Continuous volumetric imaging via an optical phase-locked ultrasound lens

Journal name:
Nature Methods
Volume:
12,
Pages:
759–762
Year published:
DOI:
doi:10.1038/nmeth.3476
Received
Accepted
Published online

In vivo imaging at high spatiotemporal resolution is key to the understanding of complex biological systems. We integrated an optical phase-locked ultrasound lens into a two-photon fluorescence microscope and achieved microsecond-scale axial scanning, thus enabling volumetric imaging at tens of hertz. We applied this system to multicolor volumetric imaging of processes sensitive to motion artifacts, including calcium dynamics in behaving mouse brain and transient morphology changes and trafficking of immune cells.

At a glance

Figures

  1. Design of the OPLUL-based imaging system.
    Figure 1: Design of the OPLUL-based imaging system.

    A femtosecond laser beam (two-photon excitation beam) and a CW-diode laser beam (reference beam), both horizontally polarized, are combined by a dichroic mirror (DM) DM1. The beams travel through a polarizing beam splitter (PBS), a quarter waveplate (QWP) and an ultrasound lens (UL). Two lenses (F1 and F2) are used to image the UL onto an end mirror that directs the beam backward to the UL for a second pass. The beams exit the PBS and are imaged to the galvo scanning mirror by a second pair of lenses (F3 and F4). A second DM (DM2) separates the CW beam from the femtosecond beam. The CW beam is spatially filtered by an iris before entering a photodiode (PD) which provides the reference signal to the phase-locked loop (PLL). All of these elements form the OPLUL that is inserted between the light source and the multiphoton microscope (MPM) (left, top).

  2. 3D in vivo calcium imaging in neurons.
    Figure 2: 3D in vivo calcium imaging in neurons.

    (a) Representative volume view of a GCaMP6s-expressing dendritic segment (of three data sets) in mouse V1 cortex (x × y × z: 60 × 3.75 × 40 μm3; at a depth of 107–147 μm under the dura). Green overlay shows dendritic spines. Several sections from two separate dendritic segments (cyan- or magenta-labeled) are shown (arrowheads) as well as dendritic spines attached to these dendrites (arrows). D1, dendrite 1; S1, spine 1; D2, dendrite 2; S2, spine 2. (b) ΔF/F traces (at 56 Hz) extracted from several regions of interest (ROIs). Traces are color coded according to the depth of the ROIs and marked as in a. Asterisk denotes the onset of a large global calcium signal. Colored boxes (orange and blue) are expanded below to highlight smaller calcium responses in a spine and dendrite, respectively. (c) Representative volume view (of eight data sets) of the S1 barrel cortex (375 × 112 × 130 μm3; at a depth of 115–245 μm under the dura) of awake mice. Maximum intensity projections (MIPs) are shown in green and the cell locations (n = 63) are indicated by brown spheres. (d) Examples of sensory stimulation–evoked neuron activity (recorded at 14 Hz) of the cells in c. The gray boxes show the time span of air puff stimulation. (e) Representative volume view (of six data sets) of the dendrites and spines in the S1 barrel cortex (78 × 20 × 40 μm3; at a depth of 40–80 μm under the dura) of awake mice. D, dendrite; S, spine. (f) ΔF/F traces (at 14 Hz) extracted from each ROI for the dendrites and spines in e. Traces are marked with their corresponding colors in e: arrowheads are dendritic segments and arrows are dendritic spines. The gray box highlights calcium responses that appear in a spine but not elsewhere along the dendrite. (g) Representative volume view (of 10 data sets) of the M1 cortex (448 × 252 × 130 μm3; at a depth of 150–280 μm under the dura) of head-restrained, behaving mice. MIPs are shown in green and the cell locations (n = 304) are indicated by spheres. The red spheres (146 in total) label the neuron cluster of significant (P < 0.05, Student's t-test) positive correlation with running, and cyan spheres label the rest. (h) Mouse running velocity and calcium dynamics (at 10 Hz) of the neuron ensemble in g. In the upper graph, blue bars show the time of air puff and the red plot shows running velocity.

  3. 3D in vivo imaging of cell dynamics.
    Figure 3: 3D in vivo imaging of cell dynamics.

