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

Frontiers in Cellular Neuroscience

Gabrielle eThériault¹  Martin eCottet¹  Annie eCastonguay¹  Nathalie eMcCarthy²  Yves eDe Koninck² 

[1] Centre de recherche de l'Institut universitaire en santé mentale de Québec;Université Laval;

关键词: Cellular Imaging;    3D imaging;    temporal resolution;    depth of field;    nonlinear microscopy;    axicon;   

DOI  :  10.3389/fncel.2014.00139

来源: DOAJ

【 摘 要 】

Two-photon microscopy has revolutionized functional cellular imaging in tissue, but although the highly confined depth of field of standard set-ups yields great optical sectioning, it also limits imaging speed 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 depth of field 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, theses 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.

【 授权许可】

Unknown