High-speed, low-photodamage nonlinear imaging using passive pulse splitters.
Pulsed lasers are key elements in nonlinear bioimaging techniques such as two-photon fluorescence excitation (TPE) microscopy. Typically, however, only a percent or less of the laser power available can be delivered to the sample before photoinduced damage becomes excessive. Here we describe a passive pulse splitter that converts each laser pulse into a fixed number of sub-pulses of equal energy. We applied the splitter to TPE imaging of fixed mouse brain slices labeled with GFP and show that, in different power regimes, the splitter can be used either to increase the signal rate more than 100-fold or to reduce the rate of photobleaching by over fourfold. In living specimens, the gains were even greater: a ninefold reduction in photobleaching during in vivo imaging of Caenorhabditis elegans larvae, and a six- to 20-fold decrease in the rate of photodamage during calcium imaging of rat hippocampal brain slices.
Ji
Na
N
Janelia Farm Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn, Virginia 20147, USA. jin@janelia.hhmi.org
Magee
Jeffrey C
JC
Betzig
Eric
E
D000818:Animals; D017173:Caenorhabditis elegans; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured / Q000166:cytology* / Q000528:radiation effects; D004867:Equipment Design; D019544:Equipment Failure Analysis; D007089:Image Enhancement / Q000295:instrumentation* / Q000379:methods; D007834:Lasers; D008027:Light; D008029:Lighting / Q000295:instrumentation* / Q000379:methods; D051379:Mice; D008856:Microscopy, Fluorescence / Q000295:instrumentation* / Q000379:methods; D017711:Nonlinear Dynamics; D051381:Rats