Photostimulation in a compact package

Abstract

A miniature microscope that incorporates laser scanning optical microscopy and arbitrary spatially patterned photo stimulation typically at a different wavelength with two optical arms. The first optical arm both delivers patterned photostimulation and performs optical collection of fluorescence from a sample. The second arm provides scanned excitation pulses to cause the fluorescence. The light from the two optical arms can be combined with a dichroic mirror or polarizer to align the beams in the field of view of the microscope objective element.

Description

[0001] PHOTOSTIMULATION IN A COMPACT PACKAGE

[0002] BACKGROUND OF THE INVENTION

[0003] This invention was made with government support under Award Nos. 1 UF1 NS116241 and R01NS118188 awarded by the National Institutes of Health and under Award Nos. BCS 1926676 and 1926668 by the National Science Foundation. The government has certain rights in the invention.

[0004] U.S. patent number 10,634,899 and U.S. patent application number 63 / 575,698 are incorporated herein by reference.

[0005] FIELD OF THE INVENTION

[0006] The present invention relates to optogenetic multiphoton targeted photostimulation in a compact package. In particular, the present invention relates to a miniature fiber- coupled two photon microscope with optogenetic stimulation.

[0007] DISCUSSION OF RELATED ART

[0008] Multiphoton microscopy combined with optogenetic photostimulation is a powerful technique in neuroscience enabling precise control of cellular activity to determine the neural basis of behavior in a live animal. Two-photon spatially patterned photostimulation has taken this further by allowing interrogation at the individual neuron level. However, it remains a challenge to implement imaging of neural activity with spatially patterned two-photon photostimulation in a freely moving animal.

[0009] SUMMARY OF THE INVENTION

[0010] Embodiments include a miniature optogenetic photostimulation enabled two-photon microscope having two branches. The first branch includes a source of, for example, 1030 nm pulses which are provided via a fiber bundle to a sample in order to photostimulate selected regions on the sample. This branch is also used for fluorescence collection. The second branch includes a source of, for example, 920 nm pulses to form an excitation beam that is scanned over the sample using a MEMS mirror (microelectromechanical systems) scanner. These branches are combined with an optical element into a common objective branch which focuses the light into the sample. In some embodiments the excitation light is provided to the sample via a polarization maintaining single-mode fiber that is scanned on the sample to collect an image, reducing or eliminating pixelation. This has the further advantage of eliminating image quality degradation resulting from differences in two- photon excitation by distortion of the excitation light through different fibers in the fiber bundle, due to differences in dispersion and birefringent properties between different fiber cores in the bundle.

[0011] In some embodiments a fiber bundle is used to provide spatial light-patterning at the sample. This bundle may remain stationary rather than being scanned.

[0012] Some embodiments include a GRIN (gradiant refractive index) relay lens, which allows for deeper imaging. Some embodiments include a liquid lens or lenses in one or both of the branches of the device, which permits axial scanning as well as the excitation beam scanning caused by the MEMS scanner.

[0013] A microscope includes an objective, a first path configured to both deliver spatial patterned light to a sample through a fiber bundle and the objective and to collect light generated by the sample back along the same route through the objective and the fiber bundle, a second path configured to deliver imaging excitation light to the sample through the objective to cause light to be generated by the sample, a scanner to scan the excitation light across the sample, and an element configured to combine the spatial patterned light and the imaging excitation light and deliver them to the sample through the objective.

[0014] Imaging excitation light is delivered to the second path through an excitation path fiber, which may comprise a single mode polarization maintaining fiber or a hollow core fiber. The excitation path fiber might also be a few mode fiber, a large mode area fiber, a photonic crystal fiber, or a fiber bundle.

[0015] The light generated by the sample might be fluorescent light.

[0016] The scanner might be a microelectromechanical systems (MEMS) mirror scanner. The combining element might be a beam splitter or a dichroic mirror. Then the dichroic mirror passes the spatially patterned light and the light generated by the sample and reflects the imaging excitation light.

[0017] Thee imaging excitation light may comprise laser pulses of 3 picoseconds to 40 femtoseconds in duration.

[0018] The microscope might further include a gradient index of refraction (GRIN) optical element having a numerical aperture in the range of 0.3 to 0.9. Then the objective could include optics for correction of GRIN aberrations. Or, the microscope might include a variable optical element configured to shift a focal plane such as a tunable liquid lens, generally placed before the objective.

[0019] An inspection system for calibrating the microscope may use a fluorescent sample and include a filter set after the fluorescent sample, a camera configured to collect light from the filter system, and a processor configured to measure offsets between fluorescence generated from the imaging excitation light from the first path and spatially patterned light from the second path.

[0020] Miniatture microscopes might have dimensions of less than 40 mm x 50 mm x 15 mm and weight under 10 grams.

[0021] The microscope may be housed in a clamshell case having a front shell and a back shell, with optical elements are press-fitted into the back shell before affixing the front shell to the back shell.