    (a) A representative neutrophil image (of six data sets) reconstructed from 2D cross-sectional imaging of a pial vein in mouse brain at a 1-kHz frame rate and a depth of 5–45 μm under the dura. The x-z frame size was 18 × 20 μm2 (25 frames). (b) Representative snapshots (of three data sets) of a neutrophil trafficking in a pial vein of mouse brain (at 39 Hz; 112 × 38 × 40 μm3; at a depth of 5–45 μm). (c) Representative image (of four data sets) of rapid morphological changes of a neutrophil trafficking through capillaries in mouse cerebral cortex (at 14Hz, 151 × 38 × 23 μm3; at a depth of 50–90 μm). Green, neutrophil (the snapshot at t0 + 71.4 ms is in yellow); magenta, SR101-stained astrocytes. Asterisk marks another neutrophil crawling in the capillary. (d) Representative images (of three data sets) of rapid morphological changes of a CD11c-EYFP–bright cell in the mouse popliteal lymph node (35 × 35 × 40 μm3; at a depth of 55–95 μm under the surface). Orange, cell morphologies in 3D; gray, MIPs. (e) Representative image (of five data sets) of spatiotemporal dynamics of lymphocytes and intracellular fluorescent clusters in the mouse popliteal lymph node (84 × 21 × 40 μm3; at a depth of 100–140 μm under the surface). The time–color-coded traces show trajectories of two intracellular fluorescent clusters. (f) Left, representative composite image (of two data sets) of transient subcellular structural changes of a microglial cell activated by BBB disruption in the mouse cerebral cortex (64 × 64 × 40 μm3; at a depth of 100–140 μm under the dura). Three snapshots separated by 14.33 s are shown in red, green and blue. Right, transient structural changes of the microglial branch marked by the white arrow in the left image (6 × 6 × 15 μm3).

