Cable movement-isolated multi-channel fluorescence measurement system

1. Field of the Invention

The present invention relates to the fields of spectroscopy and fluorescence recording, and in particular to a multi-channel fluorescence measurement system having fluorescence stimulation/detection units incorporated at the rotor of an electrical rotary joint for in vivo fluorescence recording in biological tissue for biomedical research applications.

2. Background of the Invention

Fiber photometry is an optical technique initially developed for neuroscience applications to record the activity of a specific population of neurons in behaving animals (e.g. brain activity). To become light sensitive, brain tissue is labeled with genetically encoded functional fluorescent proteins (e.g., Calcium indicators) that have fluorescence emissions which are modulated by activity of the labeled cells.

In contrast to miniaturized epi-fluorescence microscopy designed for activity monitoring of single cells, spatial resolution of fiber photometry is typically limited by the diameter of the optical fiber cable used to perform the measurement. A typical signal in fiber photometry provides a sum of the activity of hundreds of cells located close to the tip of a fiber optic implant. Early fiber photometry systems were developed to record a single brain region connected to a single optical fiber cable, and typically use photodiodes to perform measurements. When making measurements on freely moving animals, optical rotary joints are used to prevent twisting of the optical fiber cable and to reduce stress on the animal that would otherwise be caused by the pulling/twisting of the cable. In order to simultaneously record the fluorescence activity of multiple brain regions, proposed solutions use an image sensor in place of the photodiodes to image the output of several optical fibers cable that are connected to different brain regions.

However, both the existing and proposed solutions described above are limited, in that using an optical rotary joint induces signal fluctuations caused by the rotation and limits the system to two optical cables when a two-channel optical rotary joint is used, limiting the measurement to two brain regions. In the proposed image sensor solution, single channel or two-channel optical rotary joints cannot be used to transfer an optical image, so the rotary joints are not compatible with available systems for multiple site recording that use an image sensor as a detector.

Therefore, it would be desirable to provide a multi-channel fluorescence measurement system that can provide multiple simultaneous fluorescence measurements of brain regions, while releasing cable twisting when the animal is freely behaving.

The above objectives, as well as other improvements, are achieved in an optical measurement system for performing fluorescence measurements, a rotary joint and a method of performing fluorescence measurements. The rotary joint is a rotary joint forming part of the optical measurement system and the method is a method of operation of the optical measurement system.

The optical measurement system performs multiple fluorescence measurements at multiple distinct regions within a moving sample, and includes a plurality of optical fibers having first ends coupled to the moving sample. The optical system also includes an electrical rotary joint including a rotor and a stator. The rotor includes at least one optical connection for receiving second ends of the optical fibers, and at least one fluorescence stimulation/detection unit coupled to the optical connection(s) for introducing excitation light to the plurality of optical fibers and receiving returning fluorescence light from the plurality of optical fibers. The stimulation/detection units include at least one optical filter optically coupled to the connection(s) for separating the returning fluorescence light from the excitation light, at least one light source for providing the excitation light, and at least one electro-optical detector coupled to the optical connection(s) for detecting the returning fluorescence light. An output of the electro-optical detector(s) is coupled from the rotor to the stator of the rotary joint, and the stator provides at least one electrical output for providing electrical connection to the at least one electro-optical detector and power for the light source is also coupled through the electrical rotary joint. The optical measurement system also includes a data acquisition unit with one or more electrical detection inputs coupled to the electrical output(s) of the stator of the electrical rotary joint for recording an output of the electro-optical detector(s) and an output for controlling power supplied to the input of the at least one light source.

The connection from the optical fibers may be a single bundled connection of the second ends of the optical fibers, and the electro-optical detector may use an image sensor to image the ends of the optical fibers to produce measurements of the returning fluorescence emission from each fiber. Alternatively, the connection from the optical fibers may be a corresponding plurality of connections, with multiple electro-optical detectors included in or on the rotor to perform individual measurements of the returning fluorescence emission from each fiber.

The foregoing and other objectives, features, and advantages of the invention will be apparent from the following, more particular, description of the preferred embodiment of the invention, as illustrated in the accompanying drawings.

The novel features believed characteristic of the invention are set forth in the appended claims. The invention itself, however, as well as a preferred mode of use, further objectives, and advantages thereof, will best be understood by reference to the following detailed description of an illustrative embodiment when read in conjunction with the accompanying drawings, wherein like reference numerals indicate like components, and:

FIG. 1A is a block diagram of an example optical measurement system 100 A in accordance with an embodiment of the disclosure.

