Two-photon microscopy : sequential imaging studies in vivo

Abstract

Microscopists have always desired to look inside various organ tissues to study structure, function and dysfunction of their cellular constituents. In the past, this has frequently required tissue extraction and histological preparation to gain access. Traditional optical microscopy techniques, which use linear (one-photon) absorption processes for contrast generation, are limited to use near the tissue surface (< 80 µm) because at greater depths strong and multiple light scattering blurs the images. Scattering particularly strongly affects signal strength in confocal microscopy, which achieves three-dimensional resolution and optical sectioning with a detection pinhole that rejects all light that appears not to originate from the focus. New optical microscopy techniques have been developed that use nonlinear light-matter interactions to generate signal contrast only within a thin raster-scanned plane. Since its first demonstration over a decade ago, two-photon microscopy has been applied to a variety of imaging tasks and has now become the technique of choice for fluorescence microscopy in thick tissue preparations and in live animals. The gain in resolution over conventional in vivo imaging techniques has been several orders of magnitude. Neuroscientists have used it to measure calcium dynamics deep in brain slices and in live animals, blood flow measurement, neuronal plasticity and to monitor neurodegenerative disease models in brain slices and in live rodents. These types of applications define the most important niche for two-photon microscopy - high-resolution imaging of physiology, morphology and cell-cell interactions in intact tissue. Clearly the biggest advantage of two-photon microscopy is in longitudinal monitoring of rodent models of disease or plasticity over days to weeks. The aim of this article is to discuss some methodological principles, and show some applications of this technique obtained from our laboratory in the area of acute experimental stroke research.