Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Brief Communication
  • Published:

Continuous volumetric imaging via an optical phase-locked ultrasound lens

Abstract

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.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Design of the OPLUL-based imaging system.
Figure 2: 3D in vivo calcium imaging in neurons.
Figure 3: 3D in vivo imaging of cell dynamics.

Similar content being viewed by others

References

  1. Germain, R.N., Miller, M.J., Dustin, M.L. & Nussenzweig, M.C. Nat. Rev. Immunol. 6, 497–507 (2006).

    Article  CAS  Google Scholar 

  2. Grewe, B.F. & Helmchen, F. Curr. Opin. Neurobiol. 19, 520–529 (2009).

    Article  CAS  Google Scholar 

  3. Göbel, W., Kampa, B.M. & Helmchen, F. Nat. Methods 4, 73–79 (2007).

    Article  Google Scholar 

  4. Grewe, B.F., Langer, D., Kasper, H., Kampa, B.M. & Helmchen, F. Nat. Methods 7, 399–405 (2010).

    Article  CAS  Google Scholar 

  5. Cheng, A., Gonçalves, J.T., Golshani, P., Arisaka, K. & Portera-Cailliau, C. Nat. Methods 8, 139–142 (2011).

    Article  CAS  Google Scholar 

  6. Ahrens, M.B., Orger, M.B., Robson, D.N., Li, J.M. & Keller, P.J. Nat. Methods 10, 413–420 (2013).

    Article  CAS  Google Scholar 

  7. Schrödel, T., Prevedel, R., Aumayr, K., Zimmer, M. & Vaziri, A. Nat. Methods 10, 1013–1020 (2013).

    Article  Google Scholar 

  8. Bouchard, M.B. et al. Nat. Photonics 9, 113–119 (2015).

    Article  CAS  Google Scholar 

  9. Tang, J., Germain, R.N. & Cui, M. Proc. Natl. Acad. Sci. USA 109, 8434–8439 (2012).

    Article  CAS  Google Scholar 

  10. Denk, W., Strickler, J.H. & Webb, W.W. Science 248, 73–76 (1990).

    Article  CAS  Google Scholar 

  11. Botcherby, E.J. et al. Proc. Natl. Acad. Sci. USA 109, 2919–2924 (2012).

    Article  CAS  Google Scholar 

  12. Katona, G. et al. Nat. Methods 9, 201–208 (2012).

    Article  CAS  Google Scholar 

  13. Duemani Reddy, G., Kelleher, K., Fink, R. & Saggau, P. Nat. Neurosci. 11, 713–720 (2008).

    Article  CAS  Google Scholar 

  14. McLeod, E., Hopkins, A.B. & Arnold, C.B. Opt. Lett. 31, 3155–3157 (2006).

    Article  Google Scholar 

  15. Fan, Z. et al. Nat. Med. 16, 718–722 (2010).

    Article  CAS  Google Scholar 

  16. Lämmermann, T. et al. Nature 498, 371–375 (2013).

    Article  Google Scholar 

  17. Lindquist, R.L. et al. Nat. Immunol. 5, 1243–1250 (2004).

    Article  CAS  Google Scholar 

  18. Zhu, J. et al. Immunity 37, 660–673 (2012).

    Article  CAS  Google Scholar 

  19. Kastenmüller, W., Torabi-Parizi, P., Subramanian, N., Lämmermann, T. & Germain, R.N. Cell 150, 1235–1248 (2012).

    Article  Google Scholar 

  20. Jung, S. et al. Mol. Cell. Biol. 20, 4106–4114 (2000).

    Article  CAS  Google Scholar 

  21. Otsu, Y. et al. J. Neurosci. Methods 173, 259–270 (2008).

    Article  Google Scholar 

  22. Chen, T.-W. et al. Nature 499, 295–300 (2013).

    Article  CAS  Google Scholar 

  23. Nimmerjahn, A., Kirchhoff, F., Kerr, J.N. & Helmchen, F. Nat. Methods 1, 31–37 (2004).

    Article  CAS  Google Scholar 

  24. Nimmerjahn, A., Kirchhoff, F. & Helmchen, F. Science 308, 1314–1318 (2005).

    Article  CAS  Google Scholar 

  25. Guizar-Sicairos, M., Thurman, S.T. & Fienup, J.R. Opt. Lett. 33, 156–158 (2008).

    Article  Google Scholar 

  26. Kim, J. et al. Nat. Methods 9, 96–102 (2012).

    Article  CAS  Google Scholar 

  27. Adams, R. & Bischof, L. IEEE Trans. Pattern Anal. Mach. Intell. 16, 641–647 (1994).

    Article  Google Scholar 

  28. Dombeck, D.A., Graziano, M.S. & Tank, D.W. J. Neurosci. 29, 13751–13760 (2009).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