FIG. 1B is a block diagram of another example optical measurement system 100 B in accordance with another embodiment of the disclosure.

FIG. 2A is a pictorial diagram depicting an example optical cable bundle 120 of optical measurement system 100 A depicted in FIG. 1A .

FIG. 2B is a pictorial diagram depicting an example fiber optic cable 8 B of optical measurement system 100 B depicted in FIG. 1B .

FIG. 3 is a side view of an example electrical rotary joint 130 A of optical measurement system 100 A depicted in FIG. 1A .

FIG. 4 is a bottom view of example electrical rotary joint 130 A of FIG. 3 .

FIG. 5A is a side cut-away view of a fluorescence stimulation/detection unit 12 A of electrical rotary joint 130 A of FIG. 3 and FIG. 4 .

FIG. 5B is a side cut-away view of an alternative fluorescence stimulation/detection unit 12 C that may be used in electrical rotary joint 130 A of FIG. 3 and FIG. 4 .

FIG. 6A is a pictorial diagram depicting an illumination pathway within fluorescence stimulation/detection unit 12 A of FIG. 5A .

FIG. 6B is a pictorial diagram depicting an illumination pathway within fluorescence stimulation/detection unit 12 B of FIG. 10 .

FIG. 7A is a pictorial diagram depicting an example of a patterned optical filter that may be used in fluorescence stimulation/detection unit 12 A of FIG. 5A for multi-color imaging.

FIG. 7B is a graph depicting the transmission spectra of a patterned optical filter that may be used in fluorescence stimulation/detection unit 12 A of FIG. 5A for multi-color imaging.

FIG. 8 is a side view of an example electrical rotary joint 130 B of optical measurement system 100 B of FIG. 1B .

FIG. 9A is a bottom view of example electrical rotary joint 130 B of FIG. 8 .

FIG. 9B is a bottom view of another example electrical rotary joint 130 C that may be used within optical measurement system 100 B depicted in FIG. 1B .

FIG. 9C is of example electrical rotary joint 130 B of FIG. 8 with one of fluorescence stimulation/detection units 12 B removed.

FIG. 10 is a side cut-away view of a fluorescence stimulation/detection unit 12 B of electrical rotary joint 130 B of FIG. 8 .

FIG. 11 is a side view of an example of optical measurement system 100 B of FIG. 1B , including a motor-assisted electrical rotary joint 130 D.

The present disclosure provides examples of photometry systems for simultaneous monitoring of cell activity in different regions, e.g., brain, spinal cord, muscles, etc., in freely behaving animals, e.g., rodents or monkeys. To monitor the different regions simultaneously, an optical fiber cable is connected to each region of the sample and relays the fluorescence signal from the sample to the light detector, as well as providing excitation light to the sample regions. The optical fibers are connected to the rotor of an electrical rotary joint that releases cable twisting when the animal is freely behaving. Two distinct example embodiments are disclosed herein. In one embodiment, the proximal ends of all the optical fiber cables are combined and imaged on a single image sensor (e.g. CCD, CMOS, . . . ) to simultaneously monitor the activity of all the sites. Only one set of optical filters is required and the number of recording sites is only limited by the number of optical fibers that can be imaged within the field of view of the system.

In another embodiment, the fluorescence signal returning from multiple optical fibers is individually detected by a corresponding photo-detector, e.g., a photodiode, photo-transistor or photomultiplier tube (PMT). The photo-detectors are synchronized to simultaneously record the activity of all of the brain regions. In the instant configuration, one set of optical filters is required for each optical fiber.

In both of the disclosed embodiments, to release twisting of the optical fiber connected to the animal, a rotor of an electrical rotary joint incorporates the fluorescence detection/stimulation unit(s) that provide the excitation light and detect the returning fluorescence emission light. The rotor conveys electrical signals to and from the stator of the rotary joint, which is connected to a data acquisition and control unit. Free rotation is essential for multi-brain-site measurements where multiple optical fiber cables are connected to a moving sample. The rotary joint may be passive or may be torque-assisted. In a passive mode or in passive implementations, the rotor passively follows the movement of the animal using low friction bearings and low friction electrical contacts. To ensure sufficient electrical conductivity, the electrical contacts will necessarily introduce friction, which will produce resistance to rotation that is difficult to overcome for small rodents, such as mice. Therefore, in an active mode or an active implementation, a torque-assisted electrical rotary joint is provided, in which a torque sensor detects a torque applied to the rotor by the animal and triggers motor-assisted rotation. The active mode/implementation is particularly suitable for use with animals of small weight, such as mice.

