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.

  • Protocol
  • Published:

Standardized measurements for monitoring and comparing multiphoton microscope systems

Nature Protocols volume 20, pages 2171–2208 (2025)Cite this article

Subjects

Abstract

The goal of this protocol is to improve the characterization and performance standardization of multiphoton microscopy hardware across a large user base. We purposefully focus on hardware and only briefly touch on software and data analysis routines where relevant. Here we cover the measurement and quantification of laser power, pulse width optimization, field of view, resolution and photomultiplier tube performance. The intended audience is scientists with little expertise in optics who either build or use multiphoton microscopes in their laboratories. They can use our procedures to test whether their multiphoton microscope performs well and produces consistent data over the lifetime of their system. Individual procedures are designed to take 1–2 h to complete without the use of expensive equipment. The procedures listed here help standardize the microscopes and facilitate the reproducibility of data across setups.

Key points

  • The best practices for the quantification of the performance of multiphoton microscopes are described, necessary to overcome the wide user- and facility-dependent variations in microscope calibration, which inevitably affect the reproducibility of data across laboratories.

  • The procedures cover laser power, pulse width optimizations, field of view, resolution and photomultiplier tube performance and the majority should be carried out on a regular basis for maintenance of the system.

Access through your institution
Buy or subscribe

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

Access options

Access through your institution

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

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

Fig. 1: Basic schematic of a resonant-scanning multiphoton microscope.
Fig. 2: Object distortion caused by RI mismatching and the correction factor.
Fig. 3: Laser power measurement.
Fig. 4: FOV size measurement.
Fig. 5: FOV size comparison for two-photon and epifluorescent modes on a large FOV microscope (Mesoscope).
Fig. 6: FOV homogeneity.
Fig. 7: Two-photon FWHM as a function of NA and wavelengths.
Fig. 8: Example measurement of PSFs.
Fig. 9: Example image pixel histogram.
Fig. 10: Pulse width optimization measurement.
Fig. 11: A hand-made tritium light source.
Fig. 12: First-day performance for three multialkali PMT units of the same model.
Fig. 13: Change in PMT performance over time.
Fig. 14: Pixel grayscale value distributions and ROC–AUC at a series of gain settings for an example multialkali PMT.
Fig. 15: Comparison of two different GaAsP PMTs of the same model (Hamamatsu H10770PB-40).
Fig. 16: Photon transfer analysis.
Fig. 17: A different image sequence from another source.
Fig. 18: Pockels cell resonance effect.
Fig. 19: The effect of dielectric coatings on GDD.

Similar content being viewed by others

References

  1. Goppert-Mayer, M. Elementary file with two quantum fissures. Ann. Phys. 9, 273–294 (1931).

    CAS  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  3. Debarre, D., Olivier, N., Supatto, W. & Beaurepaire, E. Mitigating phototoxicity during multiphoton microscopy of live Drosophila embryos in the 1.0–1.2 mu m wavelength range. PloS ONE 9, 12 (2014).

    Article  Google Scholar 

  4. Schonle, A. & Hell, S. W. Heating by absorption in the focus of an objective lens. Opt. Lett. 23, 325–327 (1998).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Stujenske, J. M., Spellman, T. & Gordon, J. A. Modeling the spatiotemporal dynamics of light and heat propagation for in vivo optogenetics. Cell Rep. 12, 525–534 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Schmidt, E. & Oheim, M. infrared excitation induces heating and calcium microdomain hyperactivity in cortical astrocytes. Biophys. J. 119, 2153–2165 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Magidson, V. & Khodjakov, A. Circumventing photodamage in live-cell microscopy. Methods Cell Biol. https://doi.org/10.1016/B978-0-12-407761-4.00023-3 (2013).

  9. Icha, J., Weber, M., Waters, J. C. & Norden, C. Phototoxicity in live fluorescence microscopy, and how to avoid it Bioessays https://doi.org/10.1002/bies.201700003 (2017).

  10. Nadiarnykh, O., Thomas, G., Van Voskuilen, J., Sterenborg, H. & Gerritsen, H. C. Carcinogenic damage to deoxyribonucleic acid is induced by near-infrared laser pulses in multiphoton microscopy via combination of two- and three-photon absorption. J. Biomed. Opt. 17, 7 (2012).

