Patent Publication Number: US-2023152228-A1

Title: System, method and optical module for multi-fiber photometry with patterned sample stimulation

Description:
BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention relates to multi-fiber photometry and optical sample stimulation, and more particularly concerns a system for in vivo, e.g., in freely-moving/behaving animals and head-fixed animals, or in vitro sample stimulation and fluorescence detection through multiple optical fiber connections with regions of the sample. 
     2. Background of the Invention 
     Biological fluorescence detection and analysis has been extended to involve optical stimulation of biological samples, which may include operations that modify the samples. Techniques such as optogenetic stimulation, bio-molecular uncaging, and laser-ablation are performed under optical guidance and/or measurement using fluorescence measurements. Systems have been implemented that integrate optical stimulation with fluorescence measurements, such as those systems disclosed in U.S. Pat. Nos. 9,791,683 and 9,846,300. However, while such systems are capable of receiving fluorescence emissions returning from the sample along multiple optical fibers and measuring a response of different regions to the stimulation, the stimulation in such systems is performed across the sample and is typically constrained to the same aperture. 
     In fluorescence imaging, and more specifically calcium imaging, in order to render a specific population of neurons light sensitive, the population is labeled with genetically encoded functional fluorescent proteins, e.g., calcium indicators, that modulate their fluorescence emission according to the activity of the labeled cells. The labeling enables a visual observation of neuronal activity by observing a fluorescence response to excitation illumination. Typically, the observation is made with a single fiber and the spatial resolution of the measurement is typically limited by the diameter of the fiber optic cable. The collected signal aggregates the activity of hundreds of cells located close to the tip of a fiber optic implant. Early fluorescence imaging systems were developed to record one brain region coupled to one fiber optic cable and typically include light sources (e.g., lamps, LEDs, or lasers), optical fibers, one or more photo-sensors (e.g., photodiodes or image sensors), and a light-filtering assembly that spectrally separates excitation and emission light. Control of the excitation illumination sources and photo-sensors data acquisition are typically managed by a computer. When the samples are in freely behaving animals, an optical rotary joint, such as that described in U.S. Pat. No. 10,564,101, is included to remove twists that may otherwise occur in the fiber optic cable. Entirely optical fiber-based systems have also been proposed, such as those disclosed in P.R.C. Patent No. CN213309653U where spectral separation is performed in fiber bundles. 
     The above-described fluorescence imaging techniques monitor cell activity by recording temporal fluorescence variations during optical excitation of a fluorescent indicator, e.g., a calcium indicator. In order to simultaneously record the fluorescence activity of multiple brain regions, it has also been proposed to replace the photodiodes with a CMOS image sensor, such as in the systems described in U.S. Patent Application Publication No. 20180228375A1 to image signals from several optical fiber cables that are connected to, and excite different brain regions. 
     Optogenetics is a neuroscience research technique that uses specific light stimuli for activating or inhibiting cells such as neurons. To render cells light-sensitive, the cells are marked with genetically encoded light sensitive ion channels, e.g., channelrhodopsin (ChR2) for activation of neural activity or halorhodopsin for inhibition of neural activity. Frequently, it is desirable to combine fluorescence imaging with optogenetics in order to gain deeper insight and confirm correlations between specific brain regions activity and behavior of the behaving animal. However, optogenetics and fluorescence imaging do not mix well due to the significant difference between the light intensity requirements, with optogenetics stimulation requiring significantly greater intensity than that required for fluorescence excitation. Excessive optogenetics stimulation or fluorescence excitation illumination may cause photobleaching or trigger phototoxicity mechanisms, and consequently induce damage to fluorescent markers or the targeted tissue. Ideally, optogenetics and fluorescence imaging measurements should be separated spatially, whenever possible, or temporally. Since the optogenetics stimulation illumination is of greater intensity, the stimulation should be confined to the smallest area possible. 