Videos

  1. 2D cross-sectional in vivo calcium imaging of mouse V1 cortex (data used in Supplementary Fig. 8).
    Video 1: 2D cross-sectional in vivo calcium imaging of mouse V1 cortex (data used in Supplementary Fig. 8).
    The frame was 250 × 130 μm2 (at depth 100-230 μm under the dura), and the frame rate was 893 Hz. The grating patterns used for visual stimulation are also shown.
  2. 3D in vivo imaging of spontaneous neuronal network activity (dendrites and spines) at mouse V1 cortex.
    Video 2: 3D in vivo imaging of spontaneous neuronal network activity (dendrites and spines) at mouse V1 cortex.
    The volume was 60 × 3.75 × 40 μm3 (at depth 107-147 μm under the dura) and the volume rate was 56 Hz. We displayed the maximum intensity projections of each volume in the video.
  3. Neuronal ensemble in Fig. 2a.
    Video 3: Neuronal ensemble in Fig. 2a.
    Maximal intensity projections of the ensemble in Supplementary Video 2 along the time axis. The volume was 60 × 3.75 × 40 μm3 at depth 107-147 μm under the dura. The video displays the x-y cross-sectional view of the volume.
  4. 3D in vivo calcium imaging of mouse visual cortex.
    Video 4: 3D in vivo calcium imaging of mouse visual cortex.
    The volume was 375 × 375 × 130 μm3(at depth 100-230 μm under the dura) and the volume rate was 5.6 Hz. Maximum intensity projections of each volume are displayed along with the corresponding grating patterns used for the visual stimulation.
  5. Soma ensemble of V1 cortex in Supplementary Fig. 9.
    Video 5: Soma ensemble of V1 cortex in Supplementary Fig. 9.
    The volume was 375 × 375 × 130 μm3 at depth 100-230 μm under the dura. Maximum intensity projections of the ensemble in Supplementary Video 4 along the time axis. Both the x-z cross-sectional view and the maximum intensity projections along x, y and z are shown.
  6. 3D in vivo imaging of the sensory-evoked neuronal network activity in the S1 cortex of awake mice under air-puff stimulations.
    Video 6: 3D in vivo imaging of the sensory-evoked neuronal network activity in the S1 cortex of awake mice under air-puff stimulations.
    The volume was 375 × 112 × 130 μm3 (at depth 115-245 μm under the dura) and the volume rate was 14 Hz. Maximum intensity projections of each volume and the applied air-puff stimulation are shown.
  7. Soma ensemble (S1 cortex) in Fig. 2c.
    Video 7: Soma ensemble (S1 cortex) in Fig. 2c.
    Maximal intensity projections of the ensemble in Supplementary Video 6 along the time axis. The volume was 375 × 112 × 130 μm3 at depth 115-245 μm under the dura. The video shows both the x-z cross-sectional view and the maximum intensity projections.
  8. 3D in vivo imaging of the spontaneous dendritic network activity in the S1 cortex of awake mice.
    Video 8: 3D in vivo imaging of the spontaneous dendritic network activity in the S1 cortex of awake mice.
    The volume was 78 × 20 × 40 μm3 (at depth 40-80 μm under the dura) and the volume rate was 14 Hz. Maximum intensity projections of each volume are shown in the video.
  9. Dendrites/spines ensemble (S1 cortex) in Fig. 2e
    Video 9: Dendrites/spines ensemble (S1 cortex) in Fig. 2e".
    Maximal intensity projections of the ensemble in Supplementary Video 8 along the time axis. The volume was 78 × 20 × 40 μm3 at depth 40-80 μm under the dura. Both the x-y cross-sectional view and the maximum intensity projections are shown.
  10. 3D in vivo imaging of neuron ensemble activity in the M1 cortex of awake behaving mice.
    Video 10: 3D in vivo imaging of neuron ensemble activity in the M1 cortex of awake behaving mice.
    The volume was 448 × 252 × 130 μm3 (at depth 150-280 μm under the dura) and the volume rate was 10 Hz. Maximum intensity projections of each volume are shown in the video.
  11. Soma ensemble (M1 cortex) in Fig. 2g.
    Video 11: Soma ensemble (M1 cortex) in Fig. 2g.
    Average intensity projections of the ensemble in Supplementary Video 10 along the time axis. The volume was 448 × 252 × 130 μm3 at depth 150-280 μm under the dura. The video displays the x-y cross sectional view of the volume.
  12. 2D cross-sectional in vivo imaging of neutrophil trafficking in the pial vein of mouse brain.
    Video 12: 2D cross-sectional in vivo imaging of neutrophil trafficking in the pial vein of mouse brain.
    The imaging plane was 56 × 40 μm2 at depth 5-45 μm under the dura, at 90 degree with respect to the blood flow. The frame rate was 1 kHz. Green: GFP expressing neutrophil. The neutrophil image shown in Fig. 3a is from this video.
  13. 3D in vivo imaging of neutrophils trafficking in the pial vein of mouse brain.
    Video 13: 3D in vivo imaging of neutrophils trafficking in the pial vein of mouse brain.
    The volume was 112 × 38 × 40 μm3 (at depth 5-45 μm under the dura) and the volume rate was 39 Hz. Red: SR101 stained tissue, Green: GFP expressing neutrophils. The neutrophil images shown in Fig. 3b are from volume 5154-5159 of this video. The flow velocity of neutrophils in this video is 750-1243 μm/s.
  14. 3D in vivo imaging of neutrophils trafficking and rolling in the pial vein of mouse brain.
    Video 14: 3D in vivo imaging of neutrophils trafficking and rolling in the pial vein of mouse brain.
    The volume was 151 × 38 × 40 μm3 (at depth 5-45 μm under the dura) and the volume rate was 14 Hz. Red: SR101 stained tissue, Green: GFP expressing neutrophils.
  15. 3D in vivo imaging of neutrophils trafficking in mouse brain cortex (S1).
    Video 15: 3D in vivo imaging of neutrophils trafficking in mouse brain cortex (S1).
    The volume rate was 14 Hz. Red: SR101 stained astrocyte network, Green: GFP expressing neutrophils. We show the entire volume (151 × 38 × 40 μm3 at depth 50-90 μm under the dura) in the upper video and the sub-volume (z from 20 to 40 μm) in the lower video. The neutrophil images in Fig. 3c are from volume 93-95 and 97 of this video.
  16. Astrocyte network stained with SR101 in Supplementary Video 15.
    Video 16: Astrocyte network stained with SR101 in Supplementary Video 15.
    The volume was 151 × 38 × 40 μm3 at depth 50-90 μm under the dura. The video displays the x-y cross-sectional view.
  17. 3D in vivo imaging of neutrophils trafficking in mouse ear vasculature.
    Video 17: 3D in vivo imaging of neutrophils trafficking in mouse ear vasculature.
    The volume was 250 × 24 × 40 μm3 (at depth 65-105 μm under the surface) and the volume rate was 37 Hz. Red: blood plasma labeled with Q-dots (Qtracker® 655); Green: GFP expressing neutrophils. The neutrophil images shown in Supplementary Fig. 12 are from volume 7672-7679 of this video.
  18. 3D in vivo imaging of neutrophils rolling along the mouse ear vasculature of a Lyz2gfp/+ B6.Albino transgenic mouse.
    Video 18: 3D in vivo imaging of neutrophils rolling along the mouse ear vasculature of a Lyz2gfp/+ B6.Albino transgenic mouse.
    The volume was 250 × 47 × 40 μm3 (at depth 55-95 μm under the surface) and the volume rate was 19 Hz. Red: blood plasma labeled with Q-dots (Qtracker® 655); Green: GFP expressing neutrophils.
  19. 3D in vivo imaging of neutrophils rolling along the mouse ear vasculature of a DsRed+/- Lyz2gfp/+ B6.Albino transgenic mouse.
    Video 19: 3D in vivo imaging of neutrophils rolling along the mouse ear vasculature of a DsRed+/- Lyz2gfp/+ B6.Albino transgenic mouse.
    The volume was 250 × 125 × 40 μm3 (at depth 50-90 μm under the surface) and the volume rate was 3.5 Hz. Red: dsRed expressing tissue stroma cells and blood vessel endothelium; Green: GFP expressing neutrophils.
  20. 3D in vivo imaging of neutrophils in mouse ear vasculature and extravascular tissue.
    Video 20: 3D in vivo imaging of neutrophils in mouse ear vasculature and extravascular tissue.
    The volume was 300 × 150 × 40 μm3 (at depth 50-90 μm under the surface) and the volume rate was 3.5 Hz. Red: blood plasma labeled with Q-dots (Qtracker® 655); Green: GFP expressing neutrophils; Blue: second harmonic generation (SHG) signals from the extracellular matrix. Data are shown in three colors on the left column, and in two colors on the right (no SHG) to highlight neutrophil dynamics.
  21. 3D in vivo dendritic cell imaging inside mouse popliteal lymph node.
    Video 21: 3D in vivo dendritic cell imaging inside mouse popliteal lymph node.
    The volume was 500 × 250 × 40 μm3 (at depth 40-80 μm under the surface) and the volume rate was 1.7 Hz.
  22. 3D in vivo dendritic cell imaging inside mouse popliteal lymph node (data used in Fig. 3d).
    Video 22: 3D in vivo dendritic cell imaging inside mouse popliteal lymph node (data used in Fig. 3d).
    A bright cell appeared in volume 121. The volume was 125 × 125 × 40 μm3, at depth 55-95 μm under the surface. The volume rate was 1.7 Hz. The bright cell in volume 120, 121 and 149 of this video is also shown in Fig. 3d.
  23. 3D in vivo dendritic cell imaging inside mouse popliteal lymph node (data used in Supplementary Fig. 13).
    Video 23: 3D in vivo dendritic cell imaging inside mouse popliteal lymph node (data used in Supplementary Fig. 13).
    The volume was 94 × 47 × 40 μm3, at depth 50-90 μm under the surface. The volume rate was 3.5 Hz. This cell (volume 499, 549, and 599 of this video) is also shown in Supplementary Fig. 13.
  24. 3D in vivo imaging of the lymphocytes (mostly memory T cells and natural killer cells) and intracellular fluorescent clusters in the popliteal lymph node of a Tbet:ZsGreen mouse.
    Video 24: 3D in vivo imaging of the lymphocytes (mostly memory T cells and natural killer cells) and intracellular fluorescent clusters in the popliteal lymph node of a Tbet:ZsGreen mouse.
    The volume was 84 × 21 × 40 μm3 (at depth 100-140 μm under the surface) and the volume rate was 7 Hz. In this data set, the movement of two fluorescent clusters, which belong to different cells, is tracked with Imaris software and the time-color-coded traces show their trajectories. The moving speed of both clusters ranges from 0.1 to 6 μm /s, and the average speed is about 1 μm /s.
  25. 3D in vivo imaging of resting microglia.
    Video 25: 3D in vivo imaging of resting microglia.
    The volume was 150 × 150 × 40 μm3 (at depth 75-115 μm under the dura) and the volume rate was 1.7 Hz. We display the volume with the 3D voltex mode of Amira. The transient morphology of the two closely located microglial cells around the center (volume 51 of this video) is also shown in Supplementary Fig. 15.
  26. 3D in vivo imaging of resting microglia surveying the microenvironment and monocytes patrolling around the cortex.
    Video 26: 3D in vivo imaging of resting microglia surveying the microenvironment and monocytes patrolling around the cortex.
    The volume was 150 × 150 × 40 μm3 (at depth 60-100 μm under the dura) and the volume rate was 1.7 Hz. Green: microglia and monocyte, Red: astrocytes stained with SR101. We display the volume with the 3D voltex mode of Amira. The transient morphology of the monocyte in volume 71-73 of this video is also shown in Supplementary Fig. 16.
  27. 3D in vivo imaging of the microglia activated by the brain-blood-barrier disruption.
    Video 27: 3D in vivo imaging of the microglia activated by the brain-blood-barrier disruption.
    The volume was 150 × 75 × 40 μm3 (at depth 100-140 μm under the dura) and the volume rate was 3.5 Hz. Blue: microglia; Red: blood plasma labelled with Q-dots (Qtracker® 655). We show the volume with the 3D voltex mode of Amira. The bright red spot was from deposited Q-dots as a result of the brain blood barrier disruption. The microglia responded by directed subcellular structural changes. Data used in Fig. 3f was from the microglia to the left of the middle plane. The activated microglia in volume 51, 101 and 151 are shown in the left image of Fig. 3f, and those from volume 51, 56, 61 and 66 are shown in the right image of Fig. 3f.
  28. 3D in vivo imaging of the microglia before being activated by neuron damage.
    Video 28: 3D in vivo imaging of the microglia before being activated by neuron damage.
    Resting microglia dynamics and spontaneous neuron activities. The volume was 187.5 × 187.5 × 40 μm3 (at depth 60-100 μm under the dura) and the volume rate was 1.7 Hz. The microglia and neurons are shown in orange and green, respectively.
  29. 3D in vivo imaging of the microglia after being activated by neuron damage.
    Video 29: 3D in vivo imaging of the microglia after being activated by neuron damage.
    Transient structural changes of microglia after neuron damage. The volume was 187.5 × 187.5 × 40 μm3 (at depth 60-100 μm under the dura) and the volume rate was 1.7 Hz. Laser ablation was applied to the neuron that blinked frequently in the middle of the volume shown in Supplementary Video 28.
  30. Simultaneous imaging of the neuronal calcium signals and the change of blood vessel diameters in the S1 cortex of awake mice.
    Video 30: Simultaneous imaging of the neuronal calcium signals and the change of blood vessel diameters in the S1 cortex of awake mice.
    The volume was 210 × 70 × 40 μm3 (at depth 100-140 μm under the dura) and the volume rate was 14 Hz. Green: GCaMP6f-expressing neurons, Red: blood plasma labelled with Q-dots (Qtracker® 655).
  31. Neuron ensemble and vascular network of S1 cortex in Supplementary Fig. 17.
    Video 31: Neuron ensemble and vascular network of S1 cortex in Supplementary Fig. 17.
    The volume was 210 × 70 × 40 μm3 at depth 100-140 μm under the dura. In this video, maximum intensity projections along the time axis were performed to reduce the 4D data to a 3D stack and the x-y cross-sectional view is displayed.