L.K. and M.C. thank W. Gan and H. Dana for many helpful discussions on cerebral cortex imaging, T.-W. Chen for help on data analysis in the visual cortex experiments, S. Peron and N.-L. Xu (Janelia Research Campus) for providing animals during system calibration, K. Ritola (Janelia Research Campus) for preparing GCaMP6 virus, K. Morris and J. Rouchard for virus injections, J. Liu for help on cluster computing, P. Keller for suggestions on data storage, B.-C. Chen and W. Legant for suggestions on data visualization, V. Iyer for support on ScanImage, D. Milkie for developing the custom Labview program, S. Sawtelle and L. Ramasamy for making the low-noise high-speed comparator, V. Goncharov for testing high-speed photodetectors, J. Freeman and F. Amat for help on data analysis and the Howard Hughes Medical Institute for funding the project. This work was supported in part by the Intramural Program of the National Institute of Allergy and Infectious Diseases, US National Institutes of Health (NIH) (to R.N.G.), and NIH grant P41 E8015903-02S1 (to C.P.L.).

Author information

Authors and Affiliations

Authors

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.

Corresponding author

Correspondence to Meng Cui.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–18, Supplementary Table 1 and Supplementary Discussion (PDF 33384 kb)

Source data to Supplementary Figure 1

Source data to Supplementary Figure 3

Source data to Supplementary Figure 4

Source data to Supplementary Figure 5

Source data to Supplementary Figure 7

Source data to Supplementary Figure 8

Source data to Supplementary Figure 9

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. (MP4 20255 kb)

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. (MOV 2070 kb)

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. (AVI 209 kb)

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. (MOV 7585 kb)

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. (MOV 463 kb)

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. (MOV 1294 kb)

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. (MOV 230 kb)

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. (MOV 1566 kb)

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. (MOV 336 kb)

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. (MOV 8464 kb)

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. (MOV 104 kb)

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. (AVI 3001 kb)

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. (MOV 5841 kb)

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. (MOV 4048 kb)

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. (MOV 1690 kb)

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. (MOV 187 kb)

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. (MOV 6505 kb)

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. (MOV 6679 kb)

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. (MOV 2389 kb)

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. (MOV 1204 kb)

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. (MOV 1250 kb)

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. (MOV 632 kb)

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. (MOV 709 kb)

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. (AVI 4445 kb)

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. (AVI 14192 kb)

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. (AVI 5388 kb)

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. (AVI 12620 kb)

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. (AVI 14195 kb)

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. (AVI 14191 kb)

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). (MOV 2058 kb)

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. (MOV 166 kb)

Supplementary Data

Data for Fig. a and Fig. b in Supplementary Discussion (XLSX 63 kb)

Supplementary Software

Matlab codes for image reconstruction (ZIP 145050 kb)

Source data

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kong, L., Tang, J., Little, J. et al. Continuous volumetric imaging via an optical phase-locked ultrasound lens. Nat Methods 12, 759–762 (2015). https://doi.org/10.1038/nmeth.3476

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth.3476

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing