Multicolor two-photon tissue imaging by wavelength mixing

Abstract

We achieve simultaneous two-photon excitation of three chromophores with distinct absorption spectra using synchronized pulses from a femtosecond laser and an optical parametric oscillator. The two beams generate separate multiphoton processes, and their spatiotemporal overlap provides an additional two-photon excitation route, with submicrometer overlay of the color channels. We report volume and live multicolor imaging of 'Brainbow'-labeled tissues as well as simultaneous three-color fluorescence and third-harmonic imaging of fly embryos.

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Figure 1: Principles of multicolor two-photon imaging using synchronized pulses.
Figure 2: Deep imaging and colorimetric analysis of Brainbow-labeled mouse cortical samples using multicolor two-photon imaging.
Figure 3: Continuous three-color 2PEF and THG imaging of live embryonic tissue.

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Acknowledgements

We thank A. McMahon and A. Stathopoulos (California Institute of Technology) for the mcd8-GFP/H2A-RFP Drosophila line, R. Barry for transgene preparation, S. Fouquet for technical assistance and M.-C. Schanne-Klein, M. Joffre, J.-L. Martin and J.-A. Sahel for helpful comments and discussions. This work was supported by Agence Nationale de la Recherche, Fondation Louis D de l'Institut de France, Ville de Paris, Inserm Avenir and Ecole des Neurosciences de Paris.

Author information

Affiliations

Authors

Contributions

E.B., P.M., D.D., M.Z., G.L. and W.S. designed, implemented and performed imaging. J.L., K.L., X.M. and K.S.M. designed and implemented Brainbow strategies. X.M. prepared chicken embryo samples. W.S. prepared Drosophila. P.M., W.S. and K.S.M. performed image analysis. P.M., W.S., D.D., J.L. and E.B. analyzed the data and wrote the manuscript.

Corresponding author

Correspondence to Emmanuel Beaurepaire.

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Competing interests

E.B., P.M., D.D. and W.S. have applied for a patent (no. FR1250990) concerning work covered in this manuscript.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–12 (PDF 10196 kb)

Brainbow–labeled mouse cerebral cortex: 3D rendering of a 900 × 720 × 370 μm3 volume.

Three-dimensional rendering of a 400-μm slice of Brainbow-labeled mouse cortex (CFP, YFP, tdTomato/mCherry) imaged with synchronized 850/1,100 nm pulses. The image is a mosaic of six z stacks, encompassing a total volume of 900 × 720 × 370 μm3. The pixel dwell time was 5 μs, the voxel size was 0.48 × 0.48 × 1.0 μm3, each image was averaged two times, the laser power was adjusted automatically with depth and color balance with the field of view was postcorrected. (MOV 28084 kb)

Brainbow–labeled mouse cerebral cortex: progressive maximum intensity projection.

xy and xz sweeping maximum-intensity projection of a 180-μm slice of Brainbow-labeled mouse cortex (CFP, YFP, tdTomato/mCherry) imaged with synchronized 850/1,100 nm pulses. The image consists of four adjacent z stacks, encompassing a total volume of 1,450 × 430 × 170 μm3. The pixel dwell time was 5 μs, the voxel size was 0.48 × 0.48 × 1.0 μm3, each image was averaged two times, laser power was adjusted automatically with depth and color balance with the field of view was postcorrected. Scale bar, 100 μm. (AVI 26043 kb)

Neuron tracing from multicolor images.

Three-dimensional rendering of a subvolume from the 180-μm slice of Brainbow-labeled mouse cortex shown in Supplementary Video 2 (360° rotation along the y axis). Left: 2PEF images (levels and offset were postcorrected). Right: segmented neurons. The xy:z ratio was adjusted to 1:1 by interpolating long the z axis. Scale bar, 50 μm. (AVI 14421 kb)

Developing Brainbow-labeled embryonic chicken spinal cord.

Three-dimensional rendering of a stack encompassing a total volume of 450 × 500 × 230 μm3. The pixel dwell time was 5 μs, the voxel size was 0.80 × 0.80 × 2.0 μm3 and laser power was adjusted automatically with depth. The tissue was imaged continuously and one 3D stack was recorded every 430 s (including time needed for objective motion, motorized power adjustment and scanning flyback). The grid pitch is 50 μm. (MOV 3185 kb)

Developing Brainbow-labeled motor neurons in chicken embryonic tissue: 3D+time multicolor two-photon imaging.

Three-dimensional rendering of a stack encompassing a total volume of 200 × 300 × 80 μm3. The pixel dwell time was 5μs, the voxel size was 0.60 × 0.60 × 2.0 μm3 and laser power was adjusted automatically with depth. The tissue was imaged continuously and one 3D stack was recorded every 90 s. The grid pitch is 50 μm. (MOV 1452 kb)

Developing Brainbow-labeled commissural neurons in chicken embryonic tissue: 3D+time multicolor two-photon imaging.

Three-dimensional rendering of a stack encompassing a total volume of 420 × 250 × 120 μm3. The pixel dwell time was 5 μs, the voxel size was 0.60 × 0.60 × 2.0 μm3 and laser power was adjusted automatically with depth. The tissue was imaged continuously and one 3D stack was recorded every 170 s. The grid pitch is 50 μm. (MOV 6778 kb)

Developing Drosophila embryo: 3D+time multicolor two-photon imaging.

Time-lapse 3D multicolor imaging showing the rapid morphogenetic movement of germ-band extension in a living Drosophila embryo expressing EGFP in cell membranes and RFP in cell nuclei. One 3D stack recorded every 45 s with 5-μs pixel dwell time. Voxel size: 0.80 × 0.80 × 3.0 μm3. Excitation wavelengths: 820/1,175 nm. Grid pitch: 50 μm. (AVI 1132 kb)

Developing Drosophila embryo: 3D+time multicolor 2PEF imaging during mesoderm invagination (10 s per stack).

Time-lapse 3D multicolor imaging showing the rapid morphogenetic movement of germ-band extension in a living Drosophila embryo expressing EGFP in cell membranes and RFP in cell nuclei. One 3D stack recorded every 11 s with 5-μs pixel dwell time. Voxel size: 0.80 × 0.80 × 1.5 μm3. Excitation wavelengths: 820/1,100 nm. Grid pitch: 50 μm. (MOV 4732 kb)

Developing Drosophila embryo: simultaneous multicolor 2PEF/THG imaging (single plane extracted from 3D stack, raw data).

Simultaneous THG and three-channel fluorescence raw data recorded on a living Drosophila embryo with EGFP labeling of the cell membranes and RFP labeling of the cell nuclei. The pixel dwell time was 5 μs, the pixel size was 0.40 × 0.40 μm2 and each image was averaged two times. Excitation wavelengths: 820/1,175 nm. Scale bar, 50 μm. (AVI 6511 kb)

Developing Drosophila embryo: simultaneous multicolor 2PEF/THG imaging (single plane extracted from 3D stack, images after unmixing).

Simultaneous THG and three-channel fluorescence unmixed raw data recorded on a living Drosophila embryo with EGFP labeling of the cell membranes and RFP labeling of the cell nuclei. The pixel dwell time was 5 μs, the pixel size was 0.40 × 0.40 μm2 and each image was averaged two times. Excitation wavelengths: 820/1,175 nm. Scale bar, 50 μm. (AVI 6317 kb)

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Mahou, P., Zimmerley, M., Loulier, K. et al. Multicolor two-photon tissue imaging by wavelength mixing. Nat Methods 9, 815–818 (2012). https://doi.org/10.1038/nmeth.2098

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