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Speed scaling in multiphoton fluorescence microscopy

A Publisher Correction to this article was published on 29 November 2021

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Abstract

After over 30 years of advances, multiphoton microscopy (MPM) is now instrumental in a wide range of in vivo biological imaging applications. However, it has, until recently, remained not achievable or affordable to meet the unmet need for fast monitoring of biological dynamics and large-scale examination of biological heterogeneity. Only within the past few years have new strategies emerged to empower MPM at a speed that was once inconceivable, notably at kilohertz two-dimensional (2D) frame rate, and 3D rate or beyond. This Review highlights the latest high-speed innovations and discusses the potential of their synergism with other advanced, but less speed-centric MPM toolboxes. Recognizing these prospects and challenges could inspire new approaches for reprioritizing imaging speed in future MPM developments.

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Fig. 1: Advances in high-speed 2PFM.
Fig. 2: High-speed strategies enabling planar MPM at subkilohertz to kilohertz frame rate.
Fig. 3: Strategies for high-speed MPM axial access.
Fig. 4: Ongoing advances in MPM that address the challenges of imaging a FOV, SNR and tissue penetration, together with temporal and spatial resolution.

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References

  1. Hooke, R. Micrographia: or Some Physiological Descriptions of Minute Bodies Made by Magnifying Glasses. With Observations and Inquiries Thereupon (The Royal Society, 1665).

  2. Denk, W., Strickler, J. H. & Webb, W. W. Two-photon laser scanning fluorescence microscopy. Science 248, 73–76 (1990).

    Article  ADS  Google Scholar 

  3. Göppert-Mayer, M. Über Elementarakte mit zwei Quantensprüngen. Ann. Phys. 9, 273–294 (1931).

    Article  MATH  Google Scholar 

  4. Bahlmann, K. et al. Multifocal multiphoton microscopy (MMM) at a frame rate beyond 600 Hz. Opt. Express 15, 10991–10998 (2007).

    Article  ADS  Google Scholar 

  5. Zhang, T. et al. Kilohertz two-photon brain imaging in awake mice. Nat. Methods 16, 1119–1122 (2019).

    Article  Google Scholar 

  6. Sacconi, L. et al. Multiphoton multifocal microscopy exploiting a diffractive optical element. Opt. Lett. 28, 1918–1920 (2003).

    Article  ADS  Google Scholar 

  7. Nikolenko, V. et al. SLM microscopy: scanless two-photon imaging and photostimulation with spatial light modulators. Front. Neural Circuits 2, 5 (2008).

    Article  Google Scholar 

  8. Wu, J. et al. Kilohertz two-photon fluorescence microscopy imaging of neural activity in vivo. Nat. Methods 17, 287–290 (2020).

    Article  Google Scholar 

  9. Wu, J. et al. Ultrafast laser-scanning time-stretch imaging at visible wavelengths. Light Sci. Appl. 6, e16196 (2017).

    Article  Google Scholar 

  10. Karpf, S. et al. Spectro-temporal encoded multiphoton microscopy and fluorescence lifetime imaging at kilohertz frame-rates. Nat. Commun. 11, 2062 (2020).

    Article  ADS  Google Scholar 

  11. Mandracchia, B. et al. Fast and accurate sCMOS noise correction for fluorescence microscopy. Nat. Commun. 11, 94 (2020).

    Article  ADS  Google Scholar 

  12. Voleti, V. et al. Real-time volumetric microscopy of in vivo dynamics and large-scale samples with SCAPE 2.0. Nat. Methods 16, 1054–1062 (2019).

    Article  Google Scholar 

  13. Schrödel, T., Prevedel, R., Aumayr, K., Zimmer, M. & Vaziri, A. Brain-wide 3D imaging of neuronal activity in Caenorhabditis elegans with sculpted light. Nat. Methods 10, 1013–1020 (2013).

    Article  Google Scholar 

  14. Truong, T. et al. Deep and fast live imaging with two-photon scanned light-sheet microscopy. Nat. Methods 8, 757–760 (2011).

    Article  Google Scholar 

  15. Oron, D., Tal, E. & Silberberg, Y. Scanningless depth-resolved microscopy. Opt. Express 13, 1468–1476 (2005).

    Article  ADS  Google Scholar 

  16. Zhu, G., Van Howe, J., Durst, M., Zipfel, W. & Xu, C. Simultaneous spatial and temporal focusing of femtosecond pulses. Opt. Express 13, 2153–2159 (2005).

    Article  ADS  Google Scholar 

  17. Papagiakoumou, E., Ronzitti, E. & Emiliani, V. Scanless two-photon excitation with temporal focusing. Nat. Methods 17, 571–581 (2020).

    Article  Google Scholar 

  18. Podgorski, K. & Ranganathan, G. Brain heating induced by nearinfrared lasers during multiphoton microscopy. J. Neurophysiol. 116, 1012–1023 (2016).

    Article  Google Scholar 

  19. Dana, H. et al. Hybrid multiphoton volumetric functional imaging of large-scale bioengineered neuronal networks. Nat. Commun. 5, 3997 (2014).

    Article  ADS  Google Scholar 

  20. Xue, Y. et al. Scanless volumetric imaging by selective access multifocal multiphoton microscopy. Optica 6, 76–83 (2019).

    Article  ADS  Google Scholar 

  21. Hillman, E. M. C., Voleti, V., Li, W. & Yu, H. Light-sheet microscopy in neuroscience. Annu. Rev. Neurosci. 42, 295–313 (2019).

    Article  Google Scholar 

  22. Wolf, S. et al. Whole-brain functional imaging with two-photon light-sheet microscopy. Nat. Methods 12, 379–380 (2015).

    Article  Google Scholar 

  23. Mahou, P., Vermot, J., Beaurepaire, E. & Supatto, W. Multicolor two-photon light-sheet microscopy. Nat. Methods 11, 600–601 (2014).

    Article  Google Scholar 

  24. Maioli, V. et al. Fast in vivo multiphoton light-sheet microscopy with optimal pulse frequency. Biomed. Opt. Express 11, 6012–6026 (2020).

    Article  Google Scholar 

  25. Kumar, M. et al. Integrated one- and two-photon scanned oblique plane illumination (SOPi) microscopy for rapid volumetric imaging. Opt. Express 26, 13027–13041 (2018).

    Article  ADS  Google Scholar 

  26. Sapoznik, E. et al. A versatile oblique plane microscope for large-scale and high-resolution imaging of subcellular dynamics. eLife 9, e57681 (2020).

    Article  Google Scholar 

  27. Kazemipour, A. et al. Kilohertz frame-rate two-photon tomography. Nat. Methods 16, 778–786 (2019).

    Article  Google Scholar 

  28. Escobet-Montalbán, A. et al. Wide-field multiphoton imaging through scattering media without correction. Sci. Adv. 4, eaau1338 (2018).

    Article  ADS  Google Scholar 

  29. Katona, G. et al. Fast two-photon in vivo imaging with three-dimensional random-access scanning in large tissue volumes. Nat. Methods 9, 201–208 (2012).

    Article  Google Scholar 

  30. Sakaki, K. D. R., Podgorski, K., Toth, T. A. D., Coleman, P. & Haas, K. Comprehensive imaging of sensory-evoked activity of entire neurons within the awake developing brain using ultrafast AOD-based random-access two-photon microscopy. Front. Neural Circuits 14, 33 (2020).

    Article  Google Scholar 

  31. Nadella, K. M. et al. Random-access scanning microscopy for 3D imaging in awake behaving animals. Nat. Methods 13, 1001–1004 (2016).

    Article  Google Scholar 

  32. Szalay, G. et al. Fast 3D imaging of spine, dendritic and neuronal assemblies in behaving animals. Neuron 92, 723–738 (2016).

    Article  Google Scholar 

  33. Li, B. et al. Two-photon voltage imaging of spontaneous activity from multiple neurons reveals network activity in brain tissue. iScience 23, 101363 (2020).

    Article  ADS  Google Scholar 

  34. Chamberland, S. et al. Fast two-photon imaging of subcellular voltage dynamics in neuronal tissue with genetically encoded indicators. eLife 6, e25690 (2017).

    Article  Google Scholar 

  35. Villette, V. et al. Ultrafast two-photon imaging of a high-gain voltage indicator in awake behaving mice. Cell 179, 1590–1608 (2019).

    Article  Google Scholar 

  36. Geng, Q., Gu, C., Cheng, J. & Chen, S.-H. Digital micromirror device-based two-photon microscopy for three-dimensional and random-access imaging. Optica 4, 674–677 (2017).

    Article  ADS  Google Scholar 

  37. Griffiths, V. A. et al. Real-time 3D movement correction for two-photon imaging in behaving animals. Nat. Methods 17, 741–748 (2020).

    Article  Google Scholar 

  38. Kang, S., Duocastella, M. & Arnold, C. B. Variable optical elements for fast focus control. Nat. Photon. 14, 533–542 (2020).

    Article  ADS  Google Scholar 

  39. Grewe, B. F., Voigt, F. F., van’t Hoff, M. & Helmchen, F. Fast two-layer two-photon imaging of neuronal cell populations using an electrically tunable lens. Biomed. Opt. Express 2, 2035–2046 (2011).

    Article  Google Scholar 

  40. Kong, L. et al. Continuous volumetric imaging via an optical phase-locked ultrasound lens. Nat. Methods 12, 759–762 (2015).

    Article  Google Scholar 

  41. Piazza, S., Bianchini, P., Sheppard, C., Diaspro, A. & Duocastella, M. Enhanced volumetric imaging in 2-photon microscopy via acoustic lens beam shaping. J. Biophoton. 11, e201700050 (2018).

    Article  Google Scholar 

  42. Zong, W. et al. Large-field high-resolution two-photon digital scanned light-sheet microscopy. Cell Res. 25, 254–257 (2015).

    Article  Google Scholar 

  43. Žurauskas, M., Barnstedt, O., Frade-Rodriguez, M., Waddell, S. & Booth, M. J. Rapid adaptive remote focusing microscope for sensing of volumetric neural activity. Biomed. Opt. Express 8, 4369–4379 (2017).

    Article  Google Scholar 

  44. Peinado, A., Bendek, E., Yokoyama, S. & Poskanzer, K. E. Deformable mirror-based axial scanning for two-photon mammalian brain imaging. Neurophotonics 8, 015003 (2021).

    Article  Google Scholar 

  45. Rupprecht, P., Prendergast, A., Wyart, C. & Friedrich, R. W. Remote z-scanning with a macroscopic voice coil motor for fast 3D multiphoton laser scanning microscopy. Biomed. Opt. Express 7, 1656–1671 (2016).

    Article  Google Scholar 

  46. Botcherby, E. J. et al. Aberration-free three-dimensional multiphoton imaging of neuronal activity at kHz rates. Proc. Natl Acad. Sci. USA 109, 2919–2924 (2012).

    Article  ADS  Google Scholar 

  47. Sofroniew, N. J., Flickinger, D., King, J. & Svoboda, K. A large field of view two-photon mesoscope with subcellular resolution for in vivo imaging. eLife 5, e14472 (2016).

    Article  Google Scholar 

  48. Chakraborty, T. et al. Converting lateral scanning into axial focusing to speed up three-dimensional microscopy. Light Sci. Appl. 9, 165 (2020).

    Article  ADS  Google Scholar 

  49. Planchon, T. et al. Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination. Nat. Methods 8, 417–423 (2011).

    Article  Google Scholar 

  50. Shin, Y., Kim, D. & Kwon, H.-S. Oblique scanning 2‐photon light‐sheet fluorescence microscopy for rapid volumetric imaging. J. Biophoton. 11, e201700270 (2017).

    Article  Google Scholar 

  51. Cheng, A., Gonçalves, J. T., Golshani, P., Arisaka, K. & Portera-Cailliau, C. Simultaneous two-photon calcium imaging at different depths with spatiotemporal multiplexing. Nat. Methods 8, 139–142 (2011).

    Article  Google Scholar 

  52. Stirman, J. N., Smith, I. T., Kudenov, M. W. & Smith, S. L. Wide field-of-view, multi-region, two-photon imaging of neuronal activity in the mammalian brain. Nat. Biotechnol. 34, 857–862 (2016).

    Article  Google Scholar 

  53. Chen, J. L., Voigt, F. F., Javadzadeh, M., Krueppel, R. & Helmchen, F. Long-range population dynamics of anatomically defined neocortical networks. eLife 5, e14679 (2016).

    Article  Google Scholar 

  54. Beaulieu, D. R. et al. Simultaneous multiplane imaging with reverberation two-photon microscopy. Nat. Methods 17, 283–286 (2020).

