Abstract
The use of wavefront shaping to generate extended optical excitation patterns which are confined to a predetermined volume has become commonplace on various microscopy applications. For multiphoton excitation, three-dimensional confinement can be achieved by combining the technique of temporal focusing of ultra-short pulses with different approaches for lateral light shaping, including computer generated holography or generalized phase contrast. Here we present a theoretical and experimental study on the effect of scattering on the propagation of holographic beams with and without temporal focusing. Results from fixed and acute cortical slices show that temporally focused spatial patterns are extremely robust against the effects of scattering and this permits their three-dimensionally confined excitation for depths more than 500 µm. Finally we prove the efficiency of using temporally focused holographic beams in two-photon stimulation of neurons expressing the red-shifted optogenetic channel C1V1.
Highlights
With the advent of computer generated holography (CGH), arbitrary optical excitation patterns are generated routinely for many applications, including optical trapping, photostimulation, and optical lithography [1,2,3,4].In CGH, excitation patterns are formed by placing a spatial light modulator at the Fourier conjugate plane to the imaging plane, and applying an appropriate phase mask to obtain an excitation intensity pattern as close as possible to the desired one
We have recently shown that, alternatively to adaptive optics (AO) or Bessel beams, efficient in-depth generation of extended patterns can be reached by combining temporal focusing (TF) either with low-numerical aperture (NA) Gaussian beams or beams generated with generalized phase contrast (GPC) [23,24]
We show that CGH generated patterns are very robust to scattering, and that the combination with TF enhances axial confinement up to several scattering lengths deep into the excited sample
Summary
With the advent of computer generated holography (CGH), arbitrary optical excitation patterns are generated routinely for many applications, including optical trapping, photostimulation, and optical lithography [1,2,3,4]. In CGH, excitation patterns are formed by placing a spatial light modulator at the Fourier conjugate plane to the imaging plane, and applying an appropriate phase mask to obtain an excitation intensity pattern as close as possible to the desired one. For optical trapping or multisite uncaging, 2D and 3D holographic generation of near-diffraction-limited spots [4,7,8,9,10] have been used. For applications in photostimulation or lithography the use of holographic extended pattern is preferred as it permits to quickly cover large excitation areas [3,11]. When combined with temporal focusing (TF), this can be achieved with micrometric axial resolution [11,12]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
