Extended two-photon microscopy in live samples with Bessel beams: steadier focus, faster volume scans, and simpler stereoscopic imaging

Abstract

Two-photon microscopy has revolutionized functional cellular imaging in tissue, but although the highly confined depth of field (DOF) of standard set-ups yields great optical sectioning, it also limits imaging speed in volume samples and ease of use. For this reason, we recently presented a simple and retrofittable modification to the two-photon laser-scanning microscope which extends the DOF through the use of an axicon (conical lens). Here we demonstrate three significant benefits of this technique using biological samples commonly employed in the field of neuroscience. First, we use a sample of neurons grown in culture and move it along the z-axis, showing that a more stable focus is achieved without compromise on transverse resolution. Second, we monitor 3D population dynamics in an acute slice of live mouse cortex, demonstrating that faster volumetric scans can be conducted. Third, we acquire a stereoscopic image of neurons and their dendrites in a fixed sample of mouse cortex, using only two scans instead of the complete stack and calculations required by standard systems. Taken together, these advantages, combined with the ease of integration into pre-existing systems, make the extended depth-of-field imaging based on Bessel beams a strong asset for the field of microscopy and life sciences in general.

Simultaneous Multi-plane Imaging of Neural Circuits

“…Basic knowledge of the underlying physical structure of the neural circuit and sparsity of its activity are used to define constraints for the recovery of the underlying signal, and allow for higher fidelity reconstruction and increased imaging speed in complex samples. With our multiplexed SLM approach we determine the number of areas simultaneously illuminated and have direct control over the effective number of sources, in contrast to alternate extended two-photon approaches, such as Bessel beam scanning (Botcherby et al, 2006; Theriault et al, 2014), where the sample alone controls the complexity of the signal.…”

Fast volumetric calcium imaging across multiple cortical layers using sculpted light

“…Since the inception of two-photon microscopy, there have been a number of efforts to increase the imaging field-of-view (FOV), speed and the obtainable acquisition depth 11,12 . These improvements include extended depth of field excitation 13 , remote axial scanning strategies 14 and the use of electro-tunable (ETLs) 15 or ultrasound lenses 16,17 for fast focusing. Other fast sequential imaging strategies are based on acousto-optic deflectors (AODs) and only excite and detect from a limited set of diffraction limited points on neurons at high speed within a relatively large volume of tissue (~1 mm 3 ).…”

Three-photon fluorescence microscopy with an axially elongated Bessel focus

“…Several approaches have been demonstrated for generating a Bessel beam under two-photon excitation. The most common approach involves the use of an axicon (conical lens) [8, 9, 10]. Other approaches employed a phase mask [11], or alternatively a spatial light modulator (SLM) [12], with the latter allowing for great flexibility in generating different types of Bessel foci with shaped axial intensity profiles.…”