Automated focusing of nuclei for time lapse experiments on single cells using holographic optical tweezers

Abstract

Experiments on single cells are currently gaining more and more interest. Single cell studies often concerns the spatio-temporal distribution of fluorescent proteins inside living cells, visualized using fluorescence microscopy. In order to extract quantitative information from such experiments it is necessary to image the sample with high spatial and temporal resolution while keeping the photobleaching to a minimum. The analysis of the spatial distribution of proteins often requires stacks of images at each time point, which exposes the sample to unnecessary amounts of excitation light. In this paper we show how holographic optical tweezers combined with image analysis can be used to optimize the axial position of trapped cells in an array in order to bring the nuclei into a single imaging plane, thus eliminating the need for stacks of images and consequently reducing photobleaching. This allows more images to be collected, as well as increasing the time span and/or the time resolution in time lapse studies of single cells.