Multiphoton Imaging of Neurons in Living Tissue: Acquisition and Analysis of Time-Lapse Morphological Data

Abstract

Multiphoton laser scanning microscopy offers improved axial resolution over confocal microscopy for three-dimensional (3D) imaging of fluorescently labeled cells. It also greatly reduces phototoxicity and photobleaching outside the plane of focus when collecting 3D series of optical sections in living tissue. This permits images to be collected at shorter intervals with less harm to the specimen. To facilitate the transition from confocal to two-photon in vivo imaging, we have compared some of the most commonly used fluorescent vital dyes for their applicability to confocal and two-photon laser scanning microscopy. We find that many of the dyes commonly used for morphological imaging can be used for two-photon excitation microscopy. However, the optimal wavelengths for two-photon excitation cannot always be deduced from the one-photon fluorescence spectra of the fluorophores. As improvements in imaging technology are beginning to make near-real-time in vivo fluorescence imaging more feasible, flexible software that can perform temporal as well as spatial analyses of anatomical data becomes increasingly important. Here we demonstrate how we have used Object–Image, an extension of the popular public-domain NIH Image software, together with a set of custom macros for time-lapse morphometric analysis, to analyze neuronal branch growth and complexity in three dimensions over time.