipulation. Excitation light at specific wavelengths can be generated by LEDs or lasers, then filtered and directed toward the fibre patch cord 24a that connects to the animal's brain. Emission light from the fluorescent biosensor is filtered and directed to the SPAD detector 20 for acquisition of high-frame rate images.

[0168] Continuous recording mode is suitable for imaging from a single-colour fluorescence continuously. For example, continuous recording is ideal for capturing the activity of GEVI or GECI-expressing neurons in an animal model with minimal movement. This type of recording does not provide a reference channel for movement artefacts, therefore it cannot remove such artefacts from the recorded signal. However, continuous recording takes advantage of high temporal resolution and provides the best signal quality. It can be particularly useful in head-fixed experiments or simultaneously recording optical and electrophysiology signals. Movement artefacts can also be tested separately, for example by recording isosbestic signals in conditions identical to those used for recording the signals of specific experimental interest.

[0169] To achieve continuous recording, the LED driver 42 is set to continuous wave mode to provide a continuous excitation light source. The emitted light from the targeted fluorescence will be continuously captured by the SPAD array detector 20 at the sampling rate. Continuous data can be saved and analysed offline. T o introduce another light channel or utilise the isosbestic point of GCaM P, timedivision mode recording or frequency modulated recording can be used.

[0170] For time-division mode recording, this configuration enables the LED driver 42 to alternate the delivery of multiple excitation lights from the LEDs 38a, 38b, as previously described above and shown in Figures. 7 and 8.

[0171] Figure 8 shows the simulation of time-division modulated recording for 100 ms signals. The SPAD sensor sampling rate is 9,938.4 Hz in this simulation. Real signals from each channel (a). Different channels are alternately excited by using square waves to drive excitation LEDs (b). Modulated signals (c) from each channel are mixed at the readout port. The recorded mix data (d) can be demodulated by applying masks and calculating the envelopes, therefore, signals from each channel can be recovered at a lower effective sampling rate as the carrier square waves. Note that recovered signals look different from the real signal because the sampling rate is lower.

[0172] To implement frequency-modulated recording, the waveform generator 40 produces two sinusoidal waves with distinct frequencies, as described previously above and shown in Fig. 7 (b).

[0173] Figure 9 shows the simulation of frequency modulated recording for 100 ms signals. The SPAD sensor sampling rate is 9,938.4 Hz in this simulation. Real signals from each channel (a) will be modulated at a different frequency band by using sinusoidal waves at 443 Hz (green line) and 913 Hz (red line) to drive the LEDs (b). Modulated signals (c) from each channel are mixed at the readout port. The recorded mix data (d) can be demodulated by applying bandpass filters and calculating the envelopes, therefore, signal from each channel can be recovered at a lower effective sampling rate as the carrier sinusoidal waves. Note that recovered signals look different from the real signal because the sampling rate is lower.

[0174] Each frame is recorded as an X*Y binary array (320x240 for the example SPAD sensor), indicating whether there’s a photon count on a specific pixel in this frame. By aggregating multiple frames, a clear image can be revealed. In this example image, there are two fibre tips from two channels. Targeted regions of interest can be defined with this image and the coordinates of pixels. Then, by concatenating the sum of photon counts within a region of interest over time, a signal trace can be obtained to measure the fluorescence changing from the fibre tip.

[0175] Figure 10 shows the data structure and decoding process of the SPAD imaging system. A. SPAD data shown as pixel arrays (bit-planes) along the time axis, each frame contains the photon count information on each pixel, the region of interest (ROI) can be a single or multiple fibre tips. B. An example image shows two fibre tips imaged by the SPC imager; the picture is obtained by accumulating multiple frames and removing hot- pixel artefacts. C. An example calcium imaging trace from the green channel in B; signal trace was obtained by calculating the total photon count within the targeted ROI for each frame and concatenating data points over time.

[0176] Figure 11 shows an example of animal recording data. The fibre was implanted into CA1 , where GCaMP8s-aav-retro is expressed. Both GCaMP emission and isosbestic emission were recorded under time-division mode, therefore the effective sampling rate was 500Hz, as shown in the figure. With the SPAD imager, 1 ,000Hz timedivision frequency can also be achieved to review more details of fluorescent signals. In Figure 11 , an example of 10 seconds calcium transient imaged with the SPC imager and processed with the presented data analysis pipeline. GCaMP emission and isosbestic emission were imaged with time-division mode (GCaMP emission is the uppermost graph in Figure 11 (a), (b) and (c), and the isosbestic emission is the middle trace of Figure 11 (a), (b) and (c)). Zscore (bottom plot of Figures 16 (a), (b) and (c)) was calculated by detrending, smoothing, normalising and subtracting the isosbestic signal from the GCaMP channel signal. A. Raw traces obtained from the SPC imager with an effective 500 Hz sampling rate. B. Traces smoothed to 100 Hz. C. Traces smoothed to 20Hz. D. Histology image with viral expression and the implanted fibre track.