[0022] A method of calibrating the foci of a microscope includes the steps of providing a sample, axially adjusting the fiber relative to the microscope, delivering imaging light to an imaging spot in a field, providing a patterned spot to the sample from the spatial pattern arm and positioning the patterned spot laterally adjacent the imaging spot, focusing at least one of either the patterned spot or the imaging spot on the sample, axially translating the microscope relative to the sample and imaging the sample with an inspection system, measuring an offset between the imaging spot and the patterned spot, determining whether the offset falls within a desired range, where if the offset does not fall within a desired range, the fiber is axially adjusted with respect to the microscope and process repeated. If the offset does fall within a desired range, the fiber is fixed with respect to the microscope, for example by gluing. In some cases the sample is fluorescent and wherein the imaging light causes fluorescent emission at the imaging spot. The fiber and / or the microscope may be adjusted with a translation stage.

[0023] A method of calibrating microscope imaging and photostimulation coordinate systems of a microscope includes providing a fluorescent sample with easily distinguishable features It has a photostimulation arm configured to both deliver spatially patterned light through a fiber to the sample and to collect light from the sample back along the same route to microscopically image the sample, and an image light arm configured to deliver imaging light to the sample. The process images light transmitted from the sample with an inspection system, while imaging the distinguishable features with the microscope. Three photostimulation calibration spots are delivered to the sample. Coordinates of the photostimulation spots in the photostimulation coordinate system are determined and recorded. The location of the photostimulation spots relative to the distinguishable features in a microscopy image provided are determined and the photostimulation spot coordinates in the microscope imaging coordinate system are recorded. The location of the recorded spots in the two coordinate systems is used to determine a transformation between the two coordinate systems, which is saved into software and used to enable accurate targeting of the photostimulation field. In some cases the three photo stimulation calibration spots are adjusted to a desired location relative to the distinguishable features. General the transformation is tested by placing photostimulation spots at new distinguishable locations on the sample in the laser scanning microscopy field and imaging the spots with the inspection scope.

[0024] BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1A is a schematic block diagram of a microscope for two-photon imaging. Figure 1 B shows a fluorescence image of a 50 pm grid target excited using 920 nm light. Figure 10 shows an example image of spatial photostimulation pattern recorded by two-photon fluorescence from a thin rhodamine sample illuminated using 1030 nm light. Figure 2A is a schematic block diagram of a transmissive microscope for two-photon imaging which may be used for calibration. Figures 2B-2J show images used for calibration. Figure 2K shows a flowchart diagram of the axial calibration method. Figure 2L is a flow diagram showing a second calibration process.

[0026] Figure 3A is a detailed cutaway schematic diagram of a microscope for two-photon imaging and two-photon spatially patterned photostimulation within a clamshell housing. Figure 3B is an isometric view of an assembled microscope. Figure 3C is a cutaway view of the optics within the clamshell housing.

[0027] Figure 4 is a generalized schematic block diagram of a microscope for two-photon imaging.

[0028] Figures 5A, 5B, 5C, and 5D show performance of a microscope.

[0029] Figure 6 shows a microscope objective with an added GRIN lens.

[0030] Figure 7 shows a microscope objective with an added tunable liquid lens.

[0031] DETAILED DESCRIPTION OF THE INVENTION

[0032] Figure 1A is a schematic block diagram of a microscope 100 for two-photon (2P) imaging and two-photon targeted photostimulation. This embodiment is a miniature microscope using a MEMS based scanner 142 for two-photon (2P) imaging combined with spatially patterned 2P photostimulation 110, termed the Opto2P-FCM (optogenetics two-photon fiber-coupled microscope). One use for microscope 100 is as a miniature head-attached microscope for freely moving animals such as mice.

[0033] In this embodiment, microscope 100 has two branches. The top branch in Figure 1A includes a source of patterned photostimulation light 110 which are provided via a fiber bundle 102, fiber ferrule 104, and optics 108, dichroic 116, and objective 120 to a sample 124 in order to illuminate user-controlled selected regions on the sample. The spatially patterned light is relayed from the distal end of the fiber bundle to the proximal end by maintaining the spatial alignment of the fibers. In one embodiment, the fiber bundle can be an imaging fiber bundle or coherent fiber bundle.. Figure 1 B shows an example of grid target scanning from the excitation light 150 in the bottom path with emitted fluorescence 120 collected in the top path. The top branch also returns fluorescent emission 112 such as fluorescence from sample 124 along the same path. Figure 1C shows photostimulation 110 from the top branch imaged with an inspection system such as an imaging substage or inspection scope as shown in Figure 2A. Dichroic 116 is configured to allow the frequency of photostimulation 110 and the frequency of fluorescent light 112 to pass through it.

[0034] The second path at the bottom in Figure 1 provides excitation 150 to sample 124 via polarization maintaining fiber 130, fiber ferrule 132, collimating optics 138, scanner 142, scan optics 146, reflecting dichroic 116, and objective 120. Scanner 142 scans beam 150 to illuminate 128 portions of sample 124, causing fluorescence, which returns via the top path after it passes through dichroic 116.

