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 integrating miniscope recordings with multiplexed confocal spectral fingerprinting through the same GRIN lens, Neuroplex addresses key spectral limitations of head-mounted microscopy—enabling simultaneous distinction of nine neuronal populations alongside GCaMP activity.

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 integrating miniscope recordings with multiplexed confocal spectral fingerprinting through the same GRIN lens, Neuroplex addresses key spectral limitations of head-mounted microscopy—enabling simultaneous distinction of nine neuronal populations alongside GCaMP activity.

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 integrating miniscope recordings with multiplexed confocal spectral fingerprinting through the same GRIN lens, Neuroplex addresses key spectral limitations of head-mounted microscopy—enabling simultaneous distinction of nine neuronal populations alongside GCaMP activity.

Techniques: Expressing, Labeling, Imaging