[0177] To demonstrate the advantages of SPAD-based photometry, such as higher signal to noise ratios (SNRs) in high-speed imaging scenarios, tests are shown in Figure 12 comparing the open-source pyPhotometry system with two different SPAD detectors, the SPC imager and the ATLAS imager. Steady-state illumination with different light intensities was imaged by the three sensors with the configuration shown in Figure 12 (a). A 465 nm LED was used as an excitation light to illuminate a yellow paper, the emission light was filtered by a 525 nm light filter and then imaged by the detector. The theoretical true signal should be a stable flat line indicated by the mean value, with noise calculated by the standard deviation (Figure 12 (b)). SNRs under each light condition from each system were calculated based on mean values and standard deviations from 1 -second imaging data.

[0178] The sampling rates for pyPhotometry, the SPC imager, and the ATLAS imager are 1 ,000 Hz, 9,938.4 Hz, and 840 Hz, respectively. Using light powers ranging from 0.06 pW to 5 pW, it was found that the ATLAS imager has a significantly higher SNR. The SNR of raw data from the SPC imager still outperforms the pyPhotometry system under low light levels. The SPC imager has a sampling rate of 9,938.4 Hz, allowing signal quality improvement through binning for lower effective sampling rates. With effective sampling rates of 1 ,000 Hz and 500 Hz, the SPC imager shows a substantial advantage over the commonly used pyPhotometry system (Figure 12 (c)).

[0179] Different fibre image sizes on the detector can affect the SNR. Figure 12 (d) shows absolute SNR from ATLAS recording when the ROI sizes are configured differently.

[0180] Figure 12 shows the comparison of SNRs among pyPhotometry, SPC imager, and ATLAS imager under different light power conditions. (A) Experimental setup: a stable flat signal is recorded in a dark room by placing a fibre tip on yellow paper and illuminating it with 465 nm blue LED light. (B) Example trace imaged by the ATLAS camera at 5 pW blue light power; the solid trace shows the raw signal within the fibre tip region of interest in (D), with the dashed line indicating the mean signal value. (C) Absolute SNRs of the three photometry systems under low light power from 0.06 pW to 5 pW. The reflection light from yellow paper approximates green fluorescence protein magnitude. Absolute SNR is calculated by signal / noise (mean / standard deviation), with the mean signal value and the noise standard deviation. For the SPC imager, sampling at 9,938.4 Hz, the SPC_1000Hz and SPC_500Hz traces are obtained by binning 10 and 20 samples, respectively. Images from the ATLAS camera and SPC imager under different light power are shown below the SNR plot. (D) Absolute SNR of ATLAS imaging data with two different configured ROI sizes under different light powers as in C; fibre image was collimated to the sensor within a 40 x 40 pixel area and adapted directly with a certain distance to be imaged within a 80 x 80 pixel area.

[0181] Turning to Figures 13 & 14, to validate that the SPAD array detector 20 can capture GEVI signals from freely behaving animals, injection of the AAV virus carrying the voltage indicator JEDI-2P and Cre recombinase under the CamKII promoter into the hippocampal CA1 region of a wild-type mouse brain was carried out. This targeted mostly pyramidal cells at the injection site. We then simultaneously implanted an optical fibre with four electrode probes into the same region to image the JEDI-2P fluorescence signal and record the electrophysiology signal from the CA1 .

[0182] Data was obtained four weeks post-surgery to allow for virus expression and recovery. Simultaneous local field potential recording (LFP) and photometry imaging were conducted while the animal navigated an 60 cm x 40 cm arena (Figure 13) or slept in its home cage (Figure 14). Neural activity reported by GEVI was imaged using the ATLAS imager at 840 Hz or the continuous mode of the pyPhotometry system at 1 ,000 Hz to compare their efficiency in capturing in-vivo GEVI signals. During free navigation, the ATLAS imager captured GEVI signals showing theta oscillations (4-12 Hz) that corresponded closely to the electrically measured LFP (Figure 13 (c)). ATLAS imaging revealed that GEVI signals from pyramidal cells correlated with and were phase-locked to LFP during theta oscillation (Figures 13 (d), (e)). In contrast, the pyPhotometry system failed to clearly capture theta oscillations due to its relatively low SNR (Figure 13 (f) to (h)).