     For recordings over individual points of interest, fluorescence imaging and optogenetics stimulation have been combined for one or two sites as in the systems disclosed in U.S. Pat. Nos. 9,791,683 and 9,846,300. Additional sites of interest could be added by replicating the systems. However, with larger numbers of recording sites, replication becomes expensive unwieldy. Another approach that has been taken using multiple site fiber photometry and optogenetics uses a single image sensor, one optical fiber bundle, and one light source for each spectral region, significantly reducing the system complexity. However, the above approach lacks specificity, as each site receives the same excitation and optogenetics lights. In some instances, various regions of the sample may be desirably stimulated or altered optically, and those regions may or may not correspond to regions of interest in the fluorescence measurements. It may also be desirable to have control of a number of regions to be stimulated simultaneously or sequentially in a measurement, without stimulating the entire sample. 
     Therefore, it would be desirable to provide a method and system for performing fluorescence measurements with optical stimulation of a biological sample so that a simultaneous and/or sequential stimulation can be applied to different locations within a sample and that are capable of performing fluorescence measurements in those locations. 
     SUMMARY OF THE INVENTION 
     The objectives of performing optical stimulation and fluorescence measurements in different locations of a biological sample are accomplished in optical systems, optical imaging devices for use in the systems, and methods of operating the systems. 
     The systems are systems for performing optical stimulation and fluorescence measurements on a sample, and include an illumination array having multiple stimulation illumination elements that are electronically addressable to select a spatial pattern of illumination, and an optical fiber bundle including multiple optical fibers coupled to corresponding ones of the multiple stimulation illumination elements at their proximal ends. The multiple optical fibers are for coupling to target locations within or on a sample at their distal ends, so that activated ones of the multiple stimulation illumination elements provide stimulation at corresponding ones of the target locations. The systems also include a fluorescence illumination source for providing fluorescence illumination and coupled to the proximal end of the optical fiber bundle, and an imaging system coupled to the proximal end of the optical fiber bundle for measuring fluorescence returning from the target locations. 
     The optical imaging devices for use in the systems include a housing, a sample connector accessible at an exterior of the housing and configured for receiving connection with a first optical fiber bundle including multiple optical fibers. The sample connector has an indexing feature complementary with that of the received connection with the first optical fiber bundle, so that a fixed relationship between the individual ones of the multiple fibers and internal optical paths of the optical imaging device is maintained. The optical imaging devices also include a stimulation illumination input connector accessible at the exterior of the housing and having a second indexing feature, and the stimulation illumination input connector is configured for receiving connection from a second optical fiber bundle comprising second multiple optical fibers that provide individual elements of a pattern of illumination, so that a fixed relationship between the pattern of illumination and the internal optical paths of the optical imaging device is maintained. The optical imaging devices also include a fluorescence illumination input connector accessible at the exterior of the housing for receiving an optical connection from a fluorescence illumination source, and an output connector accessible at the exterior of the housing for providing fluorescence image information to an external system. The output connector may be an optical connector for providing a fluorescence image to an external optical imaging system, or the device may include an electro-optic sensor array and the output connector may be an electrical connector for connecting the sensor array to an electronic controller. 
     The summary above is provided for brief explanation and does not restrict the scope of the claims. The description below sets forth example embodiments according to this disclosure. Further embodiments and implementations will be apparent to those having ordinary skill in the art. Persons having ordinary skill in the art will recognize that various equivalent techniques may be applied in lieu of, or in conjunction with, the embodiments discussed below, and all such equivalents are encompassed by the present disclosure. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG.  1    is a pictorial diagram depicting an example system  10 , in accordance with an embodiment of the disclosure. 
         FIGS.  2 A- 2 D  are pictorial diagrams depicting various states of illumination within fiber bundle  14 B in example system  10  of  FIG.  1   , in accordance with embodiments of the disclosure. 
         FIG.  3    is a block diagram depicting an example system  10 A, in accordance with an embodiment of the disclosure and including details of an example optical combiner/splitter device  30 A, in accordance with an embodiment of the disclosure. 
         FIG.  4    is a pictorial diagram depicting details of another example optical combiner/splitter device  30 B that may be used in example system  10  of  FIG.  1    and example system  10 A of  FIG.  3   , in accordance with an embodiment of the disclosure. 
         FIG.  5    is a pictorial diagram depicting an example stimulus illumination system  50  that may be used in example system  10  of  FIG.  1    and example system  10 A of  FIG.  3   , in accordance with an embodiment of the disclosure. 
         FIG.  6    is a pictorial diagram depicting yet another stimulus illumination system  60  that may be used in example system  10  of  FIG.  1    and example system  10 A of  FIG.  3   , in accordance with an embodiment of the disclosure. 
