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Frequency-multiplexed in vivo multiphoton phosphorescence lifetime microscopy

Nature Photonics volume 7, pages 33–37 (2013)Cite this article

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Abstract

Multiphoton microscopy (MPM) is widely used in vivo for optical sectioning deep inside scattering tissue1,2. Phosphorescence lifetime imaging microscopy (PLIM)3 is a powerful technique for obtaining biologically relevant chemical information through Förster resonance energy transfer and phosphorescence quenching4,5. Point-measurement PLIM6 of phosphorescence quenching probes has recently provided oxygen partial pressure measurements in small rodent brain vasculature identified by high-resolution MPM7,8. However, the maximum fluorescence generation rate, which is inversely proportional to the phosphorescence lifetime, fundamentally limits PLIM pixel rates. Here, we demonstrate experimentally a parallel-excitation/parallel-collection MPM–PLIM system that increases the pixel rate by a factor of 100 compared with conventional configurations, while simultaneously acquiring lifetime and intensity images at depth in vivo. Full-frame, three-dimensional, in vivo PLIM imaging of phosphorescent quenching dye is presented for the first time and defines a new platform for biological and medical imaging.

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Figure 1: Schematic of the M4 microscope.
Figure 2: M4 data processing and image reconstruction.
Figure 3
Figure 4: Mouse brain vasculature containing Ru(dpp3)-pluronic-nanomicelle probes imaged by M4.
Figure 5: M4 and conventional two-photon microscopy of DsRed-labelled mouse brain vasculature, in vivo.

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Acknowledgements

This research project was supported in part by the National Science Foundation (NSF)/Division of Biological Infrastructure (DBI) (grant no. DBI-0546227) and the National Institutes of Health (NIH)/National Cancer Institute (NCI) (grant no. R01-CA133148). Nicholas G. Horton was supported by an NSF Graduate Research Fellowship (grant no. DGE-0707428). The authors would like to thank C. Schafer (Cornell University) and S. Vinogradov (Department of Biochemistry and Biophysics, University of Pennsylvania) for their helpful discussions on brain imaging and phosphorescence quenching dyes for in vivo oxygen sensing, respectively.

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Authors and Affiliations

  1. School of Applied and Engineering Physics, Cornell University, 212 Clark Hall, Ithaca, 14853, New York, USA

    Scott S. Howard, Adam Straub, Nicholas G. Horton, Demirhan Kobat & Chris Xu

  2. Department of Electrical Engineering, University of Notre Dame, 275 Fitzpatrick Hall, Notre Dame, 46556, Indiana, USA

    Scott S. Howard

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  1. Scott S. Howard
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  4. Demirhan Kobat
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Contributions

S.H. coordinated the project, designed and built the microscope, designed and fabricated the linear SLM, wrote control and image-processing software, performed simulations, wrote analysis algorithms, analysed data, carried out animal preparation and surgery, prepared dye and calibrations, and wrote the manuscript. A.S. greatly assisted with microscope design and assembly, performed simulations and analysis, wrote analysis algorithms, significantly contributed to the content of the manuscript, and performed experiments verifying pixel rate increase. N.H. and D.K. assisted in animal preparation and surgery for imaging. C.X. initiated the project, significantly contributed to the design of M4 and experimental design, and greatly contributed to the theoretical and experimental discussions. All authors contributed to editing the manuscript.

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Correspondence to Scott S. Howard or Chris Xu.

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The authors declare no competing financial interests.

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Howard, S., Straub, A., Horton, N. et al. Frequency-multiplexed in vivo multiphoton phosphorescence lifetime microscopy. Nature Photon 7, 33–37 (2013). https://doi.org/10.1038/nphoton.2012.307

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