Referring now to FIG. 1A , an example optical measurement system 100 A is shown, in accordance with an embodiment of the disclosure. Optical measurement system 100 A is a fiber photometry system for measuring fluorescence emissions returning from multiple regions within a sample 9 over multiple fiber optic cables 8 A, which may be single or multi-core. First (distal) ends of fiber optic cables 8 A are inserted into different regions of sample 9 , or are connected to further optical probes inserted into different regions of sample 9 , via connections at the distal ends thereof. Fiber optic cables 8 A conduct excitation light at one or more frequencies that stimulate fluorescence emissions from the different regions of sample 9 , as well as returning the light produced by the fluorescence emissions. Fiber optic cables 8 A are connected at their proximal ends via a connector 26 A that receives an optical cable bundle 120 that combines fiber optic cables 8 A mechanically, so that an image produced by the exit profile of optical cable bundle 120 is a composite image of the individual second ends of fiber optic cables 8 A, which correspond to different areas of the resulting image. Connector 26 A is connected to a corresponding connector 13 A of a fluorescence stimulation/detection unit 12 A, which is a module mounted on or provided internal to a rotor 4 A of an electrical rotary joint 130 A. Fluorescence stimulation/detection unit 12 A includes a set of optical filters 7 A that serve as a diplexer to separate stimulation light from the returning fluorescence light produced by sample 9 , as well as providing narrowband filtering to improve reception of the returning fluorescence light. Fluorescence stimulation/detection unit 12 A also includes one or more light sources 5 A, e.g., light-emitting diodes (LEDs) or laser diodes, that generate excitation light at the excitation wavelength(s) and provide the excitation light through optical filters 7 A to fiber optic cables 8 A. Fluorescence stimulation/detection unit 12 A also includes an image sensor 6 that captures the composite image provided by the exit profile of optical cable bundle 120 at connector 13 A, with each of fiber optic cables 8 A corresponding to a different area of the image captured by image sensor 6 .

In order to prevent twisting of fiber optic cables 8 A and resulting extraction of probes from sample 9 or other harm to sample 9 and/or the measurements, image data and control signals 11 are electrically coupled between rotor 4 A and a stator 3 A of electrical rotary joint 130 A. The image data includes a representation of the composite image of the fluorescence emissions captured by fiber optic cables 8 A, with individual areas of the image corresponding to individual ones of fiber optic cables 8 A. The intensity within each of the areas are then averaged with a weighting function to produce an intensity result for each of fiber optic cables 8 A over time. The collection and averaging of the image sensor data is performed by a computer 1 coupled to a data acquisition and control unit 2 , or alternatively, a portion or all of the weighting can be performed within data acquisition and control unit 2 , with computer 1 collecting and further processing the resulting output of data acquisition and control unit 2 . Computer 1 executes dedicated software that can be synchronized with other components via analog and/or digital inputs/outputs of data acquisition and control unit 2 . The software features a graphic interface to synchronize data acquisition operations and to specify parameters of recording and light control as performed by optical measurement system 100 A.

Referring now to FIG. 1B , an example optical measurement system 100 B is shown, in accordance with another embodiment of the disclosure. Example optical measurement system 100 B is similar to example optical measurement system 100 A described above, so only differences between the two embodiments will be described below. In example optical measurement system 100 B, fiber optic cables 8 B are not bundled, but are rather connected to multiple individual fluorescence stimulation/detection units 12 B incorporated in or on an electrical rotary joint 130 B. A single example of fluorescence stimulation/detection units 12 B is illustrated in FIG. 1B , for clarity. Fluorescence stimulation/detection units 12 B is connected to fiber optic cables 8 B by individual connectors 26 B and corresponding individual connectors 13 B, one for each of fluorescence stimulation/detection units 12 B. Fluorescence stimulation/detection units 12 B include light sources 5 B and optical filters 7 B, which perform the same functions as described above with reference to FIG. 1A as light sources 5 A and optical filters 7 A. However, each of fluorescence stimulation/detection units 12 B includes a set of light sources 5 B and optical filters 7 B for excitation and measurement of a single region within sample 9 , and a photo-detector 10 , e.g., a photodiode, phototransistor or PMT is used to measure the returning fluorescence light returned by the corresponding one of fiber optic cables 8 B. Output signals from photo-detector 10 and control signals for operating light sources 5 B are coupled from a rotor 4 B of electrical rotary joint 130 B to a stator 3 B and are sent to data acquisition and control unit 2 and to computer 1 for further processing.

