Abstract
We use an ultrafast diode-pumped semiconductor disk laser (SDL) to demonstrate several applications in multiphoton microscopy. The ultrafast SDL is based on an optically pumped Vertical External Cavity Surface Emitting Laser (VECSEL) passively mode-locked with a semiconductor saturable absorber mirror (SESAM) and generates 170-fs pulses at a center wavelength of 1027 nm with a repetition rate of 1.63 GHz. We demonstrate the suitability of this laser for structural and functional multiphoton in vivo imaging in both Drosophila larvae and mice for a variety of fluorophores (including mKate2, tdTomato, Texas Red, OGB-1, and R-CaMP1.07) and for endogenous second-harmonic generation in muscle cell sarcomeres. We can demonstrate equivalent signal levels compared to a standard 80-MHz Ti:Sapphire laser when we increase the average power by a factor of 4.5 as predicted by theory. In addition, we compare the bleaching properties of both laser systems in fixed Drosophila larvae and find similar bleaching kinetics despite the large difference in pulse repetition rates. Our results highlight the great potential of ultrafast diode-pumped SDLs for creating a cost-efficient and compact alternative light source compared to standard Ti:Sapphire lasers for multiphoton imaging.
Highlights
Over the past decades, multiphoton microscopy (MPM) methods have been widely adopted in biomedical research owing to their combination of high spatial resolution, intrinsic optical sectioning and the ability to perform deep imaging in scattering samples [1, 2]
Multiphoton imaging at high laser repetition rates As noted by Girkin and Wokosin [43], due to the short gain carrier lifetime in semiconductors passively mode-locked semiconductor disk laser (SDL) typically operate at repetition rates in the gigahertz domain compared to the approximately 80-MHz repetition rate provided by Ti:Sapphire lasers
These results demonstrate that two-photon calcium imaging in the superficial layers of mouse neocortex at 100-350 μm depths, which is a major type of experiments in current neuroscience, is feasible with an ultrafast SDL
Summary
Multiphoton microscopy (MPM) methods have been widely adopted in biomedical research owing to their combination of high spatial resolution, intrinsic optical sectioning and the ability to perform deep imaging in scattering samples [1, 2]. 2. Multiphoton imaging at high laser repetition rates As noted by Girkin and Wokosin [43], due to the short gain carrier lifetime in semiconductors passively mode-locked SDLs typically operate at repetition rates in the gigahertz domain compared to the approximately 80-MHz repetition rate provided by Ti:Sapphire lasers. The use of GHz sources based on Ti:Sapphire as a gain medium has been demonstrated in only a small number of studies for SHG microscopy [49] and two-photon microscopy [50,51,52], likely due to the high complexity and cost of these lasers. For parallel fluorescence imaging with a red and green channel (e.g. Figure 11), a filter set consisting of a Qioptic DC-Green dichroic and two emission filters (Semrock Brightline Basic 535/22 and Chroma ET605/70M) were used
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