Nature Neuroscience: doi: /nn Supplementary Figure 1. Optimized Bessel foci for in vivo volume imaging.

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1 Supplementary Figure 1 Optimized Bessel foci for in vivo volume imaging. (a) Images taken by scanning Bessel foci of various NAs, lateral and axial FWHMs: (Left panels) in vivo volume images of YFP + neurites in cortex of an awake mouse (Thy1-YFP line H); scale bar: 10 m. (Right panels) Axial point spread functions measured from 0.2- m-diameter beads; x scale bar: 5 m; z scale bar: 25 m. (b) Mean intensity projection of the same neurites (color-coded by depth below pia) imaged by scanning a 1.05-NA Gaussian focus in 3D. Higher NA Bessel foci have stronger side rings, resulting in hazy backgrounds. Objective: Olympus 25, 1.05 NA.

2 Supplementary Figure 2 Axonal varicosities and dendritic spines are resolvable in single frames of 30-Hz Bessel volume scanning. (a) Individual raw image frames during 30-Hz volumetric measurements of calcium transients in a volume extending 60 m in z. Arrows and numbers label individual axonal varicosities (putative boutons, 1,2) and dendritic spines (3-10) (same as in Fig. 1d), with their zoomed-in view shown in the panels on the right. (b) As comparison, the same synaptic structures in the average of 5 frames (i.e., effective volume rate of 6 Hz). Scale bars: left panels, 20 m; right panels, 5 m.

3 Supplementary Figure 3 Bessel focus-scanning is robust against axial motion artifacts. (a) Mean axial projection of a 3D image stack (180 m 180 m 160 m, 100 2D images) collected by Gaussian focus scanning, of

4 GCaMP6f + dendrites (0-160 m below pia) in awake mouse S1 (structures color-coded by depth). (b) Averaged image of a 266-sec time series of the same dendrites imaged with a Bessel focus (0.4 NA and 91 m axial FWHM) at 30 Hz. (c) Averaged image after registering the time series using TurboReg plugin of ImageJ. (d) Mean axial projection of a 3D image stack (80 m 40 m 25 m, 25 2D images) collected by Gaussian focus scanning of YFP + dendrites (46-70 m below pia) in awake mouse V1 (Thy1-YFP line H, structures color-coded by depth). (e) Averaged image of a 100-sec 2D-registered time series of dendrites at 53 m below pia acquired with Gaussian focus scanning at 7.4 Hz. (f) Averaged image of a 100-sec 2D-registered time series of the volume of dendrites in d imaged with a Bessel focus (0.4 NA and 19 m axial FWHM) at 7.4 Hz. (g,h) Brain motion (upper panels, quantified as the lateral image displacement with time) causes (g) large changes of fluorescence signal from two YFP + dendrites (ROI1 and ROI2) in Gaussian focus scanning mode, (h) but not Bessel focus scanning mode. Objective: Olympus 25, 1.05 NA; Post-objective power: (a) 30 mw, (b,c) 103 mw, (d,e) 9mW, (f) 21 mw; Wavelength: (a-c) 960 nm, (d-f) 940nm. Scale bars: 20 m.

5 Supplementary Figure 4 In vivo characterization of dendritic spines in ferret V1 at 30-Hz volume rate. (a,b) Two examples of applying Bessel focus scanning to the characterization of spines in ferret V1. (Left) Mean intensity projection of 3D image stacks by conventional Gaussian focus imaging in V1 of anesthetized ferrets; (Right) Images collected by scanning a Bessel beam with 0.4 NA and 50 m axial FWHM. Yellow circles highlight example spines. (c) 10-trial-averaged calcium transients of spines in a and b for 12 grating angles. (d) Tuning curves of individual spines in a and b. Objective: Nikon 16, NA 0.8; Post-objective power: 55 mw for Gaussian scanning and 84 mw for Bessel scanning; Wavelength: 960 nm; Shadow (c) and error bars (d): s.e.m; Scale bars: 10 m. Representative images from 2 ferrets.

6 Supplementary Figure 5 Removal of neuropil contamination in Bessel focus scanning mode. (a) (left) Image of a cell body obtained by scanning a Bessel beam with 0.4 NA and 78 m axial FWHM, (right) overlaid with region of interest (ROI, orange outline) and the area used for calculating neuropil background (red region). (b) The difference between the averaged signal within ROI (F 1) and the averaged signal within neuropil mask (F neuropil) is used to calculate calcium transient of the neuron. After the removal of neuropil contamination, the cell body still exhibited activity correlated with pupil size. (c) and (d) same as a and b, except that the removal of neuropil contamination was applied to a volume of neuropil, resulting in minimal calcium transients. All fluorescence traces were plotted on the same scale.