Referring now to FIG. 2A , an example optical cable bundle 120 is shown, as may be used in optical measurement system 100 A of FIG. 1A . Individual fiber optic cables 8 A are connected to the animal, i.e., coupled to probes within the animal, with miniature optical connectors 27 , e.g., ferrules and sleeves. A more robust connector type, e.g., FC-PC or SMA optical connectors is used as connector 26 A which connects to connector 13 A in optical measurement system 100 A of FIG. 1A and which combines the individual cores of fiber optic cables 8 A as shown in an interface 25 A, which produces the image received by fluorescence stimulation/detection unit 12 A, and also receives a uniform illumination by the excitation light, as will be described in further detail below. The use of miniature optical connectors 27 and connector 26 A provides optical connection repeatability and easy replacement of fiber optic cables 8 A. The cores of fiber optic cables 8 A are in the range from 10 μm to 1 mm, and the numerical aperture is above 0.20 for light collection efficiency.

Referring now to FIG. 2B , an example fiber optic cable 8 B is shown, as may be used in optical measurement system 100 B of FIG. 1B . Individual fiber optic cables 8 B are connected to the animal, i.e., coupled to probes within the animal, with miniature optical connectors 27 , e.g., ferrules and sleeves. A more robust connector type, e.g., FC-PC or SMA optical connectors are used as connector 26 B, which provides individual interfaces 25 B, one at the end of each of multiple fiber optic cables 8 B, which are connected to corresponding connectors 13 B in optical measurement system 100 B of FIG. 1B . As in optical cable bundle 120 of FIG. 2A , the cores of fiber optic cables 8 B are in the range from 10 μm to 1 mm, and the numerical aperture is above 0.20 for light collection efficiency.

Referring now to FIG. 3 and FIG. 4 , a side view and a bottom view, respectively, of electrical rotary joint 130 A of optical measurement system 100 A of FIG. 1A are shown. The positions of rotor 4 A and stator 3 A are shown, and rotor 4 A is rotationally coupled to stator 3 A with a low-friction bearing and electrical interconnects. Electrical connections 14 A are provided on stator 3 A for connection to data acquisition and control unit 2 of FIG. 1A . Fluorescence stimulation/detection unit 12 A is mounted to, or forms the body of rotor 4 A, which includes connector 13 A for attachment of connector 26 A of optical cable bundle 120 of FIG. 2A . By providing fluorescence stimulation/detection units 12 A as modules mounted to rotor 4 A, fluorescence stimulation/detection units 12 A can be interchanged for other fluorescence stimulation/detection units 12 A having different excitation and detection capabilities. For example fluorescence stimulation/detection units 12 A having different illumination sources and illumination/detection pathway filter wavelengths, or different numbers of detection and/or illumination features, as will be described for different applications below. The interchangeability provides compatibility with a multitude of fluorescent proteins having different excitation and emission spectra.

