Editing Microscopy (section)

==== Two-photon microscopy ====
A two-photon microscope is also a laser-scanning microscope, but instead of UV, blue or green laser light, a [[Ultrashort pulse|pulsed infrared laser]] is used for excitation. Only in the tiny focus of the laser is the intensity high enough to generate fluorescence by [[Two-photon excitation microscopy|two-photon excitation]], which means that no out-of-focus fluorescence is generated, and no pinhole is necessary to clean up the image.<ref>{{Cite journal|last1=Denk|first1=Winfried|last2=Svoboda|first2=Karel|date=March 1997|title=Photon Upmanship: Why Multiphoton Imaging Is More than a Gimmick|journal=Neuron|language=en|volume=18|issue=3|pages=351–357|doi=10.1016/S0896-6273(00)81237-4|pmid=9115730|s2cid=2414593|doi-access=free}}</ref> This allows imaging deep in scattering tissue, where a confocal microscope would not be able to collect photons efficiently.<ref>{{Cite journal|last1=Denk|first1=W.|last2=Delaney|first2=K.R.|last3=Gelperin|first3=A.|last4=Kleinfeld|first4=D.|last5=Strowbridge|first5=B.W.|last6=Tank|first6=D.W.|last7=Yuste|first7=R.|date=1994|title=Anatomical and functional imaging of neurons using 2-photon laser scanning microscopy|journal=Journal of Neuroscience Methods|language=en|volume=54|issue=2|pages=151–162|doi=10.1016/0165-0270(94)90189-9|pmid=7869748|s2cid=3772937}}</ref> Two-photon microscopes with wide-field detection are frequently used for functional imaging, e.g. [[calcium imaging]], in brain tissue.<ref>{{Cite journal|last1=Svoboda|first1=Karel|last2=Denk|first2=Winfried|last3=Kleinfeld|first3=David|last4=Tank|first4=David W.|date=1997|title=In vivo dendritic calcium dynamics in neocortical pyramidal neurons|url=http://www.nature.com/articles/385161a0|journal=Nature|language=en|volume=385|issue=6612|pages=161–165|doi=10.1038/385161a0|pmid=8990119|bibcode=1997Natur.385..161S|s2cid=4251386|issn=0028-0836|access-date=2020-05-20|archive-date=2021-05-25|archive-url=https://web.archive.org/web/20210525175417/https://www.nature.com/articles/385161a0|url-status=live}}</ref> They are marketed as '''Multiphoton microscopes''' by several companies, although the gains of using 3-photon instead of 2-photon excitation are marginal.