7 Supplementary Figure 6 Activity synchrony among GABAergic neurons persists in the dark. (a) Representative image of GAD2 + neurons expressing GCaMP6s (0-100 m below pia) collected by scanning a Bessel beam with 0.4 NA and 78 m axial FWHM. (b) Pupil size (red) and concurrently measured calcium transients of example neurons labeled in a. (c) Pearson s correlation coefficient (R) between neuronal calcium transients and pupil size (1st row and 1st column, sorted by decreasing R), as well as between the calcium transients of all pairs of neurons identified in a. Objective: Olympus 25, 1.05 NA; Post-objective power: 21 mw; Wavelength: 960 nm; Scale bar: 50 m.

8 Supplementary Figure 7 Activity synchrony among GABAergic neurons was also observed in conventional Gaussian focus scanning mode. (a) GABAergic neurons at three different depths (55, 73, and 93 m) imaged by scanning a conventional Gaussian focus (1.05 NA, 1.4 m axial FWHM). (b) Pupil size (red) and concurrently measured calcium transients of example neurons labeled in a. (c) Pearson s correlation coefficient (R) between neuronal calcium transients and pupil size (1st row and 1st column, sorted by decreasing R), as well as between the calcium transients of all pairs of neurons identified in a. (d) Histogram distribution and (e) scatter plot of R measured from a mouse. Red open histogram: R between pupil size and neuronal calcium transients; Gray filled histogram: R between neurons; Dashed lines: median; n: number of R s. Objective: Olympus 25, 1.05 NA; Post-objective power: 18 mw for 55 and 93 m depth and 14 mw for 73 m depth; Wavelength: 960 nm; Scale bar: 50 m.

9 Supplementary Figure 8 Engineering Bessel foci with asymmetric axial-intensity distribution with an SLM. (a) Overlaying a binary concentric ring pattern on a quadratic phase pattern on the SLM allows the shaping of axial intensity distribution of the Bessel focus. S: period of the concentric rings in units of pixels. (b) Axial images and corresponding intensity profiles of a 2- m- diameter bead using three different Bessel foci generated with periods S1, S2, S3. (c,d) (From left to right) in vivo images of neurons and neurites measured in V1 of awake mice using Gaussian focus, Bessel foci generated with S1, S2, and S3, respectively. Gaussian images are mean intensity projections with features color-coded by depth. Arrows point to deeper structures that had best image quality when the Bessel focus of increasing intensity with depth (S3 in b) was used. Objective: Olympus 25, 1.05 NA; Post-objective power: c, 14 mw for Gaussian modality and 32 mw for Bessel modality; d, 21 mw for Gaussian modality and 61 mw for Bessel modality; Wavelength: 940 nm; Scale bars: 20 m.

10 Supplementary Figure 9

11 Annular apodization mask fine-tunes the axial length and intensity distribution of Bessel foci. (a-c) and (d-f): two NA-0.4 Bessel foci with different axial lengths and intensity distributions. (a,d) Binary concentric grating patterns (phase values: 0 and ) on SLM. S indicates the period of the gratings in units of pixels. (b,e) (Top) amplitude and (bottom) phase of the electric field of the light at the plane of the annular apodization mask (0.39 mm inner radius and 0.6 mm outer radius for b, mm inner radius and 0.6 mm outer radius for e). Electric field within the annulus (areas between red dashed lines) was transmitted. (c,f) (Top) axial (xz) images of 2- m-diameter fluorescent beads and (bottom) line intensity profiles along the z axis obtained with Bessel foci generated by a and b, d and e, respectively. (g) Simulated axial PSFs and (h) their intensity profiles along z as a function of annulus width of the apodization mask. Incidence beam diameter at SLM: 2.2 mm. Magnification from mask to objective back pupil: 4.7; Objective: Olympus 25, 1.05 NA; Post-objective power: 14.2 mw and 15.5 mw for c and f, respectively; Wavelength: 900 nm; Scale bar: 10 m. See Supplementary Technical Notes for more details.

12 Video rate volumetric functional imaging of the brain at synaptic resolution Supplementary Table 1: Design parameters of Bessel modules incorporated into three 2PLSM systems. Supplementary Table 1 Parameters of Bessel modules in three 2PLSM systems Focal length of Lens 1 (mm) Focal length of Lens 2 (mm) Focal length of Lens 3 (mm) Magnification between mask and back pupil plane of objective System System System Microscope objective Olympus 25x, 1.05 NA Olympus 25x, 1.05 NA Nikon 16x, 0.8 NA

13 Supplementary Table 2. Imaging throughput comparison between Bessel and Gaussian focus scanning modes. Data Figs. 1c,d Fig. 2 Figs 3a,b, 1 st panels from top Figs 3a,b, 2 nd panels from top Figs 3a,b, 3 rd panels from top Figs 3a,b, 4 th panels from top Structures imaged Dendritic spines Dendritic spines Gaussian focus frame Axial FWHM (µm) Throughput (# of structures imaged per frame) Axial FWHM (µm) Bessel focus frame Throughput (# of structures imaged per frame) Largest vertical distance between structures (µm) Minimal Gaussian frames to image the same structures to * to * Somata to ** Somata to ** Somata to ** Somata 1,4 5 to ** Figs. 3f,g Somata to ** Figs. 3k,i Somata to ** Figs. 4a,b Somata to *** Figs. 5c,d Neurites 1.4 N/A 35 N/A 61 44* Figs. 5g,h Neurites 1.4 N/A 78 N/A * Supplementary Figs 3a,c Supplementary Fig. 4a Supplementary Fig. 4b Dendritic spines Dendritic spines Dendritic spines to * 4 0 to * 4 1 to * *The ratio of Largest vertical distance between structures and Gaussian focus axial FWHM; **The ratio of Largest vertical distance between structures and Nyquist sampling distance of 6 µm (average size of mouse V1 neuron somata taken as 12 µm); ***The ratio of Largest vertical distance between structures and Nyquist sampling distance of 5 µm (average size of zebrafish neuron somata taken as 10 µm).