Referring now to FIG. 5A , a cut-away side view of fluorescence stimulation/detection unit 12 A is shown, illustrating details of the optical and electrical components within. Connector 13 A, which receives connector 26 A of optical cable bundle 120 of FIG. 2A and interface 25 A thereby, is optically coupled by an objective lens 15 . Excitation light is provided by light sources 5 A, e.g., LEDs or laser diodes, for fluorescence protein excitation. Inclusion of light sources 5 A in fluorescence stimulation/detection unit 12 A and thus on rotor 4 A of electrical rotary joint 130 A ensures excitation signal stability, since only electrical control signals and power sources that operate light sources 5 A pass through electrical rotary joint 130 A, and not the excitation illumination beams. An electrical connector 24 A provides for electrical connections between rotor 4 A and fluorescence stimulation/detection unit 12 A, providing power/control for light sources 5 A, as well as an interface to image sensor 6 within fluorescence stimulation/detection unit 12 A. Since optical power requirements for fiber-photometry measurement are low, the heat produced by light sources 5 A is also low and the heat can be dissipated passively with a passive heat sink 19 A. If higher thermal dissipation is required, a fan may be included to move air over the fins of heat sink 19 A. To deliver excitation light to all the brain regions with the same optical parameters (light intensity and angular distribution), fluorescence stimulation/detection unit 12 A is configured to generate a uniform illumination over the total field of view at the object plane located at the proximal end of the optical fibers of fiber optic cable 8 A, i.e., at interface 25 A shown in FIG. 2A . As illustrated in FIG. 6A , to which additional reference is made, in order to accomplish the above, each of light sources 5 A is imaged at the back focal plane 50 of objective lens 15 . FIG. 6A depicts the individual illumination pathway from each of light sources 5 A to interface 25 A of fiber optic cable 8 A of FIG. 1A . Imaging light sources 5 A at back focal plane 50 ensures that each of fiber optic cables 8 A are illuminated with the same angular distribution, and discontinuities at the surface of light sources 5 A are not imaged in the object plane. In the depicted embodiment, to improve filtering efficiency by reducing the angle of incidence on an excitation bandpass filter 20 and dichroic beamsplitter, the output of each of light sources 5 A is first collimated by an aspheric lens 17 before the collimated beams pass through excitation bandpass filter 20 and dichroic beamsplitter 21 . Then, another lens 18 , e.g., a plano-convex, aspheric, or achromatic lens positioned before the back focal plane of objective lens 15 , focuses the collimated beam at back focal plane 50 of objective lens 15 , resulting in uniform illumination across interface 25 A of FIG. 2A that combines the fibers of fiber optic cables 8 A.

To receive returning fluorescence light from the samples, in the example embodiment, objective lens 15 further generates a magnified image of the object plane with a fixed tube length, with no tube lens required. In order to minimize the exterior dimensions of the system and also to optimize aberration correction, a high numerical aperture aspheric lens is used for objective lens 15 . In the example, the numerical aperture of objective lens 15 is between 0.20 and 0.80. In another embodiment, objective lens 15 is infinity-corrected and a tube lens 16 , e.g., a plano-convex lens, aspheric lens, or achromatic lens, is inserted after dichroic beamsplitter 22 and a mirror 43 before reaching image sensor 6 in order to adjust the magnification and reduce the angular divergence of the returning fluorescence light at the surface of a single-band emission filter 23 A and dichroic beamsplitter 22 , thereby improving their efficiency. Single-band emission filter 23 A removes stray light coming from excitation light reflections within fluorescence stimulation/detection unit 12 A. In yet another embodiment, the aspheric lens is replaced by a 4 X to 20 X high numerical aperture infinity-corrected microscope objective for better correction of optical aberrations (chromatic aberration, spherical aberration, field curvature, and the like). Fluorescence stimulation/detection unit 12 A is configured to image a field of view of up to 3 mm×3 mm on image sensor 6 , e.g., a CMOS or CCD sensor, with 1 to 10× magnification. The size of the field of view limits the number of brain regions that can be monitored simultaneously. In interface 25 A of FIG. 2A , the proximal end of seven optical fibers 400 microns in diameter are imaged simultaneously.

In fluorescence stimulation/detection unit 12 A as illustrated in FIG. 5A , three sets of excitation bandpass filter 20 and dichroic beamsplitter 21 are included that form a primary filter set that combine the excitation illumination in the excitation optical pathway. However, the number of excitation sources, i.e., light sources 5 A may be from a single light source 5 A up to the limits of module size that can be provided on rotor 4 A and any degradation of the excitation illumination pathway through the multiple dichroic beamsplitters 21 . The primary filter set is configured to combine one, two or three light sources 5 A. The primary filter set features: an individual excitation bandpass filter 20 for each light source 5 A that reduces the spectral bandwidth of the illumination lights, and an individual dichroic beamsplitter 21 for each light source 5 A to combine two or three bands of excitation lights in a single excitation pathway. A secondary filter set separates the one, two or three bands of fluorescence signals emitted by the sample from one, two or three bands of excitation lights. The secondary filter set varies according to number and type of fluorescent markers to be detected.