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Author information

Affiliations

  1. Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA.

    • Lingjie Kong,
    • Justin P Little,
    • Yang Yu &
    • Meng Cui
  2. National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.

    • Jianyong Tang,
    • Tim Lämmermann &
    • Ronald N Germain
  3. Center for System Biology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA.

    • Charles P Lin
  4. Wellman Center for Photomedicine, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA.

    • Charles P Lin
  5. Harvard Stem Cell Institute, Cambridge, Massachusetts, USA.

    • Charles P Lin

Contributions

M.C. devised the high-speed volumetric imaging method based on OPLUL and designed both the hardware and software. L.K. provided critical feedback to the system design. L.K. and M.C. built and calibrated the system. L.K. and J.P.L. designed the calcium imaging of the dendrites (V1 cortex). L.K. designed and performed calcium imaging of mouse cerebral cortexes. J.T., L.K., T.L. and R.N.G. designed the neutrophil experiments. C.P.L. designed the blood-flow imaging experiments. J.T. and R.N.G. designed the lymph node imaging experiments. J.T. and L.K. performed lymph node and neutrophil imaging. L.K. designed and performed microglia imaging experiments. L.K., Y.Y., J.T. and J.P.L. performed 3D data rendering and analysis. All authors contributed to the writing of the manuscript.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

