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Front Cell Neurosci. 2014 May 20;8:139. doi: 10.3389/fncel.2014.00139. eCollection 2014.

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

Author information

1
Département de Physique, de Génie Physique et d'Optique, Centre d'Optique, Photonique et Laser, Université Laval Québec, QC, Canada ; Centre de Recherche de l'Institut Universitaire en Santé Mentale de Québec Québec, QC, Canada.
2
Centre de Recherche de l'Institut Universitaire en Santé Mentale de Québec Québec, QC, Canada.
3
Département de Physique, de Génie Physique et d'Optique, Centre d'Optique, Photonique et Laser, Université Laval Québec, QC, Canada ; Centre de Recherche de l'Institut Universitaire en Santé Mentale de Québec Québec, QC, Canada ; Département de Psychiatrie et de Neurosciences, Université Laval Québec, QC, Canada.

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.

KEYWORDS:

3D imaging; axicon; cellular imaging; depth of field; functional calcium imaging; nondiffractive beam; nonlinear microscopy; temporal resolution

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