    Article  Google Scholar 

  11. Konig, K., Becker, T. W., Fischer, P., Riemann, I. & Halbhuber, K. J. Pulse-length dependence of cellular response to intense near-infrared laser pulses in multiphoton microscopes. Opt. Lett. 24, 113–115 (1999).

    Article  CAS  PubMed  Google Scholar 

  12. CFI75 water dipping series. Nikon Instruments Inc. https://www.microscope.healthcare.nikon.com/products/optics/cfi75-water-dipping-series (2024).

  13. Semi-apochromat water dipping objectives for electrophysiology. Olympus LS https://www.olympus-lifescience.com/en/objectives/lumplfln-w/ (2024).

  14. Young, M. D., Field, J. J., Sheetz, K. E., Bartels, R. A. & Squier, J. A pragmatic guide to multiphoton microscope design. Adv. Opt. Photonics 7, 276–378 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Smith, S. L. in Multiphoton Microscopy Vol. 148 (ed. Hartveit, E.) 1–16 (Humana, 2019).

  16. Pawley, J. B. Handbook of Biological Confocal Microscopy (Springer, 2006).

  17. Masters, B. R. & So, P. Handbook of Biomedical Nonlinear Optical Microscopy (Oxford University, 2008).

  18. Ji, N., Freeman, J. & Smith, S. L. Technologies for imaging neural activity in large volumes. Nat. Neurosci. 19, 1154–1164 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  19. Zinter, J. P. & Levene, M. J. Maximizing fluorescence collection efficiency in multiphoton microscopy. Opt. Express 19, 15348–15362 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  PubMed  PubMed Central  Google Scholar 

  21. Zipfel, W. R., Williams, R. M. & Webb, W. W. Nonlinear magic: multiphoton microscopy in the biosciences. Nat. Biotechnol. 21, 1369–1377 (2003).

    Article  CAS  PubMed  Google Scholar 

  22. Heintzmann, R. & Sheppard, C. J. R. The sampling limit in fluorescence microscopy. Micron 38, 145–149 (2007).

    Article  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Quirin, S., Peterka, D. S. & Yuste, R. Instantaneous three-dimensional sensing using spatial light modulator illumination with extended depth of field imaging. Opt. Express 21, 16007–16021 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  28. Yang, W. et al. Simultaneous multi-plane imaging of neural circuits. Neuron 89, 269–284 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Helmchen, F. & Denk, W. Deep tissue two-photon microscopy. Nat. Methods 2, 932–940 (2005).

    Article  CAS  PubMed  Google Scholar 

  30. Gobel, W. & Helmchen, F. In vivo calcium imaging of neural network function. Physiology 22, 358–365 (2007).

    Article  CAS  PubMed  Google Scholar 

  31. Sherman, L., Ye, J. Y., Albert, O. & Norris, T. B. Adaptive correction of depth-induced aberrations in multiphoton scanning microscopy using a deformable mirror. J. Microsc. 206, 65–71 (2002).

    Article  CAS  PubMed  Google Scholar 

  32. Turcotte, R., Liang, Y. J. & Ji, N. Adaptive optical versus spherical aberration corrections for in vivo brain imaging. Biomed. Opt. Express 8, 3891–3902 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Visser, T. D. & Oud, J. L. Volume measurements in 3-dimensional microscopy. Scanning 16, 198–200 (1994).

    Article  Google Scholar 

  34. Diel, E. E., Lichtman, J. W. & Richardson, D. S. Tutorial: avoiding and correcting sample-induced spherical aberration artifacts in 3D fluorescence microscopy. Nat. Protoc. 15, 2773–2784 (2020).

    Article  CAS  PubMed  Google Scholar 

  35. Wang, K., Liang, R. F. & Qiu, P. Fluorescence signal generation optimization by optimal filling of the high numerical aperture objective lens for high-order deep-tissue multiphoton fluorescence microscopy. Ieee Photonics J. 7, 8 (2015).

    Article  Google Scholar 

  36. Takasaki, K., Abbasi-Asl, R. & Waters, J. Superficial bound of the depth limit of two-photon imaging in mouse brain. Eneuro 7, 10 (2020).

    Article  Google Scholar 

  37. Kondo, M., Kobayashi, K., Ohkura, M., Nakai, J. & Matsuzaki, M. Two-photon calcium imaging of the medial prefrontal cortex and hippocampus without cortical invasion. eLife 6, e26839 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  38. Yamaguchi, K., Kitamura, R., Kawakami, R., Otomo, K. & Nemoto, T. In vivo two-photon microscopic observation and ablation in deeper brain regions realized by modifications of excitation beam diameter and immersion liquid. PLoS ONE 15, e0237230 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Hampson, K. M. et al. Adaptive optics for high-resolution imaging. Nat. Rev. Methods Primer 1, 68 (2021).