         FIG.  7    is a perspective view showing an example multiple fiber optic connector pair that may be used in example system  10  of  FIG.  1    and example system  10 A of  FIG.  3   , in accordance with an embodiment of the disclosure. 
     
    
    
     DETAILED DESCRIPTION OF ILLUSTRATED EMBODIMENT 
     The instant disclosure provides optical stimulation and fluorescence photometry systems and methods, along with an optical imaging device that may form part of the systems. The techniques are applicable to optogenetics and fiber photometry fluorescence recording, among other potential applications, both in vivo, e.g., in freely-moving/behaving animals and head-fixed animals, and in in vitro sample analysis. The systems enable optogenetic stimulation, laser ablation and/or biomolecular uncaging as a stimulus to a sample and at different locations within the sample, and are particularly suited to performing simultaneous in vivo fluorescence recording and optogenetic stimulation in biological tissue, for biomedical research applications. The systems and techniques are also applicable in photo-stimulated uncaging of biomolecules or laser ablation and processing. The systems and techniques combine stimulation light and multi-fiber fluorescence photometry recording. The optical stimulation targets selected sites within a predefined grid of multiple sites on the biological sample, while simultaneously enabling photometry recording across some or all of the multiple sites. Fluorescence excitation illumination is provided at different sample locations and fluorescence pattern information is returned from the locations in the sample by introducing the fluorescence excitation illumination across a plurality of fibers forming a fiber bundle, and returning the resulting fluorescence information through the fibers of the fiber bundle from the various locations in the sample, which are collected by imaging the returned fluorescence information across fiber bundle. While the terms “image” and “imaging” are used to describe the fluorescence detection path in the systems disclosed herein, it is understood that the detection of the fluorescence may be performed by a discrete detector per-fiber/location and that the “image” may effectively constitute a single pixel-per fiber (discrete detectors) or the fluorescence information may be detected by a higher-resolution image sensor, such as a CMOS or CCD camera returning a single image in which image regions correspond to the individual optical fibers in the bundle and that may then be processed with an averaging algorithm to produce an intensity value for each optical fiber or location within the sample. The optogenetic stimulation, laser ablation and/or biomolecular uncaging stimulus light is also provided through selected fibers by generating the stimulus light with an illumination array having multiple stimulation illumination elements that are electronically addressable to select a spatial pattern of illumination that is introduced to the fiber bundle. The pattern is not necessarily related to the position of the locations within the sample other than the relationship between the illumination array and the optical fibers, which may be located as needed in order to stimulate the sample in a desired manner. The disclosed device can be used in a wide variety of applications and combinations where delivery of light at different wavelengths to several sites and of various intensity is desired, with the ability to record an optical signal from the several sites. 
     The combining of the optical stimulation and fluorescence photometry is accomplished using a filtering assembly in which light provided to an optogenetics illumination port from a high-density fiber-optic array that may be arranged in a grid, is collimated and projected on a fiber-optic array at the sample port, which may also be arranged in a grid. Fluorescence excitation is introduced through another connection. Excitation and emission light are spectrally separated and routed by the filter assembly. By inserting light-source-coupled optical fibers in selected receptacles within the grid of an optogenetics illumination port, it is possible to efficiently illuminate the desired targeted sites. The optogenetics stimulation illumination on the biological sample is not necessarily a grid that matches that of the optogenetics illumination port, rather, the arrangement of fibers facing the biological sample can target specific neural centers that are not necessarily in a regular grid, and are known to be at specific positions on the sample. An important aspect of the system is the ability of the system to combine fluorescence excitation light that may target the entire biological sample, with optogenetics stimulation illumination, which is generally of greater intensity than the fluorescence excitation light, and which may target only specific sites on the biological sample while simultaneously recording the fluorescence response. 
     Referring to  FIG.  1   , a pictorial diagram is shown, depicting an example system  10 , in accordance with an embodiment of the disclosure. Example system  10  is an optical measurement system for performing measurements/experiments on a sample  3 , which may be, for example, a brain in an in vivo animal specimen or brain tissue forming an ex vivo specimen. Illustrated system  10  is capable of providing stimulus illumination, as described above, that may be for optogenetic stimulus, or may be light for performing uncaging of bio-molecules or for performing laser ablation of tissue within sample  3 . For the purpose of simplification of description of system  10  and further systems and devices included in the various Figures, it will be understood that mention of optogenetics with respect to stimulus illumination and measurement will apply also to biomolecular uncaging and laser ablation functions, as well, with selection of suitable power levels and wavelengths understood. 