In one example embodiment, the returning fluorescence measurement signal of one fluorescent protein is detected by image sensor 6 , and one light source 5 A is used for excitation. In such a configuration, the secondary filter set includes a first dichroic beamsplitter 22 that separates the fluorescence signals from the excitation, and single-band emission filter 23 A. For example, GCaMP6 fluorescent proteins having an emission wavelength of approximately 515 nm may be excited by an illumination wavelength of approximately 480 nm. In another embodiment, designed for genetically encoded calcium indicators, a second light source around the isosbestic calcium independent wavelength of the calcium indicator can be added in the system for control measurements. By subtracting the calcium-independent signal from the calcium dependant signal of the calcium indication, motion artifacts can be removed. For example, a second excitation light source at the isosbestic wavelength of the calcium indicator is added to the system for control measurements, e.g., approximately 405 nm for GCaMP6.

In another example of a multi-color configuration, the fluorescence signal of two fluorescent proteins are recorded simultaneously on image sensor 6 . In the example configuration, a first dichroic beamsplitter 22 separates the fluorescence signals from the excitations, and a dual-band bandpass filter is inserted in place of single-band emission filter 23 A, and is designed to pass the fluorescence from the two fluorescent markers is inserted in front of the image sensor 6 and removes stray excitation light from excitation light reflections within fluorescence stimulation/detection unit 12 A. In one embodiment, to separate the fluorescence signal from each marker and as shown in FIG. 7A , a matrix 28 of interleaved micro single-band-pass filters 29 , 30 is inserted in front of image sensor 6 and aligned with the pixels of image sensor 6 . FIG. 7B illustrates transmission bands 31 , 32 of the two sets of micro single-band-pass filters 29 , 30 that are centered on the fluorescence emission wavelengths of the two detected fluorescent markers. The dimension of micro single-band-pass filters 29 , 30 is a multiple of the pixel size of the sensor (1× to 10×). The example of detecting two colors with two transmission bands is not limiting, and more than two colors can be detected by interleaving additional micro-filters having other transmission bands. In still another embodiment, the fluorescence signal of two fluorescent proteins are recorded in sequence on a single image sensor 6 . In such a configuration, light sources 5 A corresponding to each fluorescent marker are turned on/off in a sequence synchronized with the acquisition rate of image sensor 6 . In one example, GCaMP6 fluorescent proteins having emission of approximately 515 nm may be excited by an illumination wavelength of approximately 480 nm, while RCaMP1 fluorescent proteins having an emission wavelength of approximately 600 nm are excited with an illumination wavelength around 565 nm. In another example, a third light source 5 A at the isosbestic wavelength of the calcium indicator is added to the system for control measurements (e.g. around 405 nm for GCaMP6).

Referring now to FIG. 5B , a cut-away side view of an alternative fluorescence stimulation/detection unit 12 C that may be used in place fluorescence stimulation/detection unit 12 A of FIG. 5A of is shown, illustrating details of the optical and electrical components within. Fluorescence stimulation/detection unit 12 C of FIG. 5B is similar to fluorescence stimulation/detection unit 12 A of FIG. 5A , so only differences between them will be described below. Two image sensors 6 A and 6 B are provided in fluorescence stimulation/detection unit 12 C and an additional (second) dichroic beamsplitter 45 is included to separate light of different wavelengths between sensors 6 A and 6 B, which provide separate image sensor outputs to data acquisition and control unit 2 of FIG. 1A via an electrical connector 24 C. A dual-band emission filter 23 B removes stray light coming from excitation light reflections within fluorescence stimulation/detection unit 12 C. Dichroic beamsplitter 22 separates the fluorescence measurement signals from the excitation illumination and second dichroic beamsplitter 45 separates the two fluorescence signals emitted by the two fluorescent markers. Image sensors 6 A, 6 B each include a single band emission filter 44 , 44 A, that removes stray excitation light from excitation light reflections within fluorescence stimulation/detection unit 12 C, and also the fluorescence measurement signal from the other markers. Fluorescence stimulation/detection unit 12 A is configured to image a field of view of up to 3 mm×3 mm on each of image sensors 6 A, 6 B, e.g., a CMOS or CCD sensor, with 1 to 10× magnification. The size of the field of view limits the number of brain regions that can be monitored simultaneously. In interface 25 A of FIG. 2A , the proximal end of seven optical fibers 400 microns in diameter are imaged simultaneously on both of image detectors 6 A, 6 B. In an example application, the fluorescence measurement signal of two fluorescent proteins are recorded separately and simultaneously on image sensors 6 A, 6 B. Single-band emission filters 44 , 44 A are single band filters centered on the fluorescence emission of each fluorescent marker.