    Article  Google Scholar 

  55. Durnin, J., Miceli, J. J. & Eberly, J. H. Diffraction-free beams. Phys. Rev. Lett. 58, 1499–1501 (1987).

    Article  ADS  Google Scholar 

  56. Lu, R. et al. Video-rate volumetric functional imaging of the brain at synaptic resolution. Nat. Neurosci. 20, 620–628 (2017).

    Article  Google Scholar 

  57. Lu, R., Tanimoto, M., Koyama, M. & Ji, N. 50-Hz volumetric functional imaging with continuously adjustable depth of focus. Biomed. Opt. Express 9, 1964–1976 (2018).

    Article  Google Scholar 

  58. Fan, J. L. et al. High-speed volumetric two-photon fluorescence imaging of neurovascular dynamics. Nat. Commun. 11, 6020 (2020).

    Article  Google Scholar 

  59. Tan, X. J. et al. Volumetric two-photon microscopy with a non-diffracting Airy beam. Opt. Lett. 44, 391–394 (2019).

    Article  ADS  Google Scholar 

  60. He, H. et al. Depth-resolved volumetric two-photon microscopy based on dual Airy beam scanning. Opt. Lett. 44, 5238–5241 (2019).

    Article  ADS  Google Scholar 

  61. Yang, W. et al. Simultaneous multi-plane imaging of neural circuits. Neuron 20, 269–284 (2016).

    Article  Google Scholar 

  62. Song, A. et al. Volumetric two-photon imaging of neurons using stereoscopy (vTwINS). Nat. Methods 14, 420–426 (2017).

    Article  Google Scholar 

  63. Pnevmatikakis, E. A. et al. Simultaneous denoising, deconvolution and demixing of calcium imaging data. Neuron 89, 285–299 (2016).

    Article  Google Scholar 

  64. Oh, S. W. et al. A mesoscale connectome of the mouse brain. Nature 508, 207–214 (2014).

    Article  ADS  Google Scholar 

  65. Dekkers, J. F. et al. High-resolution 3D imaging of fixed and cleared organoids. Nat. Protoc. 14, 1756–1771 (2019).

    Article  Google Scholar 

  66. Weeber, F., Ooft, S. N., Dijkstra, K. K. & Voest, E. E. Tumor organoids as a pre-clinical cancer model for drug discovery. Cell Chem. Biol. 24, 1092–1100 (2019).

    Article  Google Scholar 

  67. Lohmann, A. W., Dorsch, R. G., Mendlovic, D., Zalevsky, Z. & Ferreira, C. Space–bandwidth product of optical signals and systems. J. Opt. Soc. Am. A 13, 470–473 (1996).

    Article  ADS  Google Scholar 

  68. McConnell, G. et al. A novel optical microscope for imaging large embryos and tissue volumes with sub-cellular resolution throughout. eLife 5, e18659 (2016).

    Article  Google Scholar 

  69. Tsai, P. S. et al. Ultra-large field-of-view two-photon microscopy. Opt. Express 23, 13833–13847 (2015).

    Article  ADS  Google Scholar 

  70. Fan, J. et al. Video-rate imaging of biological dynamics at centimetre scale and micrometre resolution. Nat. Photon. 13, 809–816 (2019).

    Article  ADS  Google Scholar 

  71. Bumstead, J. R. et al. Designing a large field-of-view two-photon microscope using optical invariant analysis. Neurophotonics 5, 025001 (2018).

    Article  Google Scholar 

  72. Terada, S. I., Kobayashi, K., Ohkura, M., Nakai, J. & Matsuzaki, M. Superwide-field two-photon imaging with a micro-optical device moving in post-objective space. Nat. Commun. 9, 3550 (2018).

    Article  ADS  Google Scholar 

  73. Lu, R. et al. Rapid mesoscale volumetric imaging of neural activity with synaptic resolution. Nat. Methods 17, 291–294 (2020).

    Article  Google Scholar 

  74. Weisenburger, S. et al. Volumetric Ca2+ imaging in the mouse brain using hybrid multiplexed sculpted light microscopy. Cell 177, 1050–1066 (2019).

    Article  Google Scholar 

  75. Tsia, K. (ed.) Understanding Biophotonics: Fundamentals, Advances and Applications (CRC Press, 2016).

  76. Horton, N. G. et al. In vivo three-photon microscopy of subcortical structures within an intact mouse brain. Nat. Photon. 7, 205–209 (2013).

