Journal: eLife

Article Title: Functional imaging of nine distinct neuronal populations under a miniscope in freely behaving animals

doi: 10.7554/eLife.110277

Figure Lengend Snippet: Video showing animal behavior during the social memory task alongside the corresponding miniscope calcium recording from the same session. Calcium-active ROIs are pseudocolored according to their Neuroplex-assigned fluorophore identity, indicating the corresponding projection-defined neuronal population.

Article Snippet: Neuroplex enables simultaneous tracking of multiple neuronal subtypes in behaving animals through a three-step pipeline: (1) Head-mounted GRIN-lens based miniscope imaging of GCaMP activity during behavior; (2) in vivo multiplexed confocal spectral imaging through the same GRIN lens to capture fluorophore fingerprints; and (3) linear unmixing to assign projection-specific identities to functionally defined neurons ( ).

Techniques:

Journal: eLife

Article Title: Functional imaging of nine distinct neuronal populations under a miniscope in freely behaving animals

doi: 10.7554/eLife.110277

Figure Lengend Snippet: ( a ) Surgical paradigm. In a TetO-GCaMP6s × CaMKII-tTa mouse, 9 AAV retro viruses are injected into downstream brain regions and gradient-index (GRIN) lens implanted into the target region. ( b ) Simultaneous recording of GCaMP6s (top) and behavior (bottom) during a social memory task. Scale bar = 100 µm ( c ) GCaMP6s recordings are processed. Constrained non-negative matrix factorization (CNMF)-defined ROIs (top) and ΔF/F traces (bottom) are exported. Scale bar = 100 µm. ( d ) Mice are head fixed and FOV under the GRIN lens imaged using the multiplexed lambda method. ( e ) Transformations are determined using anatomical background images to co-register the two imaging platforms. The transformations are applied to CNMF-defined ROIs. Scale bar = 100 µm. ( f ) Multispectral data are collected for each ROI (top) and an average spectral fingerprint for all ROIs is generated (bottom). Mean ±1.5 SD. Scale bar = 100 µm. ( g ) A linear unmixing model is applied to determine the fluorophore contribution for each ROI. Scale bar = 100 µm. ( i ) Neural activity is sorted by cell type. Scale bars = 20 ΔF/F (vertical), 20 s (horizontal).

Article Snippet: Neuroplex enables simultaneous tracking of multiple neuronal subtypes in behaving animals through a three-step pipeline: (1) Head-mounted GRIN-lens based miniscope imaging of GCaMP activity during behavior; (2) in vivo multiplexed confocal spectral imaging through the same GRIN lens to capture fluorophore fingerprints; and (3) linear unmixing to assign projection-specific identities to functionally defined neurons ( ).

Techniques: Injection, Imaging, Generated, Activity Assay

Journal: eLife

Article Title: Functional imaging of nine distinct neuronal populations under a miniscope in freely behaving animals

doi: 10.7554/eLife.110277

Figure Lengend Snippet: ( a ) Multicolor image obtained through 1×4 mm silver-doped GRIN lens of the calibration slide highlighting the z-plane chromatic aberration. ( b ) Shift in z-focal plane as a function of excitation laser wavelength. Second-order polynomial R 2 =0.9926, n=5. ( c ) Orthogonal projection of multicolor image obtained through 1×4 mm silver-doped GRIN lens of the calibration slide. Intensity profile (below) of single ring for each excitation channel shows negligible chromatic shift along lateral axes. ( d ) Percent transmission through the GRIN lens as a function of excitation laser wavelength. Sixth order polynomial R 2 =0.9751, n=5. ( e ) Orthogonal projection of calibration slide imaged through 1×4 mm silver-doped GRIN lens overlaid (in cyan) with rectilinear grid lines. Substantial overlap of fluorescent rings from the grid indicates minimal field distortions. ( f ) Excerpt from ( e ) showing the rings focused in the sagittal plane (z=+20 µm), the circle of least confusion (z=0 µm), and the tangential focal plane (z=–17.5 µm). ( g ) Curvature of the Petzval field as a function of radial distance from center of the GRIN lens. Astigmatism results in three axially separated focal planes. Second order polynomial sagittal R 2 =0.9845, least confusion R 2 =0.8839, and tangential R 2 =0.7519, n=3. Scale bars = 100 µm.

