Abstract
Tissues are heterogeneous with respect to cellular and non-cellular components and in the dynamic interactions between these elements. To study the behaviour and fate of individual cells in these complex tissues, intravital microscopy (IVM) techniques such as multiphoton microscopy have been developed to visualize intact and live tissues at cellular and subcellular resolution. IVM experiments have revealed unique insights into the dynamic interplay between different cell types and their local environment, and how this drives morphogenesis and homeostasis of tissues, inflammation and immune responses, and the development of various diseases. This Primer introduces researchers to IVM technologies, with a focus on multiphoton microscopy of rodents, and discusses challenges, solutions and practical tips on how to perform IVM. To illustrate the unique potential of IVM, several examples of results are highlighted. Finally, we discuss data reproducibility and how to handle big imaging data sets.
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Acknowledgements
C.L.G.J.S. was supported by a European Molecular Biology Organization (EMBO) long-term postdoctoral fellowship (ALTF 1035-2020), a Federation of the European Biochemical Societies (FEBS) excellence award and an Excellence of Science (EOS) grant (project ID: 40007532) of Fonds Wetenschappelijk Onderzoek — Le Fonds de la Recherche Scientifique (FWO-FNRS). P.T. was supported by the Len Ainsworth Fellowship in Pancreatic Cancer Research and is a National Health and Medical Research Council (NHMRC) Senior Research Fellow. D.H. was supported by a Cancer Institute NSW (CINSW) Early Career Research Fellowship. R.W. was supported by the National Institutes of Health (NIH), National Cancer Institute (NCI) Center for Cancer Research Intramural Research Program (ZIA BC 011682). C.N.J. was supported by the Canada Research Chairs Program. P.F. was supported by the NIH U54 CA261694-01 and ERC-2021-ADG 101054921. J.v.R. was supported by the VICI (09150182110004) of ZonMW of the Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO) and the Doctor Josef Steiner Foundation. J.v.R. and P.F. were also funded by the CancerGenomics.nl (Netherlands Organization for Scientific Research) programme. D.E. and M.H.O. are supported by the NCI (CA255153) and The Gruss-Lipper Biophotonics Center and its associated Integrated Imaging Program, and by Jane A. and Myles P. Dempsey.
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Contributions
Introduction (C.L.C., J.v.R, C.L.G.J.S.); Experimentation (P.F., R.W., D.H., P.T., D.E.); Results (D.H., P.T.); Applications (R.W., C.L.C., E.Y., M.I., C.N.J., M.H.O., D.E.); Reproducibility and data deposition (E.Y., M.I., R.W., D.H., P.T.); Limitations and optimizations (R.W., D.H., P.T., C.L.C.); Outlook (C.L.C., J.v.R., C.L.G.J.S., F.L.B.M., D.E., M.H.O.); Overview of the Primer (C.L.G.J.S., J.v.R.).
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Competing interests
P.T. receives reagents from Kadmon, InxMed (also consultant), Redx Pharma, Équilibre Biopharmaceuticals and Amplia Therapeutics. Under a licensing agreement between Amplia Therapeutics and Garvan Institute of Medical Research, D.H. and P.T. (consultant) are entitled to milestone payments. All other authors declare no competing interests.
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Nature Reviews Methods Primers thanks Heyu Ni, Helene Moreau and the other, anonymous, reviewer for their contribution to the peer review of this work.
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Fiji: https://fiji.sc/
ImageJ: https://imagej.nih.gov/ij/
Intravital_Microscopy_Toolbox: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0053942
Principles of the 3Rs: https://nc3rs.org.uk/who-we-are/3rs
Supplementary information
Glossary
- Fluorescence lifetime imaging microscopy
-
(FLIM). An imaging technique capable of assessing the chemical environment of a fluorophore by detecting the characteristic time from excitation to emission for a fluorophore rather than the number of photons emitted.
- Intravasation
-
The entry of a cell or material from tissues into the lumen of a blood or lymph vessel.
- Pulse repetition rate
-
The frequency at which high-energy laser light is emitted, typically 1–2 MHz for low-repetition rate and 80 MHz for high-repetition rate lasers.
- Optical parametric oscillators
-
Femtosecond pulsed light sources capable of emitting light in the range of 1,100–2,000 nm, increasing the wavelength of a pulsed femtosecond laser beyond 1,000 nm.
- Group velocity dispersion compensators
-
Optical devices capable of maintaining the duration of femtosecond light pulses in the presence of dispersive optical elements such as objective lenses.
- Optical window
-
A surgically implantable device that creates an optically clear aperture through which deeper tissues (such as internal organs) may be viewed.
- Gradient-index lenses
-
Very small diameter lenses (~1–2 mm) made of a material of non-uniform index of refraction that can be positioned at physical sites that cannot be accessed by standard lenses.
- Quantum yield
-
The ratio of photons emitted to photons observed, informing on the efficiency of fluorescence emission.
- Photon budget
-
The number of emitted photons per molecule before becoming non-fluorescent, which can depend on the quantum yield and photostability of a fluorophore.
- Invadopodia
-
Plasma membrane protrusions with proteolytic activity to break down extracellular matrix proteins, which can be used by cancer cells to locally invade into adjacent tissues.
- Calvarium
-
The top part of the skull. In the mouse, this includes the frontal bones, with the central suture joining them.
- Endosteum
-
The interface between bone and bone marrow, typically lined by osteoblasts and osteoclasts.
- MLL-AF9-driven murine AML
-
A murine model of poor-prognosis acute myeloid leukaemia (AML). The model can be based on the doxycycline-induced expression of the oncogene MLL-AF9 or on retroviral transduction of MLL-AF9 into haematopoietic progenitor cells. The different variants have some differences, for example, in the expression levels of the oncogene and latency of disease, but all develop into AML.
- Acini
-
A cluster of cells that form a lobed structure that looks similar to a cluster of grapes.
- apoA-IV
-
A plasma protein that shares structural features with other apolipoproteins.
- Juxtacrine
-
Signalling that requires cell–cell or cell–matrix contact.
- Surgical engineering
-
The combination of novel surgical protocols and engineering designs to expose these tissues for short-term and longitudinal microscopic analysis.
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Scheele, C.L.G.J., Herrmann, D., Yamashita, E. et al. Multiphoton intravital microscopy of rodents. Nat Rev Methods Primers 2, 89 (2022). https://doi.org/10.1038/s43586-022-00168-w
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DOI: https://doi.org/10.1038/s43586-022-00168-w