[0035] Thus beams 106 and 114 include photostimulation 110 in the forward direction and fluorescence 112 in the reverse direction. Beam 118 includes photostimulation 110 in the forward direction and fluorescence 112 in the reverse direction, and also excitation 150 in the forward direction, as it has reflected off dichroic 116.

[0036] Beam 134 is excitation pulses provided to collimating optics 138, which collimates the excitation beam. Scanner 142 scans collimated beam 144 into scan optics 146, resulting in scanned beam 148, which illuminates different areas 128 on sample 124.

[0037] In one embodiment, microscope 100 includes two femtosecond pulsed lasers, at 920 nm and 1030 nm wavelengths, and a two-channel detection system such as two photodetectors behind the fiber bundle 102 (not shown). Excitation light at 920 nm is delivered to the head-attached microscope through a polarization maintaining (PM) fiber 130 and scanning is performed with an on-board MEMS scanning mirror 142. The 1030 nm laser is spatially shaped using, for example, a spatial light modulator (not shown) and the pattern is propagated through a flexible imaging fiber bundle (CFB) 102. The mixing and separation of the 1030 nm light from the emitted light from the fiber bundle is done by a dichroic mirror (not shown). The 1030 nm beam is combined with the 920 nm laser using a dichroic mirror 116 and both are overlapped at sample 124. Fluorescence 112 from the sample 124 is collected back through the objective 120, transmitted by the dichroic 116 and focused via optics 108 onto the CFB 102, where it is relayed to a detection system. The miniature microscope 100 uses the same optical beam path for photostimulation 110 and fluorescence 112 collection which greatly reduces its complexity and weight. 2P imaging combined with 2P targeted photostimulation in the somatosensory and visual cortex in freely moving mice expressing jGCaMP7s and ChRmine20 has been demonstrated. See Figures 5A-5C. Photostimulation from selected regions in the field of view resulted in robust Ca2+ fluorescent transients timed to the stimulation pulses 110. These results are the first demonstration of a miniature head-mounted microscope that can perform high resolution 2P imaging using a MEMS scanner 142 with 2P targeted photostimulation 110 to record and manipulate specific neurons.

[0038] In a specific example tested by the inventors, the 920 nm laser output 150 from polarization maintaining fiber (PMF) 130 (PM780) is collimated 138 and reflected off a MEMS scanner 142 and passes through a scan lens 146 having a plano-convex and achromatic lens to generate a telecentric scan across the imaging plane. Patterned photostimulation light 110 at 1030 nm exits the CFB 102, is collimated using an aspheric lens 108, combined with the 920 nm light 150 using a dichroic mirror 116 and focused through the same objective 120. The miniature objective is designed with a working distance of ~1 .5 mm to focus both 920 nm and 1030 nm light to the same imaging plane on sample 124. In vivo head-attached two-photon imaging and selective optogenetic two-photon photostimulation in a freely moving mouse was demonstrated. Imaging was performed in the visual cortex with neurons co-expressing the calcium indicator jGCaMP7s and the opsin ChRmine. Two-photon photostimulation was performed on three selected regions of interest (ROI) in the imaging field. An image time sequence was acquired whereby all three ROIs were illuminated simultaneously followed by each individual ROI sequentially. The change in fluorescence over baseline fluorescence (AF / F) traces of jGCaMP7s showed responses in the ROIs that occurred synchronously with photostimulation. The resolution of the Opto2P-FCM provided visualization of processes from somatosensory cortex neurons expressing jGCaMP7s.

[0039] Those skilled in the art will appreciate that Figure 1A shows on specific example, and many variations fall within the spirit of this invention. For example, while a polarization maintaining single mode fiber bundle 130 was used in this embodiment, other fiber configurations such as a few mode fiber, large mode area fiber, photonic crystal fiber or fiber bundle could be used. A hollow core fiber could be used in place of the polarization maintaining fiber. The beam splitter could be polarizing or partially reflective. Patterned photostimulation could be any form of spatially patterned light that could be used not only for optogenetic stimulation but for chemical modification of a sample, photo-ablation, uncaging or other processes.

[0040] In addition to two-photon microscopy, one-photon, three-photon, coherent-Raman, reflectance or other optical imaging methods can be used. This device could be modified for use in a transmissive mode.

[0041] Figure 2A is a schematic block diagram of an inspection setup 200 used for various calibrations and tests of a microscope 100. Figures 2B-J show images used for calibration. Figure 2K shows the steps of one method of calibration. Figure 2L shows the steps of a second method of calibration.

[0042] Figure 2A shows a microscope 100 combined with an inspection system 200. Microscope 100 is coupled to an xyz translation stage 242 for imaging the microscope’s 100 point spread function. Spatial patterning is delivered by, for example coherent fiber bundle 202 and excitation is delivered by, for example polarization maintaining fiber 230. Coherent fiber bundle 202 is coupled to an axial translation stage 240. Samples 224 are a thin fluorescent sample, such as spin coated rhodamine, or fluorescent patterned slide with easily distinguishable features that are also opaque to white light depending on the calibration. Light 252 is transmitted through slide 242 to objective 220, reflected off mirror 254, passes through filters 256 (when used depending on the calibration) and is focused by optics 258 to camera 260.