     System  10  includes an excitation illumination source  18  for fluorescence measurement/monitoring and a stimulation illumination source  12  for performing stimulus on or in sample  3 . In the depicted embodiment, stimulus illumination source  12  includes an illumination array  22  that produces a pattern of illumination that is mapped to individual optical fibers (or alternatively individual sub-bundles of optical fibers) within an optical fiber bundle  14 A. Alternatively, in any of the locations in which an optical fiber bundle is applied in the disclosure, a multiple core fiber may alternatively be used, with suitable coupling to introduce and receive light to and from the cores in the multiple core fiber. In the illustrated example, a 19-fiber optical fiber bundle  14 A is employed to convey stimulation light from individual illumination elements of array  22 , which may be an array of light-emitting diodes (LEDs), LED lasers, lamps, or other suitable stimulus illumination source. Sources included in stimulus illumination source  12  can vary in type, number and emitted wavelength, depending on the requirements of the experiment. Each source may be individually controlled by software executing on a controller as described in further detail below. Each of the optical fibers of optical fiber bundle  14 A are individually terminated and may be connected to any of the individual sources included in stimulus illumination source  12  as required by the experiment design. Optical fiber bundle  14 A is connected to a combiner/splitter device  30  in accordance with an embodiment of the disclosure, with a keyed connector  13 A that provides proper optical alignment via a connector key  15 A and prevents mis-rotation of optical fiber bundle  14 A with respect to a stimulus illumination mating connector  11 A provided on a housing of combiner/splitter device  30 , so that the patterned relationship between the individual fibers and the illumination source elements is maintained. Connectors  13 A and  11 A ensure that the transverse and longitudinal position of the ends of the individual fibers are correct, and that efficient coupling of light from the fibers delivering the stimulus illumination to sample  3  is achieved. 
     In order to provide proper mapping from elements of stimulus illumination array  22  to individual regions  5 A- 5 D within sample  3 , i.e. individual cells or groups of cells within sample  3 , at which ends of a number of optical fibers  16 C are implanted, sample  3  is connected to optical combiner/splitter device  30  with another keyed connector pair: connector  13 B, which receives the proximal end of another multiple fiber bundle  14 B and has a connector key  15 B, and another mating connector  11 B provided on the housing or combiner/splitter device  30 . The pattern of the stimulus illumination may be generated by using a regular array of optical fibers held together by a mechanical part defining the pattern, i.e., connector  13 A. The number, type and length of the optical fibers in the connector can vary to adapt to the experiment. Both the sample connector  13 B and the patterned stimulus illumination connector  13 A may use the same mechanical design to place and hold the fibers. The number, size and pattern of the optical fibers in the fiber array can vary depending on the requirements of the experiments. Patterns in the sample and optogenetics port can be different as long as there is an overlap and light coupling from optogenetics port to the sample port. As mentioned above, in some embodiments, a multicore fiber may be used instead of an array of single fibers at both the stimulus illumination connector  11 A and the sample connector  11 B. A tapered multicore fiber connector or a similar device is used to couple light to/from the different cores of the multicore fiber at the optogenetics port. Multicore fiber is more compact and can be easier to handle than a fiber array. 
