Integrated single- and two-photon light sheet microscopy using accelerating beams

Peeter Piksarv,Andreas Stingl,Kishan Dholakia,Melissa R Andrews,Wolfgang Drexler,Tuan Le,Angelika Unterhuber,Peter E Andersen,Dominik Marti,Lindsey H Forbes

doi:10.1038/s41598-017-01543-4

Abstract

We demonstrate the first light sheet microscope using propagation invariant, accelerating Airy beams that operates both in single- and two-photon modes. The use of the Airy beam permits us to develop an ultra compact, high resolution light sheet system without beam scanning. In two-photon mode, an increase in the field of view over the use of a standard Gaussian beam by a factor of six is demonstrated. This implementation for light sheet microscopy opens up new possibilities across a wide range of biomedical applications, especially for the study of neuronal processes.

Highlights

Light sheet fluorescence microscopy (LSFM), often termed selective plane illumination microscopy (SPIM)[1] is finding key applications in neuroscience and developmental biology
To overcome the trade-off between the field of view (FOV) and the axial resolution of the light sheet microscope usually associated with Gaussian beams in the two-photon excitation mode, double-sided illumination[7, 8] and scanning the focus of tightly focused beam[9, 10] have both been used
It can be seen that the point spread functions (PSF) for 20 fs and 200 fs pulsed Airy pulses is nearly identical and the axial resolution characteristics does not depend on the pulse length unless the pulse duration is below ~5 fs

Summary

Introduction

Light sheet fluorescence microscopy (LSFM), often termed selective plane illumination microscopy (SPIM)[1] is finding key applications in neuroscience and developmental biology. Single-photon light sheet microscopy used for studies of neuronal processes, e.g., in zebrafish, requires an extended illumination at a wavelength (488 nm) that lies within the most sensitive region of the fish’s visible spectrum. This in turn may lead to direct stimulation of the blue photoreceptors in the fish retina, amongst other photosensitive cells, if not taken into consideration by careful design of the imaging geometry[2]. Benefiting from near-infrared excitation wavelengths and its “non-diffracting” nature, two-photon Bessel beam light sheet can penetrate up to 500 μm into zebrafish embryo delivering 0.5 μm lateral and 2 μm axial resolution over 600 μm wide FOV18

Methods

Results

Conclusion