14 Supplementary Table 3. Values for bars in Figure 3 histograms. Fig. 3e, panel 1 Fig. 3e, panel 2 Fig. 3e, panel 3 Mouse strain Gad2-ires-Cre Gad2-ires-Cre Gad2-ires-Cre # of mouse # of image sections # of neurons # of pairwise correlations pupil-neuron neuron - neuron pupil-neuron neuron - neuron pupil-neuron neuron - neuron 157* * * 4885 Values of bars in the histograms % R (pupil, % R(neuron, % (pupil, % ( neuron, % (pupil, [-1,-0.8) [-0.8,-0.6) [-0.6,-0.4) [-0.4,-0.2) [-0.2,0) [0,0.2) [0.2,0.4) [0.4, 0.6) [0.6, 0.8) [0.8, 1] % ( neuron, Fig. 3e, panel 4 Fig. 3j Fig. 3o Mouse strain Gad2-ires-Cre VIP-ires-Cre SST-ires-Cre # of mouse # of image sections # of neurons # of pairwise correlations pupil-neuron neuron - neuron pupil-neuron neuron - neuron pupil-neuron 212* Values of bars in the histograms % (pupil, % ( neuron, % (pupil, % ( neuron, % (pupil, [-1,-0.8) [-0.8,-0.6) [-0.6,-0.4) [-0.4,-0.2) [-0.2,0) [0,0.2) [0.2,0.4) [0.4, 0.6) [0.6, 0.8) [0.8, 1] *Concurrent recording of pupil size was not carried out for all neural imaging sessions. neuron - neuron % ( neuron,

15 Supplementary Table 4. Performance comparison on dendrite/dendritic spine imaging between Bessel focus scanning method and other fast volumetric imaging methods 3D AOD Variable focus lens 2 Remote focusing 3 Bessel approach scanning 1 Axial scanning range 140 µm demonstrated 40 µm demonstrated 30 µm demonstrated Tunable µm Volume imaging data (for dendrites or dendritic spines) Maximal rate (volume) (rate of volume imaging) (μm 3 *Hz) Essential optical elements Additional hardware (optics) compared with a conventional two-photon microscope Additional hardware (electronics) compared with a conventional two-photon microscope Additional data processing Data acquisition In vitro: 87 locations in μm 3 volume scanned at khz. In vitro: Using the number of locations for calculation: 2,589,120 locations*hz Four custom acoustic optical devices, conjugation optics Dispersion compensation units, Custom beam position stabilizer. custom driver electronics for acoustic optical devices and beam position stabilizer Not needed Custom-written LabVIEW codes In vivo: μm 3 volume at 56 Hz; In vivo: μm 3 at 14 Hz. In vivo: 873,600 μm 3 *Hz demonstrated TAG lens, conjugation optics A continuous wave laser for axial position reference and related optics High-speed digitizer system with FPGA Sinusoidal fitting followed by linear interpolation required to obtain axial position from raw data Custom-written LabVIEW codes In vitro: 2 locations 30 μm apart axially imaged at 500 Hz. In vitro: if the number of locations is used for calculation: 1000 locations*hz An additional microscope objective, a custom-made fast moving mirror mounted on two galvanometers, and conjugation optics Optics such as quarterwave plate and dichroic beamsplitter Not needed Not needed Custom-written LabVIEW codes In vivo: µm 3 at 30 Hz; In vivo: µm 3 at 30 Hz ; In vivo: µm 3 at 7.4 Hz; In vivo: 102 x 102 x 60 µm 3 at 1.5 Hz; In vivo: 50 x 50 x 72 μm 3 at 30 Hz. In vivo: 155,520,000 μm 3 *Hz demonstrated SLM, conjugation optics, annular mask Not needed Not needed Not needed (All analysis was carried out on standalone desktop or laptop computers.) No modification needed

16 software modification Compatibilit y with commercial microscope Not demonstrated Not demonstrated Not demonstrated Demonstrated 1. Katona, G., et al. Fast two photon in vivo imaging with three dimensional random access scanning in large tissue volumes. Nat Meth 9, (2012). 2. Kong, L., et al. Continuous volumetric imaging via an optical phase locked ultrasound lens. Nat Meth 12, (2015). 3. Botcherby, E.J., et al. Aberration free three dimensional multiphoton imaging of neuronal activity at khz rates. Proceedings of the National Academy of Sciences 109, (2012).