Two-photon excitation in scattering media by spatiotemporally shaped beams and their application in optogenetic stimulation

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.

Keywords: (090.1760) Computer holography; (110.0113) Imaging through turbid media; (230.6120) Spatial light modulators; (290.0290) Scattering.

Fig. 1

Multiphoton excitation pattern generated at the focal plane of the objective by numerical simulation of the propagation of a holographic beam generating a spot of 10 µm, through 200 µm of a turbid medium consisting of 2 µm dielectric spheres randomly distributed, having a refractive index higher by 0.1 than the surrounding medium and an average concentration of 1 sphere per 1000 µm³. This gives rise to a scattering length of ~135 µm, similar to that typically observed in brain tissue [25,31]. The image of the holographic spot (A) not scattered, (B) scattered but not temporally focused and (C) scattered and temporally focused are illustrated. Simulations were performed at 800 nm, with 100 fs laser pulses. The pattern uniformity was about 0.96 for the temporally focused pattern and 0.92 for the non-temporally focused one.

Fig. 2

(A) Top: x-y cross sections of the holographic beam at the objective focal plane, without scattering tissue and after propagating through 50 and 250 µm fixed brain slices, with or without TF in the optical setup; Bottom: corresponding y-z cross sections along the yellow dashed lines. λ = 800 nm. (B) Normalized axial intensity of the y-z cross sections shown in (A) without and with TF implementation. In the absence of scattering the temporally focused cross section is shown. (C) Variation of the FWHM of the axial intensity distribution of the holographic spot in A, in respect to the scattering depth. Black and blue dots correspond to theoretical modeling at 800 and 950 nm respectively for TF-CGH, orange dots correspond to experimental data for TF-CGH, and green dots correspond to experimental data for CGH beams alone. Experimental data is given as average ± STD values from 4 different realizations for each thickness. The gray-scale level in (A) is normalized to the peak intensity of the fluorescence image without scattering.

Fig. 3

(A) x-y and y-z 2P fluorescence cross sections of a 15 μm holographic spot. From left to right: without scattering, after propagation through acute cortical brain slices of 300 µm with TF, 550 µm with TF and 550 μm without TF in the optical setup. (B) Variation of the FWHM of the axial intensity distribution of the temporally focused holographic spot in respect to the scattering depth. Data corresponds to average ± STD values from 6 different realizations for each thickness. λ = 950 nm. The gray-scale level is normalized to the peak intensity of the fluorescence image without scattering.

Fig. 4

Holographic activation of C1V1_TT. (A) Representative whole cell current recorded during a 50 ms pulse at a power of 0.5 mW/µm². (B) Single action potentials could be reliably triggered in cells expressing C1V1TT. Here is one example with 1 ms pulses at 0.5 mW/µm². (C) Example of 10 Hz trains generated with a holographic spot (2 ms pulses every 100 ms, 0.5 mW/µm²). For all panels: λ = 1040 nm, spot diameter 15 µm. Laser power is given after the objective.

Fig. 5

Frequency content of CGH vs. TF-CGH beams after scattering through brain slices of thickness 300, 400 and 550 µm. The frequency content is the fraction of energy in a given frequency.

Fig. 6

The propagation of a large beam diffracted by the grating produces an ultrafast line scanning of the sample (inset). Scattering events off the scanning line at a single moment in time cannot interfere with the ballistic photon in the line.