Three-dimensional Fluorescence Lifetime Imaging with a Single Plane Illumination Microscope provides an improved signal to noise ratio.

ABSTRACT

We designed a widefield frequency domain Fluorescence Lifetime Imaging Microscopy (FLIM)setup, which is based on a Single Plane Illumination Microscope (SPIM). A SPIM provides an inherent optical sectioning capability and reduces photobleaching compared to conventional widefield and confocal fluorescence microscopes. The lifetime precision of the FLIM was characterized with Rhodamine 6G solutions of different quencher concentrations [KI]. We demonstrate the high spatial resolution of the SPIM-FLIM combination in the intensity domain as well as in the lifetime domain with latex bead samples and multiple recordings of three-dimensional live Madine-Darby Canine Kidney (MDCK) cysts. We estimate that the bleaching rate after 600 images have been recorded is below 5%.