We are using widefield ratiometric Ca2+ imaging, high-resolution confocal and multiphoton laser scanning microscopy in vitro and in vivo in order to
While widefield ratiometric calcium imaging is a valuable tool for the quantification of spontaneous and evoked Ca2+ transients [1,2], widefield imaging intrinsically suffers from a bad spatial resolution, in particular in thick samples such as brain or spinal cord slices. This draw-back can be overcome by making use of confocal laser-scanning microscopy. Instead of illuminating the entire field of view simultaneously, as in widefield imaging, the image is point-scanned with a laser, and stray light is prevented from reaching the detector. As a result, virtually blur-free images, with a much higher spatial resolution are obtained.
In our lab, we perform both confocal and multiphoton laser-scanning microscopy using a Leica TCS SP5 system, equipped with 6 laser lines from 458 nm to 633 nm, and a tuneable multiphoton laser (Coherent Chameleon Ultra, 705-980 nm).
While confocal microscopy is advantageous for obtaining high-resolution images suitable for co-localization analysis in thin samples, the beauty of multiphoton laser-scanning microscopy is its ability to penetrate deep into the tissue [3]. Additionally, fluorescence excitation, and therefore bleaching, is limited to the focal plane, making multiphoton microscopy particularly well-suited for live-cell imaging in thick slices or in vivo [4].