    Article  CAS  Google Scholar 

  40. Hirlimann, C. Femtosecond Laser Pulses: Principles and Experiments (Springer, 2005).

  41. Dispersion (optics). Wikipedia https://en.wikipedia.org/wiki/Dispersion_(optics) (2023).

  42. 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, 20 (2016).

    Article  Google Scholar 

  43. Tai, S.-P. et al. Two-photon fluorescence microscope with a hollow-core photonic crystal fiber. Opt. Express 12, 6122–6128 (2004).

    Article  PubMed  Google Scholar 

  44. Zong, W. et al. Fast high-resolution miniature two-photon microscopy for brain imaging in freely behaving mice. Nat. Methods 14, 713–719 (2017).

    Article  CAS  PubMed  Google Scholar 

  45. Farinella, D. M., Roy, A., Liu, C. J. & Kara, P. Improving laser standards for three-photon microscopy. Neurophotonics 8, 015009 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  46. Borzsonyi, A., Kovacs, A. P. & Osvay, K. What we can learn about ultrashort pulses by linear optical methods. Appl. Sci. 3, 515–544 (2013).

    Article  Google Scholar 

  47. Hamamatsu Photonics KK. Photomultiplier Tubes: Basics and Applications (Hamamatsu Photonics K.K., 2017).

  48. Wang, T. et al. Quantitative analysis of 1300-nm three-photon calcium imaging in the mouse brain. eLife 9, e53205 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Samei, E. Performance of digital radiographic detectors: quantification and assessment methods. RSNA Categ. Course Diagn. Radiol. Phys. 37–47 (2003).

  50. Measuring the gain of your imaging system. Labrigger https://labrigger.com/blog/2010/07/30/measuring-the-gain-of-your-imaging-system/ (2010).

  51. Huang, L. et al. Relationship between simultaneously recorded spiking activity and fluorescence signal in GCaMP6 transgenic mice. eLife 10, e51675 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Lecoq, J., Orlova, N. & Grewe, B. F. Wide. Fast. Deep: recent advances in multiphoton microscopy of in vivo neuronal activity. J. Neurosci. 39, 9042–9052 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. in In Vivo Optical Imaging of Brain Function, Second Edition (ed. Frostig, R. D.) 1–472 (Crc Press, 2009); 10.1201/9781420076851

  54. Rosenegger, D. G., Tran, C. H. T., Ledue, J., Zhou, N. & Gordon, G. R. A high performance, cost-effective, open-source microscope for scanning two-photon microscopy that is modular and readily adaptable. PloS ONE 9, 16 (2014).

    Article  Google Scholar 

  55. Mayrhofer, J. M. et al. Design and performance of an ultra-flexible two-photon microscope for in vivo research. Biomed. Opt. Express 6, 4228–4237 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  56. Corbin, K., Pinkard, H., Peck, S., Beemiller, P. & Krummel, M. F. in Methods in Cell Biology Vol. 123 Ch. 8 (eds. Waters, J. C. & Wittman, T.) 135–151 (Academic, 2014).

  57. Diaspro, A., Chirico, G. & Collini, M. Two-photon fluorescence excitation and related techniques in biological microscopy. Q. Rev. Biophys. 38, 97–166 (2005).

    Article  CAS  PubMed  Google Scholar 

  58. Kerr, J. N. D. & Denk, W. Imaging in vivo: watching the brain in action. Nat. Rev. Neurosci. 9, 195–205 (2008).

    Article  CAS  PubMed  Google Scholar 

  59. So, P. T. C., Dong, C. Y., Masters, B. R. & Berland, K. M. Two-photon excitation fluorescence microscopy. Annu. Rev. Biomed. Eng. 2, 399–429 (2000).

    Article  CAS  PubMed  Google Scholar 

  60. Mostany, R., Miquelajauregui, A., Shtrahman, M. & Portera-Cailliau, C. in Advanced Fluorescence Microscopy: Methods and Protocols Vol. 1251 (ed. Verveer, P. J.) 25–42 (Humana Press, 2015).

