A two-channel four-dimensional image recording and viewing system with automatic drift correction

Four-dimensional image acquisition systems have been described to analyse v arious developmental processes, for example, the Caenorhabditis elegans cel l lineage. A practical problem that is often encountered during recordings is mechanical slippage of the microscope stage, causing the sample to drift out of focus. Furthermore, with the advent of green fluorescent protein (G FP) as an in vivo marker, affordable two-channel imaging systems are needed to correlate gene expression with changes through development. To overcome the mechanical drift a device-independent, software-only solution for the MacOS was devised that can compensate for Z-axis drifts in sample position. The software also allows recording of 4D stacks in two channels. To correc t for drift, a small reference object beside the main object to be recorded is kept in focus using a simple autofocus principle, and this automatic dr ift correction allows for effective 4D recordings. In addition to the Z-axi s drives and the shutters of the microscope, a video camera can be computer controlled to switch between two light levels. Second channel live GFP rec ordings are presently limited by the fact that the high intensity of the bl ue light heats and kills C. elegans embyros quickly. To view and annotate t he stacks a MacOS viewing application was developed.