Referring now to FIG. 8 and FIG. 9A , a side view and a bottom view, respectively, of electrical rotary joint 130 B of optical measurement system 100 B of FIG. 1B are shown. The positions of rotor 4 B and stator 3 B are shown, and rotor 4 B is rotationally coupled to stator 3 B with a low-friction bearing and electrical interconnects. Electrical connections 14 B are provided on stator 3 B for connection to data acquisition and control unit 2 of FIG. 1B . Two fluorescence stimulation/detection units 12 B are mounted to, or form the body of rotor 4 B, which includes connectors 13 B for attachment of connectors 26 B of multiple instances of fiber optic cable 8 B of FIG. 2B . FIG. 9B shows an alternative embodiment in which four fluorescence stimulation/detection units 12 B are mounted to, or form the body of rotor 4 C of an electrical rotary joint 103 C. FIG. 9C shows a bottom view of electrical rotary joint 130 B of FIG. 9A with one of fluorescence stimulation/detection units 12 B removed, exposing mounting holes 48 through which machine bolts 47 are inserted to mount fluorescence stimulation/detection units 12 B and an electrical connector 46 that connects to electrical connectors 24 A, 24 B of fluorescence stimulation/detection units 12 B (or 12 A) to provide power for light sources 5 A and receive the fluorescence detection signals from photo-detectors 10 or image sensor 6 .

Referring now to FIG. 10 , a cut-away side view of an individual one of fluorescence stimulation/detection units 12 B is shown, illustrating details of the optical and electrical components within. Fluorescence stimulation/detection units 12 B are similar to fluorescence stimulation/detection units 12 A of FIG. 5A , so only differences between them and the associated applications are discussed in detail below. Connector 13 B, which receives one connector 26 B of one of fiber optic cables 8 B of FIG. 2B and interface 25 B thereby, is optically coupled by an objective lens 33 . Fluorescence stimulation/detection units 12 B are configured to image a single optical fiber at interface 25 B of up to 1 mm in diameter. The advantage of using single point photo-detectors 10 , instead of image sensors 6 as in fluorescence stimulation/detection unit 12 A of FIG. 5A , are higher detection bandwidth and higher sensitivity. To simultaneously record the activity of multiple brain regions, optical measurement system 100 B of FIG. 1B requires one complete fluorescence stimulation/detection unit 12 B per fiber optic cable 8 B, limiting the maximum number of brain regions that can be recorded. An electrical connector 24 B provides for electrical connections between rotor 4 A and each of fluorescence stimulation/detection units 12 B, providing power/control for light sources 5 B, as well as an interface to a photo-detector 10 within fluorescence stimulation/detection unit 12 A. Since optical power requirements for fiber-photometry measurement are low, the heat produced by light sources 5 B is also low and the heat can be dissipated passively with a passive heat sink 19 B. If higher thermal dissipation is required, a fan may be included to move air over the fins of heat sink 19 B.

Excitation light is provided by light sources 5 B, e.g., LEDs or laser diodes, for fluorescence protein excitation. Each of the outputs of light sources 5 B is first collimated by an aspheric lens 34 and then focused on interface 25 B of the optical fiber of fiber optic cable 8 B by objective lens 33 . FIG. 6B , which is referred to additionally, illustrates the illumination pathway between each of light sources 5 B and interface 25 B of the optical fiber in the corresponding fiber optic cable 8 B of FIG. 2B . An excitation bandpass filter 36 and a dichroic beamsplitter 37 included in optical filters 7 B of FIG. 1B filter and direct excitation light from light sources 5 B to objective lens 33 . Objective lens 33 also collimates the returning fluorescence light received from interface 25 B of fiber optic cable 8 B, which may be an aspheric or multi-element lens, e.g., an achromatic doublet, in order to reduce the angle of incidence of the light incident on emission bandpass filter 39 and a dichroic beamsplitter 38 included in optical filters 7 B of FIG. 1B that separate the excitation illumination path from the detection path, and thereby improve filtering efficiency. The collimated fluorescence measurement beam from objective lens 33 is then focused on each photo-detector 10 by a corresponding one of focusing lenses 35 , which in the example may be another aspheric lens. In the example, the numerical aperture of both objective lens 33 and focusing lenses 35 is between 0.20 and 0.80.

