Quantitative in vivo imaging of entire embryos with Digital Scanned Laser Light Sheet Fluorescence Microscopy.
The observation of biological processes in their natural in vivo context is a key requirement for quantitative experimental studies in the life sciences. In many instances, it will be crucial to achieve high temporal and spatial resolution over long periods of time without compromising the physiological development of the specimen. Here, we discuss the principles underlying light sheet-based fluorescence microscopes. The most recent implementation DSLM is a tool optimized to deliver quantitative data for entire embryos at high spatio-temporal resolution. We compare DSLM to the two established light microscopy techniques: confocal and two-photon fluorescence microscopy. DSLM provides up to 50 times higher imaging speeds and a 10-100 times higher signal-to-noise ratio, while exposing the specimens to at least three orders of magnitude less light energy than confocal and two-photon fluorescence microscopes. We conclude with a perspective for future development.
Keller
Philipp J
PJ
Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany. keller@embl.de
Stelzer
Ernst H K
EH
D000818:Animals; D005333:Fetus / Q000033:anatomy & histology*; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007834:Lasers; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence / Q000379:methods*; D036641:Microscopy, Fluorescence, Multiphoton; D038761:Photobleaching