[0043] Inspection setup 200 may be used for various calibration tasks. One task (see Figure 2K) is an axial calibration procedure. This is to get the focus, focal plane, of the imaging, which is also referred to as laser scanning microscopy (LSM) axially, overlapped with the focus, focal plane, of the photostimulation. Having the foci overlapped is referred to as being parfocal. This calibration is done first when the device is assembled. This procedure uses the inspection scope to visualize emitted fluorescence from a thin fluorescent sample.

[0044] In addition, there is a spatial calibration procedure done to enable targeting of the photostimulation (see Figure 2M). This is done with a fully assembled device. This is needed so selected targets in the LSM field can be hit with light from the photostimulation arm. In order to target the photostimulation accurately, the software needs to know how the coordinates of the imaging (LSM) relate to the coordinates of the photostimulation. This coordinate transform is referred to as an affine transformation, which is a transform that allows for scale, rotation and translation changes. The inspection scope in this calibration is used in a transmission configuration with a fluorescent sample that also occludes light in transmission. Fluorescence is not observed with the inspection scope and room lights are used for illumination. This calibration task removes filters 256. Fluorescence from the sample is imaged with the LSM of the device and collected with the device. The grid slide is fluorescent but it also occludes the light so it can be imaged in transmission mode with the inspection scope 200. The calibration can also be performed by collecting fluorescence or transmission in the epi-direction without the need for an inspection scope. The same calibration procedure would be followed but using the imaging capabilities of the microscope.

[0045] The inspection scope 200 was used to measure the axial resolution of the excitation and photostimulation beams, perform spatial calibration of the photostimulation and imaging fields, and ensure that the fields are parfocal. The inspection scope consisted of a 10x / .4 NA Olympus objective 220, a 90 deg folding mirror 254, and a 100 mm focal length lens 258 and camera 260 (FLIR CM3-US-31S4M-CS, Edmund Optics). For imaging fluorescence emission, a series of filters 256 (BG39, short pass 745, band pass 617 / 73) were placed before the camera 260 to reject the laser light at 920 and 1030 nm. For imaging the 1030 nm beam directly, filters 256 were removed and beam was attenuated.

[0046] The imaging and photostimulation planes were set to be parfocal though adjustment of the axial position of the fiber bundle 202 in the housing. This was performed by mounting the device to a micromanipulator 242 (MP-285, Sutter Instruments) for moving the device axially relative to the sample allowing for measurement of the axial focus. The fiber bundle 202 was temporarily mounted to a manual translation stage for separate adjustment 240. A thin fluorescent sample 224 was prepared by spin coating rhodamine onto a microscope slide. The slide was placed on a stage at the focus of the inspection scope 200. The fiber bundle was released from the adjustment stage 240 and held in position with a friction fit. The position of the Opto2P-FCM foci for the 920 nm beam and the 1030 nm beam was measured simultaneously by recording the 2P fluorescence on the camera 260 at different axial positions using the Sutter stage 242 and locating the position of maximum signal as shown in Figure 2H-J. If the overlap was insufficient, the fiber bundle 202 was temporarily remounted to manual translation stage 240 for adjustment, release from the stage and the axial profile was retaken. Once sufficient overlap was obtained fiber bundle 202 was subsequently affixed within the microscope 100 housing via adhesive bonding.

[0047] Figure 2K shows the steps in a process of calibrating the axial location of fiber bundle 202 to make photostimulation parfocal with imaging. In step 272, A sample is provided, here a thin fluorescent sample. In step 274 fiber 202 (here a fiber bundle) is positioned axially with respect to microscope 100, here with stage 240. In step 276, imaging light is delivered to an imaging spot or spots in a field of view. In step 278, a patterned spot or spots (here photostimulation) is positioned laterally adjacent to the imaging spot. Preferably the spots are close but not overlapping. In step 280, the microscope is focused onto one of the spots. Here microscope 100 is mounted to an xyz stage 242. The laser shutters are opened, an imaging LSM spot is centered in the field and a diffraction limited spot from the fiber bundle is focused next to the spot. Step 282 acquires images from the inspection scope to axially profile the foci in step 284. Example axial profiles of the foci are shown in Figures 2I and 2J. Step 286 determines whether the offset falls within a desired range. If yes, path 292 is followed and fiber 202 is released from its axial stage and fixed in place with respect to microscope 100 in step 294. If the offset is not acceptable, path 288 is followed, and fiber 202 is adjusted (here with axial stage 240) with respect to microscope 100 in step 290. Then, steps 276- 284 are repeated until the offset is acceptable.

