Hostname: page-component-cd9895bd7-dk4vv Total loading time: 0 Render date: 2024-12-28T04:19:16.940Z Has data issue: false hasContentIssue false
  • English
  • Français

Multi-Photon Microscopy, High Resolution Imaging Deep in Strongly Scattering Specimens

Published online by Cambridge University Press:  02 July 2020

Winfried Denk
Winfried Denk*
Affiliation:
Biological Computation Research Department Bell Laboratories, Lucent Technologies Murray Hill, NJ, 07974
Get access

Extract

Imaging small structures substantially below the tissue surface in living specimens poses special challenges mainly because light is scattered by ever present refractive index inhomogeneities. Confocal microscoy removes the blurring caused by scattered and out-of-focus light but does so only at the expense of photodynamic damage that is often unacceptable when observing live specimens.

Multi-photon absorption microscopy[l] solves these problems because excitation is virtually limited to the focal plane. Out-of-focus photobleaching and photodamage are therefore eliminated. In scattering samples substantial improvements accrue even for the focal plane because, different from confocal microscopy, where only ballistic fluorescenc photons can be used, in the multi-photon microscope scattered photons can be utilized in addition [2-4], provided whole-field detection is used[5].

Many questions in the study of the nervous system require the investigation of intact portions of neural tissue in order to preserve the multiply branched processes of neurons, often extending over hundreds of microns, together with the local nervous circuitry.

Type
Biological Applications of Multi-photon Excitation Fluorescence Imaging
Information
Microscopy and Microanalysis , Volume 3 , Issue S2: Proceedings: Microscopy & Microanalysis '97, Microscopy Society of America 55th Annual Meeting, Microbeam Analysis Society 31st Annual Meeting, Histochemical Society 48th Annual Meeting, Cleveland, Ohio, August 10-14, 1997 , August 1997 , pp. 301 - 302
Copyright
Copyright © Microscopy Society of America 1997

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

1.Denk, W., Strickler, J.H., and Webb, W.W., Two-photon laser scanning fluorescence microscopy. Science, 1990. 248: p. 73–76.Google Scholar
2.Denk, W., et al., Anatomical and functional imaging of neurones using 2-photon laser scanning microscopy. J. Neurosci. Meth., 1994. 54(2): p. 151–162.10.1016/0165-0270(94)90189-9CrossRefGoogle Scholar
3.Denk, W., Two-Photon Excitation in Functional Biological Imaging. J. Biomed. Opt., 1996. 1(3): p. 296–304.10.1117/12.242945CrossRefGoogle ScholarPubMed
4.Denk, W. and Svoboda, K., Photon upmanship: why multiphoton imaging is more than a gimmick. Neuron, 1997. : p. in press.10.1016/S0896-6273(00)81237-4CrossRefGoogle Scholar
5.Denk, W., Piston, D.W., and Webb, WW., Two-photon molecular excitation in laser scanning microscopy, in The Handbook of ConfocalMicroscopy, Pawley, J., Editor. 1995, Plenum: New York. p. 445–458.Google Scholar
6.Yuste, R. and Denk, W., Dendritic spines as basic functional units of neuronal integration. Nature, 1995. 375(6533). p. 682–684.Google Scholar
7.Denk, W., Sugimori, M., and Llinas, R., Two types of calcium response limited to single spines in cerebellar Purkinje cells. Proc. Natl. Acad. Sci. (USA), 1995. 92(18). p. 8279–8282.10.1073/pnas.92.18.8279CrossRefGoogle ScholarPubMed
8.Svoboda, K., Tank, D.W., and Denk, W., Direct measurement of coupling between dendritic spines and shaft. Science, 1996. 272(5262): p. 716–719.Google Scholar
9.Svoboda, K., Denk, W., Kleinfeld, D., and Tank, D., In Vivo Dendritic Calcium Dynamics in Neocortical Pyramidal Neurons. Nature, 1997. 385(6612): p. 161–165.Google Scholar
10.Denk, W., Two-photon scanning photochemical microscopy: mapping ligand-gated ion channel distributions. Proc. Natl. Acad. Sci. (USA), 1994. 91: p. 6629–6633.10.1073/pnas.91.14.6629CrossRefGoogle ScholarPubMed