Article Figures & Data
Figures
- Figure 1.
Hardware and software adaptations to enable IOSI on an existing upright microscope. A, Hardware necessary to achieve IOSI includes (from left to right) an Arduino microcontroller connection to an LED ring light for illumination, an objective for light collection, and a camera to record reflected light. B, Schematic of the hardware adaptations and basic connections. C, Image of the application for illumination control. Users may set the illumination wavelength and intensity. D, Image of the application for basic image analysis. Users may input settings used for image acquisition and identify acquired images. The application will then process images, calculate ΔR/R values, and display a scaled image in the application window. Thresholding can then be done within the application, using either an absolute percentage of the maximum ΔR/R value or using z scores of ΔR/R values, and a binarized map can then be overlaid onto an image of the vasculature for localization of signals.
- Figure 2.
Mapping-evoked signals across primary somatosensory and visual cortices. A, Schematic of cranial window placement for mapping evoked signals in the primary somatosensory cortex. Approximate locations of major cortical regions are outlined in white (adapted from the Allen Mouse Brain Atlas Brain Explorer 2), with the S1FL (blue), S1HL (red), and S1BF (yellow) color coded. B, Scaled ΔR/R images of IOSI from an individual mouse performed during vibrotactile stimulation of the contralateral whiskers, forelimb, or hindlimb. Scaled ΔR/R values are ×10−4. Scale bar, 0.5 mm. C, Group-averaged images from six mice of IOSI performed during vibrotactile stimulation of the contralateral whiskers, forelimb, or hindlimb. Values are arbitrary units of 8 bit images from minimum (0) to maximum (255). Scale bar, 0.5 mm. D, Merged sensory-evoked maps from B overlaid onto the cortical vasculature. ΔR/R images were z scored and thresholded for values less than –3, binarized, pseudocolored (S1FL, blue; S1HL, red; S1BF, yellow), then merged and overlaid. Scale bar, 0.5 mm. E, Displacement of S1FL and S1HL map centers, relative to the S1BF map, from 6 individual mice. Map displacements for S1FL and S1HL were significantly different from one another (one-way MANOVA, p = 1.04 × 10−5). F, Scaled ΔR/R images of IOSI from an individual mouse performed during passive viewing of drifting sinusoidal gratings by the contralateral eye. Scaled ΔR/R values are ×10−3. Scale bar, 0.5 mm. G, Visual-evoked map from F overlaid onto the cortical vasculature. Scale bar, 0.5 mm.
- Figure 3.
IOSI of single-whisker evoked cortical activity in the S1BF. A, Schematic of stimulated whiskers (B1, C1, and D1) and somatotopic organization of the corresponding barrels in the S1BF. B, Scaled ΔR/R images of IOSI from an individual mouse performed during vibrotactile stimulation of the contralateral B1, C1, and D1 whiskers. Scaled ΔR/R values are ×10−3. Scale bar, 0.5 mm. See Extended Data Figure 3-1 for scaled ΔR/R images of IOSI performed using higher frame rate acquisition (30 Hz) or acute thinned skull preparation. C, Merged single-whisker maps from B overlaid onto the cortical vasculature. ΔR/R images were z scored and thresholded for values less than –3, binarized, pseudocolored, then merged and overlaid. Scale bar, 0.5 mm. D, Average map area by IOSI trial, as a percentage of the maximum map area following all 30 trials, of 18 single-whisker representations in 6 mice.
- Figure 4.
High-sensitivity IOSI can be achieved with CCD or CMOS cameras. A, Scaled ΔR/R images of IOSI from an individual mouse performed during vibrotactile stimulation of the contralateral C1 whisker. Scaled ΔR/R values are ×10−2. Scale bar, 0.5 mm. B, Merged single-whisker maps from A overlaid onto the cortical vasculature. ΔR/R images were z scored and thresholded for values less than –3, binarized, pseudocolored, then merged and overlaid. Scale bar, 0.5 mm. C, Quantification of the C1 single-whisker map area from 6 mice imaged with the CCD camera and 3 mice imaged with the CMOS camera. Dotted lines indicate the same mouse imaged with both cameras, with imaging using the CMOS camera performed 6 d after the start of chronic whisker trimming (Fig. 5). Two-tailed t test, p = 0.13. D, Quantification of C1 single-whisker map mean ΔR/R pixel intensity from the same mice as in C. ΔR/R values are ×10−3. Two-tailed t test, p = 0.11.
- Figure 5.
Longitudinal IOSI detects cortical map plasticity following whisker trimming. A, Scaled ΔR/R images of IOSI from an individual mouse performed during vibrotactile stimulation of the contralateral C1 whisker at baseline before (left) and 21 d after whisker trimming sparing only the C1 whisker. Scaled ΔR/R values are ×10−3. Scale bar, 0.5 mm. B, Merged single-whisker maps from A overlaid onto the cortical vasculature. ΔR/R images were z scored and thresholded for values less than –3, binarized, pseudocolored, then merged and overlaid. Scale bar, 0.5 mm. C, Merged pre-whisker trimming and post-whisker trimming maps, aligned using vasculature images, showing expansion of the C1 whisker cortical map representation. Scale bar, 0.5 mm. D, Quantification of change in C1 map area after whisker trimming from 6 mice. The dots in red represent the mouse depicted in A–C. Paired-sample, two-tailed t test, **p = 0.003.
Extended Data
Extended Data 1
Extended data includes several additional files. “ISI Parts List.xlsx” is an excel spreadsheet listing all materials required to implement the IOSI adaptations described in this manuscript. “IOSI_RingLED_IlluminationBuild.docx” is a word document providing step-by-step instructions for assembling and setting up ring LED illumination. “Ring LED Holder-2.stl” is a design file to 3D print the mount for the ring LED. “NeopixelControl.mlapp” is a MATLAB application for controlling ring LED illumination. “iosgui.mlapp” is a MATLAB application for processing IOSI data. “IOSGUI_ImageAnalysis.m” is a companion script for processing IOSI data, used by “iosgui.mlapp”. “IOSGUI_Instructions.docx” is an instruction manual for using the iosgui application for processing IOSI data. Download Extended Data 1, ZIP file.
Figure 3-1
IOSI of single whisker-evoked cortical activity in the S1BF using higher frame rates or an acute thinned skull cranial window. A, Scaled ΔR/R images of IOSI from an individual mouse performed during vibrotactile stimulation of the contralateral C1 whisker, with imaging at 10 Hz (left) or 30 Hz (right). Scaled ΔR/R values are ×10−3. Scale bar, 0.5 mm. B, Scaled ΔR/R images of IOSI through the acutely thinned skull from an individual mouse performed during vibrotactile stimulation of the contralateral D1 whisker. Scaled ΔR/R values are ×10−3. Scale bar, 0.5 mm. Download Figure 3-1, EPS file.
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