    Article  ADS  Google Scholar 

  77. Wang, T. & Xu, C. Three-photon neuronal imaging in deep mouse brain. Optica 7, 947–960 (2020).

    Article  ADS  Google Scholar 

  78. Wang, T. et al. Quantitative analysis of 1,300-nm three-photon calcium imaging in the mouse brain. eLife 9, e53205 (2020).

    Article  Google Scholar 

  79. Guesmi, K. et al. Dual-color deep-tissue three-photon microscopy with a multiband infrared laser. Light Sci. Appl. 7, 12 (2018).

    Article  ADS  Google Scholar 

  80. Dana, H. et al. Sensitive red protein calcium indicators for imaging neural activity. eLife 5, e12727 (2016).

    Article  Google Scholar 

  81. Kannan, M. et al. Fast, in vivo voltage imaging using a red fluorescent indicator. Nat. Methods 15, 1108–1116 (2018).

    Article  Google Scholar 

  82. Mohr, M. A. et al. jYCaMP: an optimized calcium indicator for two-photon imaging at fiber laser wavelengths. Nat. Methods 17, 694–697 (2020).

    Article  Google Scholar 

  83. Yildirim, M., Sugihara, H., So, P. T. & Sur, M. Functional imaging of visual cortical layers and subplate in awake mice with optimized three-photon microscopy. Nat. Commun. 10, 177 (2019).

    Article  ADS  Google Scholar 

  84. Ouzounov, D. G. et al. In vivo three-photon imaging of activity of GCaMP6-labeled neurons deep in intact mouse brain. Nat. Methods 14, 388–390 (2017).

    Article  Google Scholar 

  85. Wang, T. et al. Three-photon imaging of mouse brain structure and function through the intact skull. Nat. Methods 15, 789–792 (2018).

    Article  Google Scholar 

  86. Takasaki, K. T., Tsyboulski, D. & Waters, J. Dual-plane 3-photon microscopy with remote focusing. Biomed. Opt. Express 10, 5585–5599 (2019).

    Article  Google Scholar 

  87. Escobet-Montalbán, A. et al. Three-photon light-sheet fluorescence microscopy. Opt. Lett. 43, 5484–5487 (2018).

    Article  ADS  Google Scholar 

  88. Rodríguez, C., Liang, Y., Lu, R. & Na, J. Three-photon fluorescence microscopy with an axially elongated Bessel focus. Opt. Lett. 43, 1914–1917 (2018).

    Article  ADS  Google Scholar 

  89. Chen, B. et al. Rapid volumetric imaging with Bessel-beam three-photon microscopy. Biomed. Opt. Express 9, 1992–2000 (2018).

    Article  Google Scholar 

  90. Low, R. J., Gu, Y. & Tank, D. W. Cellular resolution optical access to brain regions in fissures: imaging medial prefrontal cortex and grid cells in entorhinal cortex. Proc. Natl Acad. Sci. USA 111, 18739–18744 (2014).

    Article  ADS  Google Scholar 

  91. Ozbay, B. N. et al. Three dimensional two-photon brain imaging in freely moving mice using a miniature fiber coupled microscope with active axial-scanning. Sci. Rep. 8, 8108 (2018).

    Article  ADS  Google Scholar 

  92. Attardo, A., Fitzgerald, J. E. & Schnitzer, M. J. Impermanence of dendritic spines in live adult CA1 hippocampus. Nature 523, 592–596 (2015).

    Article  ADS  Google Scholar 

  93. Liang, B., Zhang, L., Moffitt, C., Li, Y. & Lin, D. T. An open-source automated surgical instrument for microendoscope implantation. J. Neurosci. Methods 311, 83–88 (2019).

    Article  Google Scholar 

  94. Meng, G. et al. High-throughput synapse-resolving two-photon fluorescence microendoscopy for deep-brain volumetric imaging in vivo. eLife 8, e40805 (2019).

    Article  Google Scholar 

  95. Moretti, C., Antonini, A., Bovetti, S., Liberale, C. & Fellin, T. Scanless functional imaging of hippocampal networks using patterned two-photon illumination through GRIN lenses. Biomed. Opt. Express 7, 3958–3967 (2016).