     Also illustrated are a multiple-channel optical rotary joint  9 , which may be needed for live animal studies and a multi-fiber cannula  7  (or alternatively, multiple cannulas), which may provide detachable connections to sample  3 . Details of multi-channel rotary joints that may be used in implementing example system  10  are disclosed in U.S. Pat. No. 9,046,659, the disclosure of which is incorporated herein by reference. Regions  5 A- 5 D are illustrations of various combinations of optical fibers that show other than 1:1 correspondence between regions and physical location. For example, a high-stimulation intensity region (or a higher resolution fluorescence image measurement region)  5 C might be implemented using, for example, 7 fibers out of multiple fiber bundle  14 B as shown, while regions  5 A and  5 B are connected to only one fiber and region  5 D is connected to two. In addition to allowing for disparate regions at which individual optical fibers  16 C terminate optical fiber bundle  14 B, because stimulus illumination array  22  is electronically controlled and composed of individual illumination elements, stimulus illumination can be sequentially applied to regions  5 A- 5 D, by addressing the individual elements of stimulus illumination array  22 , or any combination of regions  5 A- 5 D may be simultaneously stimulated by stimulus light. In general, a one-to-one mapping of elements in stimulus illumination array  22  to a corresponding region is not required, as long as a specific element in stimulus illumination array  22  is coupled to a specific region of sample  3 , for those elements of stimulus illumination array  22  that are to be used in an experiment. In some embodiments, two or more separate patterned stimulation illumination ports, e.g., for optogenetic stimulation, may be provided. For example, one port may be provided for each function among multiple functions that stimulate sample  3  at different wavelengths, such as wavelengths for neuron activation or inhibition. In such embodiments, additional filters, e.g., dichroic mirrors are needed to combine light from additional input port(s) into the sample connection. Patterns of any of the stimulation illumination input ports would then coincide fully or partially with the pattern of stimulus illumination provided at the sample port. 
     The fluorescence measurement/monitoring function of system  10  is provided by a fluorescence excitation illumination source  18  that is coupled to optical combiner/splitter device  30  at a single-fiber (or fiber lightguide) connector  11 D that receives the fluorescence illumination from fluorescence excitation illumination source  18  and produces substantially uniform illumination across the proximal ends of fiber bundle  14 B at connector  11 B, via action of optical combiner/splitter device  30 , details of which will be described in further detail below. Alternatively, fluorescence excitation illumination source  18  may be connected directly to optical combiner/splitter device  30 . Multiple excitation illumination sources  18  having differing wavelengths may be coupled with individual optical fibers to multiple connectors  11 B to perform detection of different markers having different excitation wavelengths. While the embodiments disclosed herein are described with respect to fluorescence excitation, it is understood that photoluminescence stimulation generally, including fluorescence and up-conversion are contemplated for measurement using the devices and systems disclosed herein. Excitation illumination source  18  may be or include a pulsed laser for energy resonance transfer measurements/decay time measurements and may comprise one or more LED sources that are combined into a single optical fiber. The power level and activation times of the individual sources are controlled electronically by a controller or computer under software control as described in further detail below. Fluorescence measurement/monitoring of the fluorescence light returning from sample  3  through optical fiber bundle  14 B is made via an image sensor  24  within a fluorescence detector  17  that is coupled to optical combiner/splitter device  30  by an optical fiber cable (or fiber lightguide)  14 D at a connector  11 C. Alternatively, an image sensor  24 A may be integrated within optical combiner/splitter device  30 , in which case an electrical control and image data interface is provided at the exterior of optical combiner/splitter device  30 , rather than optical fiber cable  14 D. Image sensor  24  and  24 A, may be CMOS or CCD arrays, or may be an array of photodiodes or photomultiplier tubes (PMTs) if faster acquisition time is needed. Fluorescence detector  17  may be, or may include a spectrometer that measures a wavelength of the fluorescence emissions returning from sample  3 . 
     Referring now to  FIGS.  2 A- 2 D , pictorial diagrams depict various states of illumination within fiber bundle  14 B in example system  10  of  FIG.  1   , in accordance with embodiments of the disclosure.  FIG.  2 A  shows a state in which all of the stimulus illumination elements in array  22  of  FIG.  1    are off, and no optogenetic stimulus or fluorescence excitation light is provided through optical fiber bundle  14 B.  FIG.  2 B  illustrates a state in which fluorescence excitation, shown as an upward-right hashing, is provided to optical fiber bundle  14 B, which is substantially uniform across the entire set of fibers.  FIG.  2 C  illustrates stimulus illumination provided to only some of the optical fibers of optical fiber bundle  14 B, as illustrated by a downward-right hashing.  FIG.  2 D  illustrates simultaneous application of the fluorescence illumination of  FIG.  2 B  and the stimulus illumination of  FIG.  2 C . 
