Two-photon excitation microscopy (2PM) is an alternative to confocal microscopy that provides attractive features for imaging deep into intact tissues and reducing overall photo-toxicity. This approach enables dynamic cellular measurements, and the depth penetration opens up a broad range of experiments that rely on non-invasive intra-vital imaging of cellular and sub-cellular processes. This chapter will detail what two-photon excitation is, how it enable imaging in practice, and how its underlying photophysics leads to both its advantages and limitations. The chapter will also cover how instrumentation design can be optimized for 2PM, and show how this approach can be extended to other related non-linear microscopies, each of which offer particular advantages. Finally, a few recent examples of 2PM will be presented to highlight its potential impact in neuroscience, developmental biology, immunology, cancer and endocrinology.

Original languageEnglish
Title of host publicationFluorescence Microscopy
Subtitle of host publicationFrom Principles to Biological Applications: Second Edition
PublisherWiley-VCH Verlag
Number of pages40
ISBN (Electronic)9783527687732
ISBN (Print)9783527338375
StatePublished - Apr 5 2017


  • 2-photon microscopy
  • Autofluorescence
  • Deep tissue imaging
  • Harmonic generation
  • In vivo imaging
  • Raman microscopy


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