    Article  Google Scholar 

  96. Sato, M. et al. Fast varifocal two-photon microendoscope for imaging neuronal activity in the deep brain. Biomed. Opt. Express 8, 4049–4060 (2017).

    Article  Google Scholar 

  97. Qin, Z. et al. Adaptive optics two-photon endomicroscopy enables deep-brain imaging at synaptic resolution over large volumes. Sci. Adv. 6, eabc6521 (2020).

    Article  ADS  Google Scholar 

  98. Chien, Y. F. et al. Dual GRIN lens two-photon endoscopy for high-speed volumetric and deep brain imaging. Biomed. Opt. Express 12, 162–172 (2021).

    Article  Google Scholar 

  99. Antonini, A. et al. Extended field-of-view ultrathin microendoscopes for high-resolution two-photon imaging with minimal invasiveness. eLife 9, e58882 (2020).

    Article  Google Scholar 

  100. Wang, C. & Ji, N. Characterization and improvement of three-dimensional imaging performance of GRIN-lens-based two-photon fluorescence endomicroscopes with adaptive optics. Opt. Express 21, 27142–27154 (2013).

    Article  ADS  Google Scholar 

  101. Wang, C. & Ji, N. Pupil-segmentation-based adaptive optical correction of a high-numerical-aperture gradient refractive index lens for two-photon fluorescence endoscopy. Opt. Lett. 37, 2001–2003 (2012).

    Article  ADS  Google Scholar 

  102. Ji, N. Adaptive optical fluorescence microscopy. Nat. Methods 14, 374–380 (2017).

    Article  Google Scholar 

  103. Booth, M. Adaptive optical microscopy: the ongoing quest for a perfect image. Light Sci. Appl. 3, e165 (2014).

    Article  ADS  Google Scholar 

  104. Wang, K. et al. Direct wavefront sensing for high-resolution in vivo imaging in scattering tissue. Nat. Commun. 6, 7276 (2015).

    Article  ADS  Google Scholar 

  105. Liu, R., Li, Z., Marvin, J. S. & Kleinfeld, D. Direct wavefront sensing enables functional imaging of infragranular axons and spines. Nat. Methods 16, 615–618 (2019).

    Article  Google Scholar 

  106. Qin, Z. et al. Adaptive optics two-photon microscopy enables near-diffraction-limited and functional retinal imaging in vivo. Light Sci. Appl. 9, 79 (2020).

    Article  ADS  Google Scholar 

  107. Ji, N., Milkie, D. E. & Betzig, E. Adaptive optics via pupil segmentation for high-resolution imaging in biological tissues. Nat. Methods 7, 141–147 (2010).

    Article  Google Scholar 

  108. Park, J. H., Kong, L., Zhou, Y. & Cui, M. Large-field-of-view imaging by multi-pupil adaptive optics. Nat. Methods 14, 581–583 (2017).

    Article  Google Scholar 

  109. Rodríguez, C. et al. An adaptive optics module for deep tissue multiphoton imaging in vivo. Preprint at bioRxiv https://doi.org/10.1101/2020.11.25.397968 (2020).

  110. Charan, K., Li, B., Wang, M., Lin, C. P. & Xu, C. Fiber-based tunable repetition rate source for deep tissue two-photon fluorescence microscopy. Biomed. Opt. Express 9, 2304–2311 (2018).

    Article  Google Scholar 

  111. Ji, N., Magee, J. C. & Betzig, E. High-speed, low-photodamage nonlinear imaging using passive pulse splitters. Nat. Methods 5, 197–202 (2008).

    Article  Google Scholar 

  112. Perillo, E. et al. Two-color multiphoton in vivo imaging with a femtosecond diamond Raman laser. Light Sci. Appl. 6, e17095 (2017).

    Article  Google Scholar 

  113. Perillo, E. P. et al. Deep in vivo two-photon microscopy with a low cost custom built mode-locked 1,060-nm fiber laser. Biomed. Opt. Express 7, 324–334 (2016).

    Article  Google Scholar 

  114. Chen, B. et al. Robust hollow-fiber-pigtailed 930-nm femtosecond Nd:fiber laser for volumetric two-photon imaging. Opt. Express 25, 22704–22709 (2017).

    Article  ADS  Google Scholar 

  115. Stachowiak, D. et al. Frequency-doubled femtosecond Er-doped fiber laser for two-photon excited fluorescence imaging. Biomed. Opt. Express 11, 4431–4442 (2020).