  61. Negrean, A. & Mansvelder, H. D. Optimal lens design and use in laser-scanning microscopy. Biomed. Opt. Express 5, 1588–1609 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  62. Gaudreault, N. et al. Illumination power and illumination stability. protocols.io https://doi.org/10.17504/protocols.io.5jyl853ndl2w/v2 (2022).

  63. Nelson, G. et al. Monitoring the point spread function for quality control of confocal microscopes. protocols.io https://doi.org/10.17504/protocols.io.bp2l61ww1vqe/v1 (2022).

  64. Faklaris, O. et al. Quality assessment in light microscopy for routine use through simple tools and robust metrics. J. Cell Biol. 221, e202107093 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Cole, R. W., Jinadasa, T. & Brown, C. M. Measuring and interpreting point spread functions to determine confocal microscope resolution and ensure quality control. Nat. Protoc. 6, 1929–1941 (2011).

    Article  CAS  PubMed  Google Scholar 

  66. Jonkman, J., Brown, C. M., Wright, G. D., Anderson, K. I. & North, A. J. Tutorial: guidance for quantitative confocal microscopy. Nat. Protoc. 15, 1585–1611 (2020).

    Article  CAS  PubMed  Google Scholar 

  67. Llopis, P. M. et al. Best practices and tools for reporting reproducible fluorescence microscopy methods. Nat. Methods 18, 1463–1476 (2021).

    Article  Google Scholar 

  68. Demmerle, J. et al. Strategic and practical guidelines for successful structured illumination microscopy. Nat. Protoc. 12, 988–1010 (2017).

    Article  CAS  PubMed  Google Scholar 

  69. Saidi, S. & Shtrahman, M. Evaluation of compact pulsed lasers for two-photon microscopy using a simple method for measuring two-photon excitation efficiency. Neurophotonics 10, 044303 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Stack, R. F. et al. Quality assurance testing for modern optical imaging systems. Microsc. Microanal. 17, 598–606 (2011).

    Article  CAS  PubMed  Google Scholar 

  71. Campbell, R. A. A., Marbach, F., Pichler, B. & Walling, M. measurePSF. GitHub https://github.com/SWC-Advanced-Microscopy/measurePSF (2024).

  72. Distortion (optics). Wikipedia https://en.wikipedia.org/wiki/Distortion_(optics) (2023).

  73. 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  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Buchholz, T.-O., Eglinger, J. & Ehrenfeuchter, N. fmi-faim/napari-psf-analysis. GitHub https://github.com/fmi-faim/napari-psf-analysis (2024).

  76. Pedregosa, F. et al. Scikit-learn: machine learning in Python. J. Mach. Learn. Res. 12, 2825–2830 (2011).

    Google Scholar 

  77. Tang, S., Krasieva, T. B., Chen, Z., Tempea, G. & Tromberg, B. J. Effect of pulse duration on two-photon excited fluorescence and second harmonic generation in nonlinear optical microscopy. J. Biomed. Opt. 11, 3 (2006).

    Article  Google Scholar 

  78. Pogue, B. W. & Patterson, M. S. Review of tissue simulating phantoms for optical spectroscopy, imaging and dosimetry. J. Biomed. Opt. 11, 16 (2006).

    Article  Google Scholar 

  79. Kane, S. & Squier, J. Grism-pair stretcher-compressor system for simultaneous second- and third-order dispersion compensation in chirped-pulse amplification. J. Opt. Soc. Am. B 14, 661–665 (1997).

    Article  CAS  Google Scholar 

  80. Chauhan, V., Bowlan, P., Cohen, J. & Trebino, R. Single-diffraction-grating and grism pulse compressors. J. Opt. Soc. Am. B 27, 619–624 (2010).

    Article  CAS  Google Scholar 

  81. Yakovlev, I. V. Stretchers and compressors for ultra-high power laser systems. Quantum Electron. 44, 393 (2014).

    Article  Google Scholar 

  82. McFadden, D., Amos, B. & Heintzmann, R. Quality control of image sensors using gaseous tritium light sources. Philos. Transact. A 380, 20210130 (2022).

    CAS  Google Scholar 

  83. Tritium radioluminescence. Wikipedia https://en.wikipedia.org/wiki/Tritium_radioluminescence (2023).

  84. Janesick, J. R. Photon Transfer: DN (Lambda) (SPIE Bellingham, 2007).

  85. Consortium, T. Mic. et al. Functional connectomics spanning multiple areas of mouse visual cortex. Preprint at bioRxiv https://doi.org/10.1101/2021.07.28.454025 (2023).