     Referring now to  FIG.  3   , a block diagram depicts an example system  10 A, in accordance with an embodiment of the disclosure. Example system  10 A is illustrated to show control of systems such as example system  10 , and details of an example combiner/splitter device  30 A that may be used to implement optical combiner/splitter device  30  in example system  10  of  FIG.  1   . Therefore, example system  10 A is similar to, and has common elements with, example system  10 , so only differences between example system  10 A and example system  10  will be described in detail below. A system controller  32  provides power to and control of fluorescence illumination source  18  so that fluorescence measurements can enabled by timed application of fluorescence excitation illumination. System controller  32  also controls stimulation illumination source  12 , to select whether or not stimulus illumination is provided to optical fiber bundle  14 A, and if so, which fibers within optical fiber bundle  14 A will receive stimulus illumination, by selecting which elements in stimulus illumination array  22  of  FIG.  1    are active. Optical combiner/splitter device  30 A includes a stimulation illumination optical path s containing the selected pattern of stimulus illumination, a fluorescence excitation optical path e, which carries the single-spot fluorescence excitation illumination, and an imaging optical path i, along with the image of returning fluorescence is guided to optical combiner/splitter device  30 A to image sensor  24 A. A power, control and data interface  39  is provided at the exterior of optical combiner/splitter device  30 A to interface image sensor  24 A with system controller  32 , so that system controller  32  can provide fluorescence detection data output for further processing/analysis. 
     Within example optical combiner/splitter device  30 A, the combining and separation of light at different wavelengths is performed by a pair of dichroic mirrors  35 A,  35 B. Connector  11 B receives an interchangeable pattern of fibers forming an array within optical fiber bundle  14 B that can be inserted into the sample port connector  11 B, which positions the patterned fiber optic array longitudinally at the focal plane of an infinity corrected optical lens  31 E. Connector  11 D receives the optical fiber connection from fluorescence illumination source  18 . The fluorescence excitation light is received by a pair of optical lenses  31 B,  31 C, that, together with infinity corrected optical lens  31 E project uniform illumination at the proximal end of optical fiber bundle  14 B using the Kohler illumination method. The spot-size of the uniform illumination arriving at the sample port connector  11 B is larger than the receptacle having the fiber array arranged in the pattern at the sample port, and thus couples to all fibers in all possible fiber array patterns. A band-pass filter  33 B is used to select only the wavelength range of the excitation light received at connector  11 D that is needed to generate the fluorescent signal. In some embodiments, the fluorescence excitation illumination may be frequency modulated, in order to produce a signal modulated at the same frequency. Such modulation techniques enable use of lock-in techniques to isolate and detect the signal with a greatly improved signal-to-noise ratio (SNR) in the electrical domain. In some embodiments, the fluorescence excitation illumination may lie in the infrared (IR) or near-infrared (NIR) region. The fluorescence excitation illumination may thereby penetrate deeper into tissue and may be used to excite unconverting nanoparticles (UCNPs), as disclosed in U.S. Pat. No. 9,522,288, which then emit light in the ultraviolet (UV) to visible (VIS) region of the spectrum. Excitation of UCNPs usually requires higher light intensity than does excitation of fluorophores. Therefore, such excitation may not necessarily be projected evenly into all optical fibers of the fiber array in the sample port, but instead may target one or a selected group of fibers in the sample port. 
     A dichroic mirror  35 B separates the returning fluorescence pattern, i.e., the fluorescence signal or resultant image, from the illumination pathways so that the light returning from sample  3  via sample connector  11 B is directed toward image sensor  24 A, which, in the depicted embodiment is a CMOS sensor array. Image sensor  24 A is at the focal point of an infinity corrected imaging lens  31 D. In some embodiments, the image of the signal may be inverted or magnified by the imaging optics. A band-pass filter  33 C selects the wavelength range of the fluorescence signal. The pattern of the multiple fibers of fiber bundle  14 B connected to sample connector  11 B is imaged on image sensor  24 A and the signal of each individual optical fiber within the pattern can be distinguished. The data from the imaging sensor is transferred to system controller  32 , and processed by image processing software. A dichroic mirror  35 A combines the stimulus light pattern received from optical fiber bundle  14 A at connector  11 A, which is at the focal point of an infinity-corrected optical lens  31 A. A bandpass filter  33 A selects the wavelength range needed to stimulate sample  3 . In other embodiments, dichroic mirrors  35 A,  35 B may be replaced with other types of combiners/splitters. For example, combining of excitation illumination from fluorescence illumination source  18  with the stimulation illumination received from connector  11 A may be performed by a spectral combiner, a polarization combiner, or by a beam splitter. 