[0183] Figure 13 shows imaging of voltage signals reported by GEVI using the ATLAS imager and the pyPhotometry system during theta oscillation. (A) Viral strategy and implant location. (B) Example histology image of hippocampal slices expressing JEDI- 2P. (C) Example of a three-second GEVI signal and LFP signal obtained while the animal was moving in an arena; colour maps show the power spectrum in the theta band from GEVI imaging and LFP recording. (D) Averaged cross-correlation of GEVI signal and LFP during hippocampal theta oscillation periods. (E) Average GEVI signal and LFP from all theta cycles from a two-minute recording. (F-H) Same as C, D, and E, with data from the same animal recorded using the pyPhotometry system.

[0184] Referring to Figure 14, during sleep and rest, the ATLAS imager captured GEVI signals correlated with sharp wave-ripples (150-250 Hz) in the CA1 , indicating that pyramidal cells tend to fire after the ripple peaks (Figure 14(b), 14(c)). In contrast, the pyPhotometry system failed to capture this ripple-related neuronal activity due to its relatively low SNR (Figure 14 (d-f)).

[0185] Figure 14 includes imaging of voltage signals reported by GEVI using the ATLAS imager and the pyPhotometry system during high-frequency oscillations. (A) Example of a 200-millisecond window showing a ripple event captured by GEVI signal (lighter line) and LFP signal (darker line) while the animal was sleeping; colour maps display the power spectrum in the ripple band from GEVI imaging and LFP recording; bottom light and dark traces show filtered ripple band signals from GEVI and LFP. (B) Average GEVI signal (smaller peak to peak) and LFP (larger peak to peak) from all ripple events in a two-minute recording; shaded areas indicate 95% confidence intervals; the vertical bar marks ripple power peak time; the dots below the graph mark GEVI signal peak times relative to ripple peaks. (C) Averaged cross-correlation of GEVI signal and LFP in 400 ms windows covering ripple epochs. (D-F) Same as A, B, and C, with data from the same animal recorded using the pyPhotometry system during awake resting.

[0186] Figure 15 shows simulated sinusoidal waves tested on different detectors. The theoretical study predicted that SPAD-based imagers outperform others in low light and high frequency conditions. A) In low light and low frequency (5Hz sinusoidal input signal with an average of 100 photons / second), both SPAD-based imagers (ATLAS and SPC) and CMOS imagers accurately reconstructed the original signal. However, photoreceiver-based light detectors (PR) failed to detect the signal due to their ineffectiveness at low light levels. B) In low light and high frequency (200Hz sinusoidal waves with an average amplitude of 100 photons / second), the high effective frame rate and sensitivity of SPAD-based imagers offered a significant advantage in detecting high- frequency oscillations. C) A detailed examination of the signals from B reveals that SPAD-based imagers successfully detected the original high-frequency signal. In contrast, conventional CMOS cameras and photoreceivers were unable to do so due to their limited dynamic range and effective frame rate.

[0187] SPAD-based detectors are therefore effective in detecting high frequency neurological activity.

[0188] Figure 16 shows a comparison of temporal resolution between two SPAD detectors (Atlas and SPC) and pyPhotometry with square waves of different frequency. Figure 16 A), B) and C) show ten cycles of square waves of different frequencies recorded by the Atlas imager, the SPC imager and the pyPhotometry system. Square waves of 10 Hz, 50 Hz, 100 Hz, 200 Hz, 300 Hz and 500 Hz are shown from the top row to the bottom row.

[0189] Figure 17 shows a comparison of temporal resolution among the Atlas imager, SPC imager and pyPhotometry with sinusoidal waves of different frequency. A), B) and C) show ten cycles of square waves of different frequencies recorded by the Atlas imager, the SPC imager and the pyPhotometry system. Sinusoidal waves of 10 Hz, 50 Hz, 100 Hz, 200 Hz, 300 Hz and 500 Hz are shown from the top row to the bottom row.

[0190] Although the disclosure has been described in terms of embodiments as set forth above, it should be understood that these embodiments are illustrative only and that the claims are not limited to those embodiments. Those skilled in the art will be able to make modifications and alternatives in view of the disclosure, which are contemplated as falling within the scope of the appended claims. Each feature disclosed or illustrated in the present specification may be incorporated in any embodiments, whether alone or in any appropriate combination with any other feature disclosed or illustrated herein.

Claims

CLAIMS:1 . A photometric system for measuring one or more neurophysiological parameters of a target, the photometric system comprising: an optical arrangement comprising: a first optical input for receiving input light from a light source; a first optical output for providing output light to a target; a first light path from the first optical input to the first optical output; a second optical input for receiving light from the target; a second optical output; a second light path from the second optical input to the second optical output; and a single-photon avalanche detector configured to receive light from the second optical output; and a processor element configured to receive data from the single-photon avalanche detector and to carry out single-photon counting.

