Fast Volumetric Imaging in Two-Photon Microscopy and Enhanced Background Rejection using an Acoustic Lens

Abstract

Two-photon excitation (2PE) microscopy has been adopted as the de facto standard for imaging thick or scattering biological samples at high resolution and at depths up to several hundreds of microns [1]. However, as the study of complex biological phenomena in fields as important as neuroscience demands for higher temporal resolutions over ever increasing penetration depths, 2PE faces two important challenges. First, the point scanning nature of 2PE seriously limits three-dimensional (3D) imaging speed. Second, in thick tissues or turbid media, out-of-focus background generated by 2PE scattered photons can degrade image quality and resolution by loss of signal to background (S/B) [2].Here, we present a method that enables enhanced S/B 2PE microscopy and/or volumetric imaging at high-rates. Our approach relies on coupling an acoustic optofluidic lens [3] into a standard 2PE setup. The lens enables phase modulation of the incident beam at microsecond time scales, leading to ultrafast beam shaping on a pixel by pixel basis. By using high-speed readout [4], the detected photons can be sorted according to the corresponding phase induced by the lens. When a parabolic phase profile is deployed, high-speed axial focus shifting occurs and continuous volumetric imaging is achieved. For other phase profiles, controlled aberrations can be induced just on the neighboring focal planes, and out-of-focus fluorescence can be removed through a subtractive algorithm. We demonstrate our approach by imaging a 150 um thick mouse brain slice and by comparing our results with traditional 2PE microscopy.[1] Diaspro, Wiley, 978-0-471-40920-5 (2001).[2] Helmchen et al., Nature Methods 2, 932 - 940 (2005).[3] Duocastella et al., J. Biomed. Opt. 17(5), 050505 (2012).[4] Duocastella et al., Opt. Express 22, 19293-19301 (2014).