     Referring now to  FIG.  4   , a pictorial diagram depicting details of another example optical combiner/splitter device  30 B that may be used in example system  10  of  FIG.  1    and example system  10 A of  FIG.  3   , is shown, in accordance with an embodiment of the disclosure. Combiner/splitter device  30 B is similar to combiner/splitter device  30 A of  FIG.  3   , so only differences between them will be described in detail below. Combiner/splitter device  30 B illustrates a different arrangement of components, and it is understood that additional other embodiments and arrangements are possible within combiner/splitter device  30  of  FIG.  1   . In combiner/splitter device  30 B, a dichroic mirror  35 C directs the stimulus illumination received from connector  11 A via an infinity-corrected optical lens  31 G, which is filtered by bandpass filter  33 A, to a dichroic mirror  35 D which combines the stimulus illumination with the fluorescence excitation illumination received at connector  11 C. Dichroic mirror  35 C separates the fluorescence signal, i.e., the fluorescent image returning from sample connector  11 B and that is passed through dichroic mirror  35 D. Bandpass filter  33 C selects the wavelength range of the fluorescence signal, which is coupled to an external imaging system via connector  11 C, which is provided with the image through an infinity-corrected lens  31 F. 
     Referring now to  FIG.  5   , a pictorial diagram depicts an example stimulus illumination system  50  that may be used in example system  10  of  FIG.  1    and example system  10 A of  FIG.  3   , in accordance with an embodiment of the disclosure. The depicted embodiment illustrates a use of optical combiner/splitter device  30 B that does not require a patterned stimulus array or other arrangement of stimulus sources, and which applies to other optical combiner splitter devices disclosed herein. Stimulus illumination is provided by a stimulus illumination source  12 A that includes a source element  42 , e.g., an LED or laser diode, etc., as described above. The output of source element  42  is demultiplexed by an optical switch that selects one of several optical fiber connections provided to a connector  43  that receives an end of optical fiber bundle  14 A. The depicted embodiment allows for sequential selection of individual ones of optical fibers  16 C in the system of  FIG.  1   , to illuminate a single region of sample  3 . 
     Referring now to  FIG.  6   , a pictorial diagram depicts an example stimulus illumination system  60  that may be used in example system  10  of  FIG.  1    and example system  10 A of  FIG.  3   , in accordance with an embodiment of the disclosure. The depicted embodiment illustrates a use of optical combiner/splitter device  30 B that does not require a patterned stimulus array or other arrangement of stimulus sources, and which applies to other optical combiner splitter devices disclosed herein. Stimulus illumination is provided by a stimulus illumination source  12 B that includes source element  42  and a beam splitter  46  that divides the stimulus illumination among several optical fiber connections provided to connector  43 . The depicted embodiment allows for simultaneous stimulus illumination provided to individual optical fibers  16 C in the system of  FIG.  1   , to illuminate multiple regions of sample  3  simultaneously. 
       FIG.  7    is a perspective view showing an example multiple fiber optic connector pair  70  that may be used in example system  10  of  FIG.  1    and example system  10 A of  FIG.  3   , in accordance with an embodiment of the disclosure. A plurality of polished ends of optical fibers  73  are inserted in a “chassis-type” multiple-position connector  11 A, 11 B, which are then brought into proximity with polished ends of optical fiber bundle  14 A,  14 B that provide the pattern that is conveyed through the multiple fiber optic connector pair  70  as described above for the stimulus illumination and sample connections. Connector keys  15 A,  15 B provide a rotationally fixed indexing system that rigidly holds connector  72 A in a fixed rotational position by insertion in mating index groove  74  of connector  72 B. A threaded portion  75  of connector  72 A receives a threaded knurled sheath  71  that secures  72 B to connector  72 A. 