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Supplementary information

Video

  1. Video 1: 2D cross-sectional in vivo calcium imaging of mouse V1 cortex (data used in Supplementary Fig. 8). (19.78 MB, Download)
    The frame was 250 × 130 μm2 (at depth 100-230 μm under the dura), and the frame rate was 893 Hz. The grating patterns used for visual stimulation are also shown.
  2. Video 2: 3D in vivo imaging of spontaneous neuronal network activity (dendrites and spines) at mouse V1 cortex. (2.02 MB, Download)
    The volume was 60 × 3.75 × 40 μm3 (at depth 107-147 μm under the dura) and the volume rate was 56 Hz. We displayed the maximum intensity projections of each volume in the video.
  3. Video 3: Neuronal ensemble in Fig. 2a. (209 KB, Download)
    Maximal intensity projections of the ensemble in Supplementary Video 2 along the time axis. The volume was 60 × 3.75 × 40 μm3 at depth 107-147 μm under the dura. The video displays the x-y cross-sectional view of the volume.
  4. Video 4: 3D in vivo calcium imaging of mouse visual cortex. (7.4 MB, Download)
    The volume was 375 × 375 × 130 μm3(at depth 100-230 μm under the dura) and the volume rate was 5.6 Hz. Maximum intensity projections of each volume are displayed along with the corresponding grating patterns used for the visual stimulation.
  5. Video 5: Soma ensemble of V1 cortex in Supplementary Fig. 9. (464 KB, Download)
    The volume was 375 × 375 × 130 μm3 at depth 100-230 μm under the dura. Maximum intensity projections of the ensemble in Supplementary Video 4 along the time axis. Both the x-z cross-sectional view and the maximum intensity projections along x, y and z are shown.
  6. Video 6: 3D in vivo imaging of the sensory-evoked neuronal network activity in the S1 cortex of awake mice under air-puff stimulations. (1.26 MB, Download)
    The volume was 375 × 112 × 130 μm3 (at depth 115-245 μm under the dura) and the volume rate was 14 Hz. Maximum intensity projections of each volume and the applied air-puff stimulation are shown.
  7. Video 7: Soma ensemble (S1 cortex) in Fig. 2c. (230 KB, Download)
    Maximal intensity projections of the ensemble in Supplementary Video 6 along the time axis. The volume was 375 × 112 × 130 μm3 at depth 115-245 μm under the dura. The video shows both the x-z cross-sectional view and the maximum intensity projections.
  8. Video 8: 3D in vivo imaging of the spontaneous dendritic network activity in the S1 cortex of awake mice. (1.52 MB, Download)
    The volume was 78 × 20 × 40 μm3 (at depth 40-80 μm under the dura) and the volume rate was 14 Hz. Maximum intensity projections of each volume are shown in the video.
  9. Video 9: Dendrites/spines ensemble (S1 cortex) in Fig. 2e". (336 KB, Download)
    Maximal intensity projections of the ensemble in Supplementary Video 8 along the time axis. The volume was 78 × 20 × 40 μm3 at depth 40-80 μm under the dura. Both the x-y cross-sectional view and the maximum intensity projections are shown.
  10. Video 10: 3D in vivo imaging of neuron ensemble activity in the M1 cortex of awake behaving mice. (8.26 MB, Download)
    The volume was 448 × 252 × 130 μm3 (at depth 150-280 μm under the dura) and the volume rate was 10 Hz. Maximum intensity projections of each volume are shown in the video.
  11. Video 11: Soma ensemble (M1 cortex) in Fig. 2g. (104 KB, Download)
    Average intensity projections of the ensemble in Supplementary Video 10 along the time axis. The volume was 448 × 252 × 130 μm3 at depth 150-280 μm under the dura. The video displays the x-y cross sectional view of the volume.
  12. Video 12: 2D cross-sectional in vivo imaging of neutrophil trafficking in the pial vein of mouse brain. (2.93 MB, Download)
    The imaging plane was 56 × 40 μm2 at depth 5-45 μm under the dura, at 90 degree with respect to the blood flow. The frame rate was 1 kHz. Green: GFP expressing neutrophil. The neutrophil image shown in Fig. 3a is from this video.
  13. Video 13: 3D in vivo imaging of neutrophils trafficking in the pial vein of mouse brain. (5.7 MB, Download)
    The volume was 112 × 38 × 40 μm3 (at depth 5-45 μm under the dura) and the volume rate was 39 Hz. Red: SR101 stained tissue, Green: GFP expressing neutrophils. The neutrophil images shown in Fig. 3b are from volume 5154-5159 of this video. The flow velocity of neutrophils in this video is 750-1243 μm/s.
  14. Video 14: 3D in vivo imaging of neutrophils trafficking and rolling in the pial vein of mouse brain. (3.95 MB, Download)
    The volume was 151 × 38 × 40 μm3 (at depth 5-45 μm under the dura) and the volume rate was 14 Hz. Red: SR101 stained tissue, Green: GFP expressing neutrophils.
  15. Video 15: 3D in vivo imaging of neutrophils trafficking in mouse brain cortex (S1). (1.65 MB, Download)
    The volume rate was 14 Hz. Red: SR101 stained astrocyte network, Green: GFP expressing neutrophils. We show the entire volume (151 × 38 × 40 μm3 at depth 50-90 μm under the dura) in the upper video and the sub-volume (z from 20 to 40 μm) in the lower video. The neutrophil images in Fig. 3c are from volume 93-95 and 97 of this video.
  16. Video 16: Astrocyte network stained with SR101 in Supplementary Video 15. (187 KB, Download)
    The volume was 151 × 38 × 40 μm3 at depth 50-90 μm under the dura. The video displays the x-y cross-sectional view.
  17. Video 17: 3D in vivo imaging of neutrophils trafficking in mouse ear vasculature. (6.35 MB, Download)
    The volume was 250 × 24 × 40 μm3 (at depth 65-105 μm under the surface) and the volume rate was 37 Hz. Red: blood plasma labeled with Q-dots (Qtracker® 655); Green: GFP expressing neutrophils. The neutrophil images shown in Supplementary Fig. 12 are from volume 7672-7679 of this video.
  18. Video 18: 3D in vivo imaging of neutrophils rolling along the mouse ear vasculature of a Lyz2gfp/+ B6.Albino transgenic mouse. (6.52 MB, Download)
