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Quantitating 1‐photon and 2‐photon Microscopic Images
Author(s) -
Nyengaard Jens Randel
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a3-b
Subject(s) - photobleaching , two photon excitation microscopy , microscopy , microscope , förster resonance energy transfer , fluorescence correlation spectroscopy , photon , fluorescence , fluorescence microscope , fluorescence lifetime imaging microscopy , optics , laser , materials science , chemistry , physics
Bioscience has within the last decade made many discoveries using 1‐photon and 2‐photon microscopy. One‐photon laser sources are available from ~400 to ~650 nm and two‐photon lasers have a bandwidth of ~720 nm to ~980 nm. Previously 1‐photon and 2‐photon microscopy was primarily used for making beautiful images of biological examples. Recently focus has changes towards various quantitative applications with these microscopes. This presentation will focus on the advantages and disadvantages of 1‐photon and 2‐photon microscopy with regards to: Fluorescence resonance energy transfer (FRET); Two channel spectral fluorescence lifetime imaging (FLIM); Two channel fluorescence correlation spectroscopy (FCS); Fluorescence recovery after photobleaching (FRAP); Fluorescence loss in photobleaching (FLIP); Uncaging of dyes, metabolites and ions as well as time‐lapse imaging; Rapid switch sequences, ratiometric and physiological measurements; Reduction of bleed‐through when using multiple‐stained specimens; Spectral separation/unmixing of many different fluorophores/colours; 3D measurements including 3D sampling of cells and organelles when the microscope stage is computer‐controlled in the x‐, y‐, and z‐axis.