[0048] Figure 2L is a flow diagram showing the second calibration process. To perform spatial calibration of the photostimulation patterning, the Opto2P-FCM was mounted to the Sutter micromanipulator 242 and the inspection scope 200 was used. As shown in Figures 2E-G, a grid slide was used as the sample 224 and brought into the axial focus of the inspection scope 200. The grid slide of Figure 2B was translated to one corner of the grid for unique identification. The same corner was then located by imaging with the Opto2P-FCM and the Sutter stage 242 was translated to put the corner in the top left of the imaging field. The photostimulation spatial calibration function in software was used to illuminate three calibration spots 1 , 2, 3 on different easily identifiable locations of the grid slide. The position of the spots were visualized with the inspection scope. The coordinates of the individual spots were adjusted in the software to overlap the distinct features pointed to in Figure 2C by visualizing their position with the inspection scope. Visualization of the spots at the final overlapped positions is shown in Figure 2F. The coordinates of these spots were then identified in the imaging field of the Opto2P-FCM. Once located, the software computed an affine transformation between the laser scanning microscopy coordinate system and holographic coordinate system needed for accurate targeting. The calibration was tested by placing targeted holographic spots 4 on known positions of the grid slide in Figure 2D and observing their location on the inspection scope Figure 2G.

[0049] Figures 3A and 3B show a specific example of a miniature microscope 300. Figure 3A is a cutaway front view, with the optical elements housed in body 302. Here front shell 302 is removed to show the optical elements. Figure 3B is an isometric view of microscope 300 with clamshell body 302 assembled. Laser 300 has several advantages including small size, light weight, and stability. Figure 3C shows clamshell body 302 with front shell 304 and back shell 306 separated to show the optical elements. Figure 3A shows the optical elements from Figure 1A tucked into recessed areas formed in back shell 306. The recessed areas are sized and configured to hold the various elements in place, maintaining spacing and providing stability. In this example, the elements are symmetrical, so front shell 304 is almost a mirror image of back shell 306, but either could be adjusted to accommodate various sizes and shapes of elements.

[0050] Fiber ferrule 104 is affixed to body 302 at the top, for example with glue after axial calibration. It could also be fixed in place with a set screw or other mechanical fasteners. Fiber bundle 102 provides photostimulation light 110 via ferrule 104. Similarly, fiber ferrule 132 is affixed to the left side of body 302 and provides excitation light via polarization maintaining fiber 103. The optics operate as shown in Figure 1A.

[0051] Figure 3B is an isometric view of an assembled clamshell body 302. The body 302 comprises a front shell 304 and a back shell 306 which close around the elements in a clamshell fashion. A clamshell design optical system is a configuration where optical components are arranged within a housing 302 that opens and closes like a clamshell. This design approach offers specific advantages, particularly in simplifying the alignment of multiple elements.

[0052] Figure 3C shows clamshell body 302 before joining front shell 304 and back shell 306. Optics are in place in back shell 306. In one example, the optical elements are press-fitted into one half 306 of the housing shell 302, while the other half is secured in place using 0-80 screws in openings 310. The 920 nm excitation light is delivered by a polarization maintaining fiber (PM780) 130 with a 2.5 mm bare ceramic ferrule on one end and an FC / APC connector on the other end. The light emerging from the PM fiber 130 is collimated by an aspheric lens. The PM fiber is axially adjusted to ensure the outgoing beam is collimated by observing the output at both near and far distances (~10 cm and ~2 meters). After mounting the MEMS mirror, its position is fine-tuned by observing the reflected beam at far distances and then glued in the housing. Front shell 304 is, for example, attached to back shell 306 by 0-80 screws(not shown). In one example, housing 302, having front shell 304 and back shell 306, was designed in SolidWorks to simplify the installation of the optics. It was 3D- printed using stereolithography with a Formlabs Form 3+ with Tough 1500 resin, enabling fast prototyping. This design allows for small, lightweight devices, such as at most 40 mm x 50 mm x15 mm, and under 10 grams. The overall size here is only on the order of 32 mm wide by 42 mm high by 8 mm deep, as indicated by the 10 mm scale bar at the bottom. One experimental head piece was 40x22x12 mm and weighed about 5 grams. Shell 302 is inserted into base 314, and MEMS scanner 316 is mounted (e.g. glued) to 142.

[0053] For one specific example, the parts used in Opto2P-FCM 300 are shown in Table 1, and the Laser specifications are shown in Table 2. Those skilled in the art will appreciate that many other configurations fall within the scope of the invention. E.g., the two arms could be swapped.

[0054] Table 1 : Parts of Microscope 300 able 2: Laser specifications

[0055] Figure 4 is a generalized schematic block diagram of a microscope 400 for two- photon imaging. This example shows a configuration like those of Figure 1A and 3A, but arms 408 and 446 could also be swapped in an alternate embodiment.

[0056] Photostimulation (spatially patterned light) is provided at 102 to top arm 408, which provides the photostimulation light 110 to sample 124 through objective 420. The emitted fluorescence light 112 from sample 124 travels in the reverse direction through objective 420 and top arm 408. In the case of a transmissive configuration, fluorescence and patterned photostimulation may both exit at the bottom of sample 424 to a recording device (not shown).

[0057] Light to excite sample 424 enters through polarization maintaining fiber 130 through left arm 446 and is reflected off element 416 (for example a dichroic) through objective 420 to sample 124.

