Abstract
Significance:Recent advances in nonlinear optics in neuroscience have focused on using two ultrafast lasers for activity imaging and optogenetic stimulation. Broadband femtosecond light sources can obviate the need for multiple lasers by spectral separation for chromatically targeted excitation.Aim:We present a photonic crystal fiber (PCF)-based supercontinuum source for spectrally resolved two-photon (2P) imaging and excitation of GCaMP6s and C1V1-mCherry, respectively.Approach:A PCF is pumped using a 20-MHz repetition rate femtosecond laser to generate a supercontinuum of light, which is spectrally separated, compressed, and recombined to image GCaMP6s (930 nm excitation) and stimulate the optogenetic protein, C1V1-mCherry (1060 nm excitation). Galvanometric spiral scanning is employed on a single-cell level for multiphoton excitation and high-speed resonant scanning is employed for imaging of calcium activity.Results:Continuous wave lasers were used to verify functionality of optogenetic activation followed by directed 2P excitation. Results from these experiments demonstrate the utility of a supercontinuum light source for simultaneous, single-cell excitation and calcium imaging.Conclusions:A PCF-based supercontinuum light source was employed for simultaneous imaging and excitation of calcium dynamics in brain tissue. Pumped PCFs can serve as powerful light sources for imaging and activation of neural activity, and overcome the limited spectra and space associated with multilaser approaches.
Highlights
The use of multiple optical tools for imaging and excitation of neurons and neural tissue has grown dramatically in recent years
We developed a multiband 2P excitation system based on supercontinuum generation for simultaneous 2P calcium imaging and 2P optogenetic excitation of neural tissue
We present a single-source approach for imaging and optogenetic control of neural activity using supercontinuum generation
Summary
The use of multiple optical tools for imaging and excitation of neurons and neural tissue has grown dramatically in recent years. With a large palette of optogenetic proteins, calcium indicators, voltage indicators, and other custom-tailored indicators to monitor and induce neural activity[8,9,10,11,12,13] comes the added need of multiple laser sources for bidirectional probing of neural tissue This becomes even more striking with the ubiquitous use of ultrafast laser sources, which are needed to induce 2P absorption events, and elicit responses from individual neurons and brain regions due to the reduced out-of-focus excitation.[3,14,15,16,17,18,19] the reduced out-of-focus excitation, which can be exploited with high spatiotemporal resolution for single-cell interrogation of neural circuits, provides an added level of control for brain research that is otherwise not possible with continuous wave (CW) illumination. The broad spectrum of fluorophores of interest to neuroscientists requires spectral tuning to realize the versatility that comes with this diversity
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