     In summary, this disclosure shows and describes systems and methods for performing optical stimulation and fluorescence measurements on a sample. The methods are methods of operation of the systems. The systems may include an illumination array having multiple stimulation illumination elements that are electronically addressable to select a spatial pattern of illumination, an optical fiber bundle including multiple optical fibers coupled to corresponding ones of the multiple stimulation illumination elements at a proximal end of the optical fiber bundle and for coupling to target locations within or on a sample at a distal end of the optical fiber bundle, so that activated ones of the multiple stimulation illumination elements provide stimulation at corresponding ones of the target locations. The systems may also include a fluorescence illumination source for providing fluorescence illumination coupled to the proximal end of the optical fiber bundle, and an imaging system coupled to the proximal end of the optical fiber bundle for measuring fluorescence returning from the target locations. 
     In some example embodiments, the system may include a combiner as an optical device integrated in the system and located at the proximal end of the optical fiber bundle. The combiner may have a first input coupled to the illumination array, a second input coupled to the fluorescence illumination source and an output coupled to the imaging system. In some example embodiments, the optical fiber bundle includes a first connector at the proximal end, and the first connector may have at least one first indexing feature having a fixed spatial relationship with individual ones of the multiple optical fibers. The combiner may further include a housing, a sample connector accessible at an exterior of the housing for receiving connection of the first connector, the sample connector may have at least one first complementary indexing feature, so that a fixed correspondence between the individual ones of the multiple fibers and the multiple stimulation elements is maintained. The optical fiber bundle may be a first optical fiber bundle, the combiner may further include a stimulation illumination input connector accessible at an exterior of the housing and having a second indexing feature, and the system may further include a second optical fiber bundle comprising second multiple optical fibers coupled to corresponding elements of the illumination array at a distal end thereof and coupled to the stimulation illumination input connector by a second connector. The second connector may have at least one second complementary indexing feature complementary to the second indexing feature, so that a fixed correspondence between the individual ones of the second multiple fibers and the elements of the illumination array is maintained. 
     In some example embodiments, the combiner may further include a fluorescence illumination input connector accessible at an exterior of the housing for receiving an optical connection from the fluorescence illumination source, and an output connector for receiving a connection conveying fluorescence image information to the imaging system. In some example embodiments, the output connector is an electrical connector, and the imaging system may include an opto-electronic image sensor mounted inside the housing and having data and control signals coupled to the output connector, so that the opto-electronic image sensor receives an image of fluorescence returning from the multiple optical fibers at the sample connector. In some example embodiments, the output connector may be an optical connector for receiving a connection to an external optical connection to convey an image of fluorescence returning from the multiple optical fibers at the sample connector to an image sensor of the imaging system. In some example embodiments, the fluorescence illumination source provides a spot size larger than the optical aperture of the optical fiber bundle at the sample connector, so that the fluorescence illumination is coupled substantially uniformly to the multiple optical fibers at the sample connector. 
     In some example embodiments, the combiner further includes a first lens system for imaging the spatial pattern of illumination received from the stimulation illumination input connector on the sample connector, and a second lens system for imaging the light returning from the sample through the first dichroic plate at the output connector. In some example embodiments, the system includes an electronic controller coupled to the illumination array for controlling provision of illumination from one or more of the multiple stimulation illumination elements to select the spatial pattern of illumination. The illumination array may include one or more optical switches coupled to the electronic controller for controlling the provision of illumination from the multiple stimulation illumination elements to select the spatial pattern of illumination, whereby the multiple stimulation illumination elements may remain active and selectable via the one or more optical switches to provide or not provide a contribution to the spatial pattern of illumination. 
     It should be understood, especially by those having ordinary skill in the art with the benefit of this disclosure, that the various operations described herein, particularly in connection with the figures, may be implemented by other components. The order in which each operation of a given method is performed may be changed, and various elements of the systems illustrated herein may be added, reordered, combined, omitted, modified, etc. It is intended that this disclosure embrace all such modifications and changes and, accordingly, the above description should be regarded in an illustrative rather than a restrictive sense. Similarly, although this disclosure makes reference to specific embodiments, certain modifications and changes may be made to those embodiments without departing from the scope and coverage of this disclosure. Moreover, any benefits, advantages, or solutions to problems that are described herein with regard to specific embodiments are not intended to be construed as a critical, required, or essential feature or element. 
     While the disclosure has shown and described particular embodiments of the techniques disclosed herein, it will be understood by those skilled in the art that the foregoing and other changes in form, and details may be made therein without departing from the spirit and scope of the disclosure.