[0058] **(Other variations we might like to cover here)

[0059] Figures 5A, 5B, 5C, and 5D show performance of a miniature two photon microscope with optogenetic stimulation. This data was collected with a clamshell microscope 300 and came from a demonstration of cell-specific 2P patterned photostimulation and simultaneous 2P imaging in a freely moving mouse.

[0060] The experiments demonstrated simultaneous MEMS based 2P calcium imaging and 2P patterned optogenetic stimulation in fiber-tethered freely moving mice with the Opto2P-FCM 300. Multi-region patterned optogenetic stimulation was performed on 6 regions of interest (ROIs) with different timing patterns that included stimulation of just one ROI at a time and stimulation of all ROIs simultaneously. The fluorescence signal from jGCaMP7s was simultaneously recorded with the device. Imaging data was analyzed using CalmAn24 to identify regions of jGCaMP7s transients in Figures 5A and 5C and extract AF / F traces in Figures 5B and 5D. These results showed good overlap with the stimulation timings shown at the bottom of the traces and timing of fluorescence transients. Robust responses to individual ROI stimulations were consistently observed, while stimulations of all ROIs simultaneously resulted in varying response between cells. The experiments were performed over multiple days with the ability to return to the same imaging field.

[0061] The stimulation sequence consists of all ROIs together followed by stimulation of each ROI individually. Figures 5A and 5B were taken using a 0.5 m length fiber bundle in visual cortex, with 5 ms, 3.4 Hz stimulation for 2 seconds. Figures 5C and 5D were taken using a 1 m length fiber bundle in somatosensory cortex, with 10 ms, 3.4 Hz stimulation for 700 ms. (a) and (d) show the fields and targeted / detected ROIs, with solid line circular ROIs indicating the targeted stimulation regions and dashed line ROIs indicating those detected by CalmAn. Figures 5A and 5C show AF / F traces of jGCaMP7s activity during the recording timelapse. Solid lines represent AF / F denoised traces from CalmAn and overlaid dashed lines are raw AF / F traces of the stimulation ROI. Numbered bars at the bottom represent stimulation times and durations, with bars labelled “AH” representing the stimulation of all ROIs.

[0062] The experiments accomplished simultaneous two-photon imaging and two-photon patterned photostimulation of neural activity in visual and somatosensory cortex of freely behaving animals using Opto2P-FCM microscope 300, a MEMS based miniature microscope. It showed repeated imaging of the same cell field over multiple days. The ability to simultaneously image and photostimulate neural activity in neuronal soma of freely moving animals using this MEMS scanner-based device represents a significant advancement over previous miniature microscope designs. The MEMS based scanner enables the Opto2P-FCM to resolve neuronal features such as dendritic structures. The novel design of the Opto2P-FCM optics combines the photostimulation and fluorescence collection pathways making the device lighter, more compact and requiring fewer optics. Short femtosecond pulses after the standard PM fiber 130 were used for efficient 2P excitation. In addition to being much lower cost and commercially available, PM fibers have reduced stiffness and are less brittle compared to the hollow core fibers used in other multiphoton miniature microscopes. The PM fiber also allows the Opto2P-FCM to be adapted to different excitation wavelengths, which are not supported in hollow core fibers that are designed for a narrow range of wavelengths. Additionally, collection of the fluorescence emission through the fiber bundle 102 allows multiple color channels to be detected simultaneously without adding the weight of a second photodetector on the head attachment. The Opto2P-FCM with a MEMS scanner overcomes limitations of previous devices by more fully utilizing the resolution of the objective.

[0063] The 920 nm excitation light is delivered through a single mode PM fiber (PM780-HP, Thorlabs) and collimated using an aspheric lens and reflected from a MEMS scanner (Mirrorcle A3I12.2-1200AL) mounted on a custom designed PCB with flex cable. A scan lens comprising a plano-convex and achromatic lenses generated a telecentric scan, for scan angles of -4, 0 and 4 degrees followed by a dichroic and an imaging objective lens. The Opto2P-FCM is designed with an NA of 0.4 for 920 and 1030 nm with a magnification of -5.5X and 2.8X respectively and overlapping FOV of 250 pm x 250 pm. The design has a working distance of -1 .5 mm while maintaining a Strehl ratio of ~0.8 across the scanning angles of -4 to 4 degrees. The point-spread function for scanning angles of 0, 2.5 and 5 degrees have a Strehl ratio of 0.8 and the focal spot within the Airy disk radius of 1 .38 pm for all scanning angles, demonstrating diffraction-limited performance. A custom dichroic (T > 90% 330-870, 975-1200 nm - R > 90% 895-935nm) separates the excitation and photostimulation / emission beam paths. The photostimulation / emission path is configured to be chromatically corrected and focuses the 1030 nm photostimulation light from the fiber bundle (Fujikura FIGH-15-600N) to the same imaging plane as the 920 nm light. The point-spread function (PSF) of the 1030 nm light at the imaging plane resulted from an input beam launched at -200, 0, and 200 pm at the entrance facet of the fiber bundle with 0.2 NA. The Strehl ratio of the 1030 nm photostimulation beam varied across the imaging field with a range of 0.6 to 0.7. The root-mean square (RMS) radius of the spot was determined for imaging (920 nm) and photostimulation (1030 nm) as a function of working distance with zero position on the minimum RMS spot at 920 nm. The minima of the RMS spots were offset by less than ~0.5 m. The system showed a 2 pm axial displacement between the 920 and 1030 nm light when scanning to 4-degree angle relative to on-axis position.