    Article  Google Scholar 

  116. Li, B., Wu, C., Wang, M., Charan, K. & Xu, C. An adaptive excitation source for high-speed multiphoton microscopy. Nat. Methods 17, 163–167 (2020).

    Article  Google Scholar 

  117. Kong, C. et al. High-contrast, fast chemical imaging by coherent Raman scattering using a self-synchronized two-colour fibre laser. Light Sci. Appl. 9, 25 (2020).

    Article  ADS  Google Scholar 

  118. Qian, Y. et al. A genetically encoded near-infrared fluorescent calcium ion indicator. Nat. Methods 16, 171–174 (2019).

    Article  Google Scholar 

  119. Shemetov, A. A. et al. A near-infrared genetically encoded calcium indicator for in vivo imaging. Nat. Biotechnol. 39, 368–377 (2021).

    Article  Google Scholar 

  120. Zhang, K., Zuo, W., Chen, Y., Meng, D. & Zhang, L. Beyond a Gaussian denoiser: residual learning of deep CNN for image denoising. IEEE Trans. Image Process. 26, 3142–3155 (2017).

    Article  ADS  MathSciNet  MATH  Google Scholar 

  121. Weigert, M. et al. Content-aware image restoration: pushing the limits of fluorescence microscopy. Nat. Methods 15, 1090–1097 (2018).

    Article  Google Scholar 

  122. Batson, J. & Royer, L. Noise2Self: blind denoising by self-supervision. Proc. 36th International Conference on Machine Learning (PMLR) 97, 524–533 (2019).

    Google Scholar 

  123. Zhu, J., Park, T., Isola, P. & Efros, A. A. Unpaired image-to-image translation using cycle-consistent adversarial networks. In Proc. 2017 IEEE International Conference on Computer Vision (ICCV) 2242–2251 (IEEE, 2017).

  124. Moen, E. et al. Deep learning for cellular image analysis. Nat. Methods 16, 1233–1246 (2019).

    Article  Google Scholar 

  125. Belthangady, C. & Royer, L. A. Applications, promises and pitfalls of deep learning for fluorescence image reconstruction. Nat. Methods 16, 1215–1225 (2019).

    Article  Google Scholar 

  126. Voigt, F. F. et al. Multiphoton in vivo imaging with a femtosecond semiconductor disk laser. Biomed. Opt. Express 8, 3213–3231 (2017).

    Article  Google Scholar 

  127. Pitrone, P. G. et al. OpenSPIM: an open-access light-sheet microscopy platform. Nat. Methods 10, 598–599 (2013).

    Article  Google Scholar 

  128. Pachitariu, M. et al. Suite2p: beyond 10,000 neurons with standard two-photon microscopy. Preprint at bioRxiv https://doi.org/10.1101/061507 (2017).

  129. Marshall, G. F. & Stutz, G. E. (eds) Handbook of Optical and Laser Scanning (Taylor & Francis, 2012).

  130. Grewe, B. F., Langer, D., Kasper, H., Kampa, B. M. & Helmchen, F. High-speed in vivo calcium imaging reveals neuronal network activity with near-millisecond precision. Nat. Methods 7, 399–405 (2010).

    Article  Google Scholar 

  131. Cossell, L. et al. Functional organization of excitatory synaptic strength in primary visual cortex. Nature 518, 399–403 (2015).

    Article  ADS  Google Scholar 

  132. Katona, G. et al. Roller coaster scanning reveals spontaneous triggering of dendritic spikes in CA1 interneurons. Proc. Natl Acad. Sci. USA 108, 2148–2153 (2011).

    Article  ADS  Google Scholar 

Download references

Acknowledgements

This work is supported by the Research Grants Council of the Hong Kong Special Administrative Region of China (grants 17208918, 17209017 and 17259316 to J.W. and K.K.T.; RFS2021-7S06 and C7047-16G to K.K.T.) and NIH BRAIN Initiative grants (1UF1NS107696 to J.W., N.J. and K.K.T.).

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Wu, J., Ji, N. & Tsia, K.K. Speed scaling in multiphoton fluorescence microscopy. Nat. Photon. 15, 800–812 (2021). https://doi.org/10.1038/s41566-021-00881-0

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