  86. Bae, J. A. et al. MICrONS Two Photon Functional Imaging (Version 0.230307.2132) [Data set]. DANDI https://doi.org/10.48324/dandi.000402/0.230307.2132 (2023).

  87. Keller, U. Ultrafast Lasers: A Comprehensive Introduction to Fundamental Principles with Practical Applications (Springer, 2021); https://doi.org/10.1007/978-3-030-82532-4

  88. Rupprecht, P. Pulse dispersion, dielectric mirrors and fluorescence yield: a case report of a two-photon microscope. Zenodo https://doi.org/10.5281/zenodo.2592465 (2019).

Download references

Acknowledgements

This research was funded in part by the Wellcome Trust [204651/Z/16/Z, 220273/Z/20/Z]. For the purpose of Open Access, the author has applied a CC BY public copyright license to any Author Accepted Manuscript version arising from this submission. This work was also supported by funding from the European Research Council under the European Union’s Horizon 2020 research and innovation program (grant agreement no. 852765), the National Institutes of Health (R01EY035378 and RF1NS121919 to S.L.S., U24NS116470 to D.Y. and U19NS104649 and U01NS113273 to D.S.P.) and the National Science Foundation (1934288 to S.L.S.).

Author information

Authors and Affiliations

  1. Science and Technology Facilities Council, Octopus imaging facility, Research Complex at Harwell, Harwell Campus, Oxfordshire, UK

    Robert M. Lees

  2. Department of Neuroscience, Physiology and Pharmacology, University College London, London, UK

    Isaac H. Bianco

  3. Sainsbury Wellcome Centre, University College London, London, UK

    Robert A. A. Campbell

  4. Allen Institute, Seattle, WA, USA

    Natalia Orlova

  5. Mortimer B. Zuckerman Mind Brain Behavior Institute, Columbia University, New York, NY, USA

    Darcy S. Peterka

  6. Independent NeuroScience Services INSS Ltd, Lewes, UK

    Bruno Pichler

  7. Department of Electrical and Computer Engineering, University of California Santa Barbara, Santa Barbara, CA, USA

    Spencer LaVere Smith & Che-Hang Yu

  8. DataJoint Inc., Houston, TX, USA

    Dimitri Yatsenko

  9. Department of Physiology, Anatomy, and Genetics, University of Oxford, Oxford, UK

    Adam M. Packer

Authors
  1. Robert M. Lees
    View author publications

    Search author on:PubMed Google Scholar

  2. Isaac H. Bianco
    View author publications

    Search author on:PubMed Google Scholar

  3. Robert A. A. Campbell
    View author publications

    Search author on:PubMed Google Scholar

  4. Natalia Orlova
    View author publications

    Search author on:PubMed Google Scholar

  5. Darcy S. Peterka
    View author publications

    Search author on:PubMed Google Scholar

  6. Bruno Pichler
    View author publications

    Search author on:PubMed Google Scholar

  7. Spencer LaVere Smith
    View author publications

    Search author on:PubMed Google Scholar

  8. Dimitri Yatsenko
    View author publications

    Search author on:PubMed Google Scholar

  9. Che-Hang Yu
    View author publications

    Search author on:PubMed Google Scholar

  10. Adam M. Packer
    View author publications

    Search author on:PubMed Google Scholar

Contributions

Investigation and reviewing/editing: All authors. Original draft of Laser power at the sample: R.M.L., R.A.A.C., N.O. FOV size: R.A.A.C. and N.O. FOV homogeneity: R.A.A.C., N.O. and R.M.L. Spatial resolution: S.L.S. and C.-H.Y. Pulse width control and optimization: D.S.P. and R.A.A.C. Photomultiplier tube performance: I.H.B. and B.P. Estimating absolute magnitudes of fluorescence signals: D.Y.

Corresponding author

Correspondence to Adam M. Packer.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Protocols thanks the anonymous reviewer(s) for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Lees, R.M., Bianco, I.H., Campbell, R.A.A. et al. Standardized measurements for monitoring and comparing multiphoton microscope systems. Nat Protoc 20, 2171–2208 (2025). https://doi.org/10.1038/s41596-024-01120-w

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue date:

  • DOI: https://doi.org/10.1038/s41596-024-01120-w

Search

Advanced 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