[0064] SlideBook 2024 (Intelligent Imaging Innovations, Inc.) software was used to control the MEMS scanning mirror 142, acquire images, calibrate and compute the spatial hologram patterns, and control the timing of photo-stimulation. The x and y galvo output channels were connected to the analog input board (BDQ PicoAmp 5.4, Mirrorcle Technologies Inc.) which connects to the MEMS mirror. The acquisition frame rate was 3.4 Hz. The photostimulation duration and power were controlled by an analog output from Slidebook connected to the power modulation input on the 1030 nm laser. The pulse timing signal of the photo-stimulation is controlled by a function generator triggered by the frame trigger output from SlideBook to provide one pulse per frame. The pulse timing signal goes to the pulse enable line on the laser. Pulse durations of 5 and 10 ms corresponding to duty cycles of 1.7% and 3.4%, were used in the experiments. The same timing signal was also used to gate the PMTs during photo-stimulation to prevent overload from background signal. The stimulation power measured at the focus of the Opto2P-FCM for individual ROIs was between 2.7 and 7.8 mW when modulated, which corresponds to 153 mW to 229 mW unmodulated.

[0065] Figure 6 shows a microscope objective 120 with an added GRIN (gradiant refractive index) relay lens 622, which allows for deeper imaging.

[0066] Figure 7 shows a microscope objective 120 with an added tunable liquid lens 722, which allows axial scanning as well as the excitation beam scanning caused by the MEMS scanner 142.

[0067] Opto2P-FCM microscopes are anticipated to provide a novel neuroscience tool that can advance our ability to conduct detailed mechanistic investigations of neural activity in freely behaving animals. By allowing researchers to observe and manipulate neural circuits in real-time, while an animal engages in natural behaviors, this technology will provide deeper insights into the neural basis of behavior. The Opto2P-FCM could lead to a better understanding of how specific neural pathways contribute to cognitive processes, sensory perception, motor control, emotion, cognition and learning, ultimately opening new avenues for research in neuroscience and related fields. An example of a future application of this device for a behavior requiring freely moving animal interactions is the study of the neural basis underlying social bonding and cognition. How these social bonds are represented in the brain is a question that has been previously tackled using cellular resolution Ca2+imaging. To move from descriptive to functional approaches, a method is needed to specifically activate or inhibit subsets of neurons in the context of behavior with prospective mates. Another application is the study of place cells in the hippocampus that encode for spatial information during navigation and are key to understand the neural basis of learning and memory. Major advances in understanding place cells have been done by imaging their activity through two photon imaging in head fixed animals navigating a scene in virtual reality (VR), but two dimensional place tuning was found to be profoundly impaired in VR navigation and the theta rhythm frequency has been found to be slower in VR environments. The role of place cells in spatial navigation has been studied in freely moving mice with one photon and two-photon miniature microscopes but did not include capabilities for optogenetic modulation. Combining imaging with optogenetics can provide an understanding the involvement of cellular processes in signal processing in place cells.

[0068] Opto2P-FCM can enable optogenetic modulation of specific subsets of neurons or specific dendritic processes and simultaneous imaging of circuit activity at timed to behavioral epochs, allowing investigators to ask how activation or inhibition of cells or cellular processes affects behaviors such as social bonding and spatial navigation. One such application would be in the investigation of CA1 pyramidal neurons in spatial and epoch behavior. A recent study performed targeted holographic stimulation in subsets of cells in CA1 in head-fixed mice undergoing one dimensional spatial navigation to show that place cells drive memory-guided spatial behavior. However, it remains a question if the activity of small subsets of neurons is causal in 2D spatial navigation. Using Opto2P-FCM, it would be possible to identify cells whose activity can be used to decode animal decisions in 2D spatial navigation tasks and stimulate these cells in a manner recreating the naturally observed activity.

[0069] The Opto2P-FCM further advances the development of optical tools for neuroscience studies in freely behaving animals by allowing high resolution MEMS 2P imaging with cell-specific patterned 2P photostimulation for the first time. Miniature microscope designs have opened new possibilities for neuroscience studies no longer confined to head fixation under large benchtop microscopes. Developments of these miniature microscopes are rapidly progressing and the Opto2P-FCM adds new capabilities to these systems. What is claimed is:

Claims

CLAIMS1. A microscope comprising: an objective; a first path configured to both deliver spatial patterned light to a sample through a fiber bundle and the objective and to collect light generated by the sample back along the same route through the objective and the fiber bundle; a second path configured to deliver imaging excitation light to the sample through the objective to cause light to be generated by the sample; a scanner to scan the excitation light across the sample; and an element configured to combine the spatial patterned light and the imaging excitation light and deliver them to the sample through the objective.