Article Snippet: Neuroplex enables simultaneous tracking of multiple neuronal subtypes in behaving animals through a three-step pipeline: (1) Head-mounted GRIN-lens based miniscope imaging of GCaMP activity during behavior; (2) in vivo multiplexed confocal spectral imaging through the same GRIN lens to capture fluorophore fingerprints; and (3) linear unmixing to assign projection-specific identities to functionally defined neurons ( ).

Techniques: Transmission Assay

Journal: eLife

Article Title: Functional imaging of nine distinct neuronal populations under a miniscope in freely behaving animals

doi: 10.7554/eLife.110277

Figure Lengend Snippet: ( a ) Shift in z-focal plane as a function of excitation laser wavelength in a 1×4 mm silver-doped GRIN lens. Second-order polynomial R 2 =0.9970. ( b ) Shift in z-focal plane as a function of excitation laser wavelength in 0.6×7 mm GRIN lenses doped with either silver or lithium. Second-order polynomials: Silver R 2 =0.9983, Lithium R 2 =0.9928. ( c ) Percent transmission through a 1×4 mm silver-doped GRIN lens as a function of excitation laser wavelength. Sixth-order polynomial R 2 =0.9926. ( d ) Percent transmission through 0.6×7 mm GRIN lenses doped with either silver or lithium as a function of excitation laser wavelength. Sixth-order polynomial: silver, R 2 =0.9979; lithium, R 2 =0.9549.

Article Snippet: Neuroplex enables simultaneous tracking of multiple neuronal subtypes in behaving animals through a three-step pipeline: (1) Head-mounted GRIN-lens based miniscope imaging of GCaMP activity during behavior; (2) in vivo multiplexed confocal spectral imaging through the same GRIN lens to capture fluorophore fingerprints; and (3) linear unmixing to assign projection-specific identities to functionally defined neurons ( ).

Techniques: Transmission Assay

Journal: eLife

Article Title: Functional imaging of nine distinct neuronal populations under a miniscope in freely behaving animals

doi: 10.7554/eLife.110277

Figure Lengend Snippet: ( a ) In vivo multiplexed spectral imaging paradigm. Schematic of multiplexed spectral imaging (left). Depiction of overlapping fluorophore spectral emissions for each excitation laser wavelength (middle). Depiction of multiplexed spectral images which create a 204-dimensional dataset (right). ( b ) Automated co-registration of miniscope and laser scanning confocal microscope (LSM) images. Top: A calibration slide used to measure scaling between modalities. Bottom: Experimental FOV showing brain vasculature. Miniscope and confocal images of the same FOV and automated co-registration overlay with zoomed-in regions of interest. ( c ) Example calcium-activity regions of interest (ROI) derived from miniscope data co-registered and overlaid on confocal LSM image. ( d ) Spectral fingerprint of the example ROI, with the solid blue line showing the example ROI and the dashed line depicting the average spectral profile of the animal. ( e ) Beta multiplier from the example ROI, depicting the deviation from the mean beta value for all ROIs from the same animal. ( f ) Empirically measured spectral profiles from pure fluorophore samples, shown as beta-weighted contributors to ROI fingerprints. Scale bar: 100 µm ( a, b ), 10 µm ( b inset and c ).

Article Snippet: Neuroplex enables simultaneous tracking of multiple neuronal subtypes in behaving animals through a three-step pipeline: (1) Head-mounted GRIN-lens based miniscope imaging of GCaMP activity during behavior; (2) in vivo multiplexed confocal spectral imaging through the same GRIN lens to capture fluorophore fingerprints; and (3) linear unmixing to assign projection-specific identities to functionally defined neurons ( ).