    The volume was 250 × 47 × 40 μm3 (at depth 55-95 μm under the surface) and the volume rate was 19 Hz. Red: blood plasma labeled with Q-dots (Qtracker® 655); Green: GFP expressing neutrophils.
  19. Video 19: 3D in vivo imaging of neutrophils rolling along the mouse ear vasculature of a DsRed+/- Lyz2gfp/+ B6.Albino transgenic mouse. (2.33 MB, Download)
    The volume was 250 × 125 × 40 μm3 (at depth 50-90 μm under the surface) and the volume rate was 3.5 Hz. Red: dsRed expressing tissue stroma cells and blood vessel endothelium; Green: GFP expressing neutrophils.
  20. Video 20: 3D in vivo imaging of neutrophils in mouse ear vasculature and extravascular tissue. (1.17 MB, Download)
    The volume was 300 × 150 × 40 μm3 (at depth 50-90 μm under the surface) and the volume rate was 3.5 Hz. Red: blood plasma labeled with Q-dots (Qtracker® 655); Green: GFP expressing neutrophils; Blue: second harmonic generation (SHG) signals from the extracellular matrix. Data are shown in three colors on the left column, and in two colors on the right (no SHG) to highlight neutrophil dynamics.
  21. Video 21: 3D in vivo dendritic cell imaging inside mouse popliteal lymph node. (1.22 MB, Download)
    The volume was 500 × 250 × 40 μm3 (at depth 40-80 μm under the surface) and the volume rate was 1.7 Hz.
  22. Video 22: 3D in vivo dendritic cell imaging inside mouse popliteal lymph node (data used in Fig. 3d). (632 KB, Download)
    A bright cell appeared in volume 121. The volume was 125 × 125 × 40 μm3, at depth 55-95 μm under the surface. The volume rate was 1.7 Hz. The bright cell in volume 120, 121 and 149 of this video is also shown in Fig. 3d.
  23. Video 23: 3D in vivo dendritic cell imaging inside mouse popliteal lymph node (data used in Supplementary Fig. 13). (709 KB, Download)
    The volume was 94 × 47 × 40 μm3, at depth 50-90 μm under the surface. The volume rate was 3.5 Hz. This cell (volume 499, 549, and 599 of this video) is also shown in Supplementary Fig. 13.
  24. Video 24: 3D in vivo imaging of the lymphocytes (mostly memory T cells and natural killer cells) and intracellular fluorescent clusters in the popliteal lymph node of a Tbet:ZsGreen mouse. (4.34 MB, Download)
    The volume was 84 × 21 × 40 μm3 (at depth 100-140 μm under the surface) and the volume rate was 7 Hz. In this data set, the movement of two fluorescent clusters, which belong to different cells, is tracked with Imaris software and the time-color-coded traces show their trajectories. The moving speed of both clusters ranges from 0.1 to 6 μm /s, and the average speed is about 1 μm /s.
  25. Video 25: 3D in vivo imaging of resting microglia. (13.85 MB, Download)
    The volume was 150 × 150 × 40 μm3 (at depth 75-115 μm under the dura) and the volume rate was 1.7 Hz. We display the volume with the 3D voltex mode of Amira. The transient morphology of the two closely located microglial cells around the center (volume 51 of this video) is also shown in Supplementary Fig. 15.
  26. Video 26: 3D in vivo imaging of resting microglia surveying the microenvironment and monocytes patrolling around the cortex. (5.26 MB, Download)
    The volume was 150 × 150 × 40 μm3 (at depth 60-100 μm under the dura) and the volume rate was 1.7 Hz. Green: microglia and monocyte, Red: astrocytes stained with SR101. We display the volume with the 3D voltex mode of Amira. The transient morphology of the monocyte in volume 71-73 of this video is also shown in Supplementary Fig. 16.
  27. Video 27: 3D in vivo imaging of the microglia activated by the brain-blood-barrier disruption. (12.32 MB, Download)
    The volume was 150 × 75 × 40 μm3 (at depth 100-140 μm under the dura) and the volume rate was 3.5 Hz. Blue: microglia; Red: blood plasma labelled with Q-dots (Qtracker® 655). We show the volume with the 3D voltex mode of Amira. The bright red spot was from deposited Q-dots as a result of the brain blood barrier disruption. The microglia responded by directed subcellular structural changes. Data used in Fig. 3f was from the microglia to the left of the middle plane. The activated microglia in volume 51, 101 and 151 are shown in the left image of Fig. 3f, and those from volume 51, 56, 61 and 66 are shown in the right image of Fig. 3f.
  28. Video 28: 3D in vivo imaging of the microglia before being activated by neuron damage. (13.86 MB, Download)
    Resting microglia dynamics and spontaneous neuron activities. The volume was 187.5 × 187.5 × 40 μm3 (at depth 60-100 μm under the dura) and the volume rate was 1.7 Hz. The microglia and neurons are shown in orange and green, respectively.
  29. Video 29: 3D in vivo imaging of the microglia after being activated by neuron damage. (13.85 MB, Download)
    Transient structural changes of microglia after neuron damage. The volume was 187.5 × 187.5 × 40 μm3 (at depth 60-100 μm under the dura) and the volume rate was 1.7 Hz. Laser ablation was applied to the neuron that blinked frequently in the middle of the volume shown in Supplementary Video 28.
  30. Video 30: Simultaneous imaging of the neuronal calcium signals and the change of blood vessel diameters in the S1 cortex of awake mice. (2 MB, Download)
    The volume was 210 × 70 × 40 μm3 (at depth 100-140 μm under the dura) and the volume rate was 14 Hz. Green: GCaMP6f-expressing neurons, Red: blood plasma labelled with Q-dots (Qtracker® 655).
  31. Video 31: Neuron ensemble and vascular network of S1 cortex in Supplementary Fig. 17. (167 KB, Download)
    The volume was 210 × 70 × 40 μm3 at depth 100-140 μm under the dura. In this video, maximum intensity projections along the time axis were performed to reduce the 4D data to a 3D stack and the x-y cross-sectional view is displayed.

PDF files

  1. Supplementary Text and Figures (34,185 KB)

    Supplementary Figures 1–18, Supplementary Table 1 and Supplementary Discussion

Excel files

  1. Supplementary Data (65,181 KB)

    Data for Fig. a and Fig. b in Supplementary Discussion

Zip files

  1. Supplementary Software (148 KB)

    Matlab codes for image reconstruction

Additional data