In fluorescence stimulation/detection unit 12 B as illustrated in FIG. 10 , two sets of excitation bandpass filters 36 and dichroic beamsplitters 37 are shown that form a primary filter set that separates the excitation illumination from the detection optical pathway. However, the number of excitation sources, i.e., light sources 5 B may be from a single light source 5 B up to the limits of module size that can be provided on rotor 4 B and any degradation of the excitation illumination pathway through the multiple dichroic beamsplitters 37 , 38 . The primary filter set is configured to combine excitation illumination light from one, two, or three light sources 5 B, which in the example of FIG. 10 is two. The primary filter set features: an individual excitation bandpass filter 36 for each light source 5 B that reduces the spectral bandwidth of the illumination lights, and an individual dichroic beamsplitter 37 for each light source 5 B to combine two or three bands of excitation lights in a single excitation pathway. A secondary filter set separates the one, two or three bands of fluorescence signals emitted by the sample from one, two or three bands of excitation lights. The secondary filter set varies according to number and type of fluorescent markers to be detected.

In one example embodiment, the returning fluorescence measurement signal of one fluorescent protein is detected by photo-detector 10 , and one light source 5 B is used for excitation. In such a configuration, a filter set includes a first dichroic beamsplitter 37 for light source 5 B that reflects the excitation light from each of light sources 5 B to connector 13 B, an excitation band-pass filter 36 and an aspheric lens 34 for light source 5 B, a second dichroic beamsplitter 38 that separates the returning fluorescence measurement signal for photo-detector 10 , and detection band-pass filter 39 having a pass-band centered on the emission wavelength of the fluorescent marker that removes stray light coming from excitation light reflections within fluorescence stimulation/detection unit 12 B. As mentioned above, a second excitation light source 5 B at the isosbestic wavelength of the calcium indicator may be added to the system for control measurements, e.g., approximately 405 nm for GCaMP6.

In another example multi-color configuration, the fluorescence measurement signal of multiple markers having two different excitation and emission spectra are recorded simultaneously by providing one light source 5 B and one photo-detector 10 for each marker. As in the single-color example given above, the filter set includes first dichroic beamsplitter 37 for each of light sources 5 B that reflects the excitation light from each of light sources 5 B to connector 13 B, an individual excitation band-pass filter 36 for each of light sources 5 B, a second dichroic beamsplitter 38 for each photo-detector 10 that separates the returning fluorescence measurement signal for each photo-detector 10 , and individual detection band-pass filter 39 for each photo-detector. Detection band-pass filter 39 has a pass-band centered on the emission wavelength of the corresponding fluorescent marker that removes stray light coming from excitation light reflections within fluorescence stimulation/detection unit 12 B, and also the fluorescence measurement signal from the other markers. To minimize lead of excitation light reflections directly to photo-detectors 10 , it is important to avoid spectral overlap between the pass-bands of excitation bandpass filter 36 and detection band-pass filter 39 .

As mentioned above, electrical rotary joints 130 A and 130 B of FIG. 3 and FIG. 8 , respectively are generally low-friction rotary joints having low-friction electrical connections, e.g., liquid metal, magnetic transmission, slip rings, etc. In one embodiment, the rotary joints 130 A, 130 B passively follows movement of sample 9 . Referring now to FIG. 11 , in another embodiment designed for small rodents, fiber optic cables 8 A in optical measurement system 100 A of FIG. 1A , or fiber optic cables 8 B in optical measurement system 100 B of FIG. 1B , are mechanically coupled to a torque sensor 40 by a guide 42 that rotates independently from electrical rotary joint 130 D. Torque sensor 40 triggers motor-assisted rotation of an electrical rotary joint 130 D that includes a motor, resulting in friction-free rotation when fiber optic cables 8 A, 8 B rotate guide 42 . The proximal ends of fiber optic cables 8 A, 8 B are connected to fluorescence stimulation/detection units 12 A or 12 B disposed on electrical rotary joints 130 A, 130 B. To maintain sufficient torque sensing, fiber optic cables 8 A, 8 B are formed in loops 41 between guide 42 and fluorescence stimulation/detection units 12 A, 12 B.

While the invention has been particularly shown and described with reference to the preferred embodiment thereof, 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 invention.