FIG. 1. Schematic diagram of the high-speed two-photon and confocal laser scanning microscope.
FIG. 2. Timing diagram of the H-sync and V-sync control signals and the analog angular position voltage signal to the galvanometer.
FIG. 3. Representative images of auto-fluorescent structures found in auricle skin and scanned at an imaging rate of 180 FPS, and averaged over 30 frames, for subsequent layers in ex vivo mouse tissue. All images are 512 × 220 pixels (220 μm × 95 μm FOV).
FIG. 4. Stationary ex vivo DiI-labeled erythrocytes translated at a fixed speed of 1200 μm/s via a motorized sample stage.
FIG. 5. Sample image of in vivo low blood flow captured at 180 FPS. (a) A single frame composite of the confocal channel, shown in red, and the two-photon DiI selection channel shown in blue. (b) The same single frame after being despeckled and isolated to the confocal channel. Individual cells are visible within the drawn-in outline of the blood vessel. (c) The same single frame but isolated to the DiI selection channel. A single labeled erythrocyte is visible within the drawn-in outline of the blood vessel as well as a weakly overlapping auto-fluorescent signal from the collagen in the blood vessel’s wall.
FIG. 6. Two-photon excited fluorescent DiI-labeled erythrocyte moving from right to left at ~130 μm/s through a blood vessel. The vessel’s walls are marked in red outline.
FIG. 8. Two-photon excited fluorescent DiI-labeled erythrocyte moving from right to left at ~1200 μm/s through a blood vessel. The vessel’s walls are marked in red outline.
FIG. 7. Two-photon excited fluorescent DiI-labeled erythrocyte moving from right to left at ~600 μm/s through a blood vessel. The vessel’s walls are marked in red outline.
References
- M. Goppert-Mayer, "Uber Elementarakte mit zwei Quantensprungen," Ann. Phys. 9, 273-295 (1931).
- W. Denk, J. H. Strickler, and W. W. Webb, "Two-photon laser scanning fluorescence microscopy," Sci. 248, 73-76 (1990). https://doi.org/10.1126/science.2321027
- C. P. Sabino, A. M. Deana, T. M. Yoshimura, D. F. T. da Silva, C. M França, M. R. Hamblin, and M. S. Ribeiro, "The optical properties of mouse skin in the visible and near infrared spectral regions," J. Photochem. Photobiol. 160, 72-78 (2016). https://doi.org/10.1016/j.jphotobiol.2016.03.047
- C. Xu, R. M. Williams, W. Zipfel, and W. W. Webb, "Multiphoton excitation cross-sections of molecular fluorophores," Bioimaging 4, 198-207 (1996). https://doi.org/10.1002/1361-6374(199609)4:3<198::AID-BIO10>3.3.CO;2-O
- G. H. Patterson and D. W. Piston, "Photobleaching in twophoton excitation microscopy," Biophys. J. 78, 2159-2162 (2000). https://doi.org/10.1016/S0006-3495(00)76762-2
- W. R. Zipfel, R. M. Williams, and W. W. Webb, "Nonlinear magic: multiphoton microscopy in the biosciences," Nat. Biotechnol. 21, 1369-1377 (2003). https://doi.org/10.1038/nbt899
- S. Choi, P. Kim, R. Boutilier, M. Y. Kim, Y. J. Lee, and H. Lee, "Development of a high speed laser scanning confocal microscope with an acquisition rate up to 200 frames per second," Opt. Express 21, 23611-23618 (2013). https://doi.org/10.1364/OE.21.023611
- J. P. Rickgauer and D. W. Tank, "Two-photon excitation of channelrhodopsin-2 at saturation," Proc. Natl. Acad. Sci. 106, 15025-15030 (2009). https://doi.org/10.1073/pnas.0907084106
- M. Hellmann, M. Roustit, J.-L. Cracowski, "Skin microvascular endothelial function as a biomarker in cardiovascular diseases?," Pharmacol. Rep. 67, 803-810 (2015). https://doi.org/10.1016/j.pharep.2015.05.008
- I. Veilleux, J. A. Spencer, D. P. Biss, D. Cote, and C. P. Lin, "In vivo cell tracking with video rate multimodality laser scanning microscopy," IEEE J. Sel. Topics Quantum Electron. 14, 10-18 (2008). https://doi.org/10.1109/JSTQE.2007.912751
- S. H. Choi, W. H. Kim, Y. J. Lee, W. J. Lee, J. D. Yang, J. W. Shim, J. W. Kim, and H. Lee, "Visualization of epidermis and dermal cells in ex vivo human skin using confocal and two-photon microscopy," J. Opt. Soc. Korea 15, 61-67 (2011). https://doi.org/10.3807/JOSK.2011.15.1.061
- S. Huang, A. A. Heikal, and W. W. Webb, "Two-photon fluorescence spectroscopy and microscopy of NAD(P)H and flavoprotein," Biophys. J. 82, 2811-2825 (2002). https://doi.org/10.1016/S0006-3495(02)75621-X
- C. Ince, J. M. C. C. Coremans, and H. A. Bruining, "In vivo NADH fluorescence," in Oxygen Transport to Tissue XIV (Plenum Press, New York, 1992), pp. 277-296.
- A. Zoumi, A. Yeh, and B. J. Tromberg, "Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence," Proc. Natl. Acad. Sci. 99, 11014-11019 (2002). https://doi.org/10.1073/pnas.172368799
- E. R. Tkaczyk, C. F. Zhong, J. Y. Ye, A. Myc, T. Thomas, A. Cao, R. Duran-Struuck, K. E. Luker, G. D. Luker, T. B. Norris, J. R. Baker Jr., "In vivo monitoring of multiple circulating cell populations using two-photon flow cytometry," Opt. Commun. 281, 888-894 (2008). https://doi.org/10.1016/j.optcom.2007.10.106
- P. T. C. So, H. Kim and I. E. Kochevar, "Two-photon deep tissue ex vivo imaging of mouse dermal and subcutaneous structures," Opt. Express 3, 339-350 (1998). https://doi.org/10.1364/OE.3.000339
- W. H. Jang, S. Shim, T. Wang, Y. Yoon, W.-S. Jang, J. K. Myung, S. Park, and K. H. Kim, "In vivo characterization of early-stage radiation skin injury in a mouse model by two-photon microscopy," Sci. Rep. 6, 19216 (2016). https://doi.org/10.1038/srep19216
- K. Namdee, M. Carrasco-Teja, M. B. Fish, P. Charoenphol, and O. Eniola-Adefesco, "Effect of variation in hemorheology between human and animal blood on the binding efficacy of vascular-targeted carriers," Sci. Rep. 5, 11631 (2015). https://doi.org/10.1038/srep11631
- C. Li, R. K. Pastila, C. Pitsillides, J. M. Runnels, M. Puoris'haag, D. Cote, and C. P. Lin, "Imaging leukocyte trafficking in vivo with two-photon-excited endogenous tryptophan fluorescence," Opt. Express 18, 988-999 (2010). https://doi.org/10.1364/OE.18.000988
- W. Zheng, D. Li, Y. Zeng, Y. Luo, and J. Y. Qu, "Twophoton excited hemoglobin fluorescence," Biomed. Opt. Express 2, 71-79 (2011). https://doi.org/10.1364/BOE.2.000071