Techniques: In Vivo, Imaging, Microscopy, Activity Assay, Derivative Assay

Journal: eLife

Article Title: Functional imaging of nine distinct neuronal populations under a miniscope in freely behaving animals

doi: 10.7554/eLife.110277

Figure Lengend Snippet: ( a–r ) Each panel shows an regions of interest (ROI) that exceeded threshold for a single fluorophore identity assignment. (Left:) Functional ROIs identified from miniscope recordings during behavior, co-registered and overlaid on corresponding in vivo confocal images. (Center:) Spectral fingerprint of the ROI (solid line), compared to the animal’s average spectral background (dashed line). Excitation-emission bins are color-coded to excitation laser wavelength. Right inset: Beta multiplier values for all fluorophores from the same ROI, plotted as standard deviations above the animal-specific baseline. Scale bars = 10 µm.

Article Snippet: Neuroplex enables simultaneous tracking of multiple neuronal subtypes in behaving animals through a three-step pipeline: (1) Head-mounted GRIN-lens based miniscope imaging of GCaMP activity during behavior; (2) in vivo multiplexed confocal spectral imaging through the same GRIN lens to capture fluorophore fingerprints; and (3) linear unmixing to assign projection-specific identities to functionally defined neurons ( ).

Techniques: Functional Assay, In Vivo

Journal: eLife

Article Title: Functional imaging of nine distinct neuronal populations under a miniscope in freely behaving animals

doi: 10.7554/eLife.110277

Figure Lengend Snippet: ( a ) Proportion of functionally defined ROIs classified as expressing one or two fluorophores based on thresholded beta multipliers. ( b ) Frequency of dual fluorophore assignments across the dataset. Left: Heatmap showing co-assignment rates between fluorophore pairs. Right: Total frequency of dual hits per individual fluorophore. ( c ) Frequency of dual-labeled ROIs by brain region. Left: Heatmap showing co-occurrence between projection-defined populations. Right: Total frequency of dual hits per primary brain region. (n=1327 ROIs) ( d ) Example ROIs from miniscope imaging co-registered with confocal lambda stacks. ROIs are overlaid on three excitation channels (405, 561, 639 nm). ( e ) Z-scored beta multipliers across all fluorophores for each example ROI, with above-threshold values circled. Dual fluorophores were assigned to two ROIs (28, 98), and only a single fluorophore to ROI 142. ( f ) Spectral fingerprints for each example ROI (solid line) plotted against the average background spectrum for the animal (dashed line). ROI 28 (top) was assigned two spectrally distinct fluorophores (mTagBFP2+mNeptune2.5); ROI 98 (middle) shows co-assignment of more spectrally overlapping fluorophores (mOrange2+mNeptune2.5); ROI 142 (bottom) is included as a single-label example with a strong match to mTagBFP2 only. Scale bars = 10 µm.

Article Snippet: Neuroplex enables simultaneous tracking of multiple neuronal subtypes in behaving animals through a three-step pipeline: (1) Head-mounted GRIN-lens based miniscope imaging of GCaMP activity during behavior; (2) in vivo multiplexed confocal spectral imaging through the same GRIN lens to capture fluorophore fingerprints; and (3) linear unmixing to assign projection-specific identities to functionally defined neurons ( ).

Techniques: Expressing, Labeling, Imaging

Journal: eLife

Article Title: Functional imaging of nine distinct neuronal populations under a miniscope in freely behaving animals

doi: 10.7554/eLife.110277

Figure Lengend Snippet: Video showing animal behavior during the social memory task alongside the corresponding miniscope calcium recording from the same session. Calcium-active ROIs are pseudocolored according to their Neuroplex-assigned fluorophore identity, indicating the corresponding projection-defined neuronal population.

Article Snippet: By combining functional recordings from a head-mounted miniscope with multiplexed spectral confocal imaging in the same animal, Neuroplex supports the assignment of fluorophore identity to functionally defined neuronal locations without relying on post-fixation tissue processing.

