title: Fluorescence microscopy gets faster and clearer: roles of photochemistry and selective illumination.
abstract: Significant advances in fluorescence microscopy tend be a balance between two competing qualities wherein improvements in resolution and low light detection are typically accompanied by losses in acquisition rate and signal-to-noise, respectively. These trade-offs are becoming less of a barrier to biomedical research as recent advances in optoelectronic microscopy and developments in fluorophore chemistry have enabled scientists to see beyond the diffraction barrier, image deeper into live specimens, and acquire images at unprecedented speed. Selective plane illumination microscopy has provided significant gains in the spatial and temporal acquisition of fluorescence specimens several mm in thickness. With commercial systems now available, this method promises to expand on recent advances in 2-photon deep-tissue imaging with improved speed and reduced photobleaching compared to laser scanning confocal microscopy. Superresolution microscopes are also available in several modalities and can be coupled with selective plane illumination techniques. The combination of methods to increase resolution, acquisition speed, and depth of collection are now being married to common microscope systems, enabling scientists to make significant advances in live cell and in situ imaging in real time. We show that light sheet microscopy provides significant advantages for imaging live zebrafish embryos compared to laser scanning confocal microscopy.
authors.affiliation: Research Scientist and Lecturer, Director of Confocal Microscope Core Imaging Facility, Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut.
authors.forename: Joseph S
authors.initials: JS
authors.lastname: Wolenski
authors.affiliation: Associate Research Scientist, Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut.
authors.forename: Doerthe
authors.initials: D
authors.lastname: Julich
mesh_terms: D000818:Animals; D004625:Embryo, Nonmammalian / Q000378:metabolism / Q000648:ultrastructure; D005456:Fluorescent Dyes / Q000737:chemistry; D008027:Light; D008163:Luminescent Measurements / Q000295:instrumentation / Q000379:methods*; D008164:Luminescent Proteins / Q000737:chemistry / Q000378:metabolism; D018613:Microscopy, Confocal / Q000295:instrumentation / Q000379:methods*; D036641:Microscopy, Fluorescence, Multiphoton / Q000295:instrumentation / Q000379:methods*; D010777:Photochemistry / Q000295:instrumentation / Q000379:methods*; D015203:Reproducibility of Results; D015027:Zebrafish / Q000196:embryology / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D008164:Luminescent Proteins
keywords: 2-photon microscopy; confocal microscopy; light sheet microscopy; photoactivatable GFP; selective plane illumination microscopy; superresolution microscopy