2. The system of claim 1 , wherein the single-photon detector comprises one or more single-photon avalanche diodes (SPADs).

3. The system of claim 1 or claim 2, wherein the system is configured for use with a free moving organism, such as a free moving human or animal.

4. The system of any preceding claim, wherein the first optical output is configured to be implantable at least partially within the target.

5. The system of any preceding claim, wherein the system comprises a first output element having a first end and a second end, wherein the first optical output and the second optical input are located at the first end, and the first output element comprises a cannula.

6. The system of any preceding claim, wherein the optical arrangement comprises a stationary part, a movable part, and a coupling element connecting the stationary part to the movable part.

7. The system of claim 5 or claim 6, wherein the first output element includes one or more, or a single optical conduit to optically connect the first end thereof to the second end thereof.

8. The system of claim 6 or claim 7, wherein the coupling element is connected between one or more stationary optical conduits and the second end of the first output element to permit at least partial rotation of the first output element relative to the one or more stationary optical conduits.

9. The system of claim 7 or claim 8, wherein the, or each optical conduit is a fibre optic conduit.

10. The system of any of claims 6 to 9, wherein the system comprises, in terms of optical components, only one or more optical conduits between the coupling element and the first optical output; and / or wherein the system comprises, in terms of optical components, only one or more optical conduits between the coupling element and the second optical input; and / or wherein the system comprises, in terms of optical components, only one or more optical conduits between the coupling element and the cannula.

11. The system of any preceding claim, wherein the system comprises a first light source for providing input light to a first optical input, and a second light source for providing input light to another first optical input.

12. The system of claim 11 , wherein the system is operable to modulate the first and second light sources.

13. The system of any preceding claim, wherein the system comprises a plurality of second optical outputs, wherein the optical arrangement is configured to divide received light from the second optical input into output sub-paths, wherein the optical arrangement is configured to provide each of the output sub-paths to at least one second optical output, wherein the optical arrangement is configured to provide each output sub-path to a sub-region of the detector.

14. The system of claim 13, wherein the sub-regions are non-overlapping or partially overlapping.

15. The system of claim 13 or claim 14, wherein the sub-regions include one or more discrete pixels of the detector, or two or more, or three or more, or four or more, or ten or more discrete pixels, optionally between 1 and 1 ,000 pixels, optionally between 1 and 100 pixels, between 1 and 10 pixels, optionally between 1 and 5 pixels, optionally between 1 and 4 pixels, optionally wherein the, or each subregion is approximately less than or equal to 50% of the pixels of the detector, optionally less than or equal to 40%, optionally less than or equal to 30 %, optionally less than or equal to 20%, optionally less than or equal to 10%, optionally less than or equal to 5%, optionally less than or equal to 1 %.

16. The system of any preceding claim, wherein the system comprises an adapter for positioning the second optical output relative to the detector, wherein the adapter is configured to maintain the second optical output a fixed distance to the detector.

17. The system of claim 16, wherein the adapter comprises an adjustment mechanism configured to allow the distance between the second optical output and the detector to be adjusted prior to or during use.

18. The system of any preceding claim, wherein the optical arrangement comprises a second output element including the second optical output at a first end thereof, and wherein the optical arrangement comprises one or more light focussing elements configured to focus light to the detector or detecting elements thereof.

19. The system of claim 18, wherein the system, in terms of optical components, comprises only one or more optical conduits and the light focussing element between the first and second ends of the second output element.

20. The system of claim 18 or claim 19, wherein the optical arrangement comprises one or more second optical components configured to divide the received light into the output sub-paths, and wherein the system, in terms of optical components between the second optical input and the second optical output, andin the second light path, comprises only one or more optical conduits, the one or more second optical components, and the light focussing element, and optionally one or more first optical components.

21. The system of any preceding claim, wherein the processor element is operable to display data from the detector, such as intensity data or temporal data, in real time.

22. The system of any preceding claim, wherein the processor element is operable to display temporal data of photon counts for a pixel or for a region of interest of pixels of the detector.

23. An imaging system comprising: a first optical input for receiving input light from a light source; a first optical output for providing output light to a target; a first light path from the first optical input to the first optical output; a second optical input for receiving light from the target; a second optical output; a second light path from the second optical input to the second optical output; and a detector configured to receive light from the second optical output.

24. A method of carrying out photometry on a target, the method comprising: providing light to at least a part of the target; receiving light from at least a part of the target; and performing single-photon counting on at least a portion of the received light from the target.

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