2. The microscope of claim 1 wherein imaging excitation light is delivered to the second path through an excitation path fiber.

3. The microscope of claim 2 wherein the excitation path fiber comprises a single mode polarization maintaining fiber.

4. The microscope of claim 2 wherein the excitation path fiber comprises a hollow core fiber.

5. The microscope of claim 2 wherein the excitation path fiber comprises one of the following: a few mode fiber; a large mode area fiber; a photonic crystal fiber; a fiber bundle.

6. The microscope of claim 1 wherein the light generated by the sample is fluorescent light.

7. The microscope of claim 1 wherein the scanner is a microelectromechanical systems (MEMS) mirror scanner.

8. The microscope of claim 1 wherein the element configured to combine is a beam splitter.

9. The microscope of claim 8 wherein the element configured to combine is a dichroic mirror.

10. The microscope of claim 9 wherein the dichroic mirror passes the spatially patterned light and the light generated by the sample and reflects the imaging excitation light.11 . The microscope of claim 1 wherein the imaging excitation light comprises laser pulses of 3 picoseconds to 40 femtoseconds in duration.

12. The microscope of claim 1 further comprising a gradient index of refraction (GRIN) optical element having a numerical aperture in the range of 0.3 to 0.9 and the objective includes optics for correction of GRIN aberrations.

13. The microscope of claim 1 further comprising a variable optical element configured to shift a focal plane.

14. The microscope of claim 13 wherein the variable optical element comprises a tunable liquid lens.

15. The microscope of claim 13 wherein the variable optical element is placed before the objective.

16. The microscope of claim 1, further comprising an inspection system for calibrating the microscope wherein the sample is fluorescent and wherein the inspection system includes a filter set after the fluorescent sample, a camera configured to collect light from the filter system, and a processor configured to measure offsets between fluorescence generated from the imaging excitation light from the first path and spatially patterned light from the second path.

17. The microscope of claim 1 configured as a miniature microscope having dimensions of less than 40 mm x 50 mm x 15 mm.

18. The microscope of claim 16 having a weight under 10 grams.

19. The microscope of claim 1 further comprising a clamshell case having a front shell and a back shell wherein optical elements are press-fitted into the back shell and the front shell is affixed to the back shell.

20. The method of calibrating the foci of a microscope comprising the steps of:(a) providing a sample;(b) providing a microscope having - a spatial pattern arm configured to both deliver spatially patterned light through a fiber to the sample and to collect light from the sample back along the same route, and an image light arm configured to deliver imaging light to the sample;(c) axially adjusting the fiber relative to the microscope;(d) delivering imaging light to an imaging spot in a field;(e) providing a patterned spot to the sample from the spatial pattern arm and positioning the patterned spot laterally adjacent the imaging spot;(f) focusing at least one of either the patterned spot or the imaging spot on the sample;(g) axially translating the microscope relative to the sample and imaging the sample with an inspection system;(h) measuring an offset between the imaging spot and the patterned spot;(i) determining whether the offset falls within a desired range;(j) if the offset does not fall within a desired range, adjusting the fiber axially with respect to the microscope and repeating steps (d) through (i)(k) if the offset does fall within a desired range, fixing the fiber with respect to the microscope.21 . The method of claim 20 wherein the sample is fluorescent and wherein the imaging light causes fluorescent emission at the imaging spot.

22. The method of claim 20 wherein the step of axially adjusting the fiber is performed by a translation stage.

23. The method of claim 20 wherein step (g) is performed by a translation stage.

24. The method of claim 20 wherein step (k) glues the fiber to the microscope.

25. The method of calibrating microscope imaging and photostimulation coordinate systems of a microscope comprising the steps of:(a) providing a fluorescent sample with easily distinguishable features;(b) providing a microscope having - a photostimulation arm configured to both deliver spatially patterned light through a fiber to the sample and to collect light from the sample back along the same route to microscopically image the sample, and an image light arm configured to deliver imaging light to the sample;(c) imaging light transmitted from the sample with an inspection system;(d) imaging the distinguishable features with the microscope while performing step (c);(e) delivering three photostimulation calibration spots to the sample;(f) recording the coordinates of the photostimulation spots in the photostimulation coordinate system;(g) determining the location of the photostimulation spots relative to the distinguishable features in a microscopy image provided in step (b) and recording the photostimulation spot coordinates in the microscope imaging coordinate system;(h) using the location of the recorded spots in the two coordinate systems to determine a transformation between the two coordinate systems;(i) saving this transformation into software;(j) using this saved transformation to enable accurate targeting of the photostimulation field.

26. The method of claim 25 further comprising the step of adjusting the three photo stimulation calibration spots to a desired location relative to the distinguishable features.

27. The method of claim 25 further comprising the step of testing the transformation by placing photostimulation spots at new distinguishable locations on the sample in the laser scanning microscopy field and imaging the spots with the inspection scope.

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