Techniques:

Journal: eLife

Article Title: Functional imaging of nine distinct neuronal populations under a miniscope in freely behaving animals

doi: 10.7554/eLife.110277

Figure Lengend Snippet: ( a ) In vivo multiplexed spectral imaging paradigm. Schematic of multiplexed spectral imaging (left). Depiction of overlapping fluorophore spectral emissions for each excitation laser wavelength (middle). Depiction of multiplexed spectral images which create a 204-dimensional dataset (right). ( b ) Automated co-registration of miniscope and laser scanning confocal microscope (LSM) images. Top: A calibration slide used to measure scaling between modalities. Bottom: Experimental FOV showing brain vasculature. Miniscope and confocal images of the same FOV and automated co-registration overlay with zoomed-in regions of interest. ( c ) Example calcium-activity regions of interest (ROI) derived from miniscope data co-registered and overlaid on confocal LSM image. ( d ) Spectral fingerprint of the example ROI, with the solid blue line showing the example ROI and the dashed line depicting the average spectral profile of the animal. ( e ) Beta multiplier from the example ROI, depicting the deviation from the mean beta value for all ROIs from the same animal. ( f ) Empirically measured spectral profiles from pure fluorophore samples, shown as beta-weighted contributors to ROI fingerprints. Scale bar: 100 µm ( a, b ), 10 µm ( b inset and c ).

Article Snippet: By combining functional recordings from a head-mounted miniscope with multiplexed spectral confocal imaging in the same animal, Neuroplex supports the assignment of fluorophore identity to functionally defined neuronal locations without relying on post-fixation tissue processing.

Techniques: In Vivo, Imaging, Microscopy, Activity Assay, Derivative Assay

Journal: eLife

Article Title: Functional imaging of nine distinct neuronal populations under a miniscope in freely behaving animals

doi: 10.7554/eLife.110277

Figure Lengend Snippet: ( a–r ) Each panel shows an regions of interest (ROI) that exceeded threshold for a single fluorophore identity assignment. (Left:) Functional ROIs identified from miniscope recordings during behavior, co-registered and overlaid on corresponding in vivo confocal images. (Center:) Spectral fingerprint of the ROI (solid line), compared to the animal’s average spectral background (dashed line). Excitation-emission bins are color-coded to excitation laser wavelength. Right inset: Beta multiplier values for all fluorophores from the same ROI, plotted as standard deviations above the animal-specific baseline. Scale bars = 10 µm.

Article Snippet: By combining functional recordings from a head-mounted miniscope with multiplexed spectral confocal imaging in the same animal, Neuroplex supports the assignment of fluorophore identity to functionally defined neuronal locations without relying on post-fixation tissue processing.

Techniques: Functional Assay, In Vivo

Journal: eLife

Article Title: Functional imaging of nine distinct neuronal populations under a miniscope in freely behaving animals

doi: 10.7554/eLife.110277

Figure Lengend Snippet: ( a ) Proportion of functionally defined ROIs classified as expressing one or two fluorophores based on thresholded beta multipliers. ( b ) Frequency of dual fluorophore assignments across the dataset. Left: Heatmap showing co-assignment rates between fluorophore pairs. Right: Total frequency of dual hits per individual fluorophore. ( c ) Frequency of dual-labeled ROIs by brain region. Left: Heatmap showing co-occurrence between projection-defined populations. Right: Total frequency of dual hits per primary brain region. (n=1327 ROIs) ( d ) Example ROIs from miniscope imaging co-registered with confocal lambda stacks. ROIs are overlaid on three excitation channels (405, 561, 639 nm). ( e ) Z-scored beta multipliers across all fluorophores for each example ROI, with above-threshold values circled. Dual fluorophores were assigned to two ROIs (28, 98), and only a single fluorophore to ROI 142. ( f ) Spectral fingerprints for each example ROI (solid line) plotted against the average background spectrum for the animal (dashed line). ROI 28 (top) was assigned two spectrally distinct fluorophores (mTagBFP2+mNeptune2.5); ROI 98 (middle) shows co-assignment of more spectrally overlapping fluorophores (mOrange2+mNeptune2.5); ROI 142 (bottom) is included as a single-label example with a strong match to mTagBFP2 only. Scale bars = 10 µm.

Article Snippet: By combining functional recordings from a head-mounted miniscope with multiplexed spectral confocal imaging in the same animal, Neuroplex supports the assignment of fluorophore identity to functionally defined neuronal locations without relying on post-fixation tissue processing.

Techniques: Expressing, Labeling, Imaging