Abstract
High-speed (video-rate) fluorescence lifetime imaging (FLIM) is reported using two different time-domain approaches based on gated optical image intensifier technology. The first approach utilizes a rapidly switchable variable delay generator with sequential image acquisition, while the second employs a novel segmented gated optical imager to acquire lifetime maps in a single shot. Lifetimes are fitted using both a non-linear least-squares fit analysis and the rapid lifetime determination method. Monte Carlo simulations were used to optimize the acquisition parameters and a comparison between theory and experiment is presented. The importance of single-shot imaging to minimize the deleterious impact of sample movements is highlighted. Real-time FLIM movies of multi-well plate samples and tissue autofluorescence are presented.
Highlights
DEUTSCHE PHYSIKALISCHE GESELLSCHAFT autofluorescence [17]
We present here a novel single-shot time-domain wide-field fluorescence lifetime imaging (FLIM) system that simultaneously acquires four time-gated images using a segmented gated optical imager (SGOI)
For any high-speed FLIM instrument, it is important to understand the trade-offs between the image acquisition rate and the achievable accuracy of the fitted lifetime values and we present here a study of how the parameters for our FLIM instrument affect the accuracy of the fitted lifetimes
Summary
The basis of the instrumentation used in wide-field time-domain FLIM is described in detail elsewhere (see e.g. [11]). For each pixel in the series of time-gated images, a lifetime value can be computed using either an iterative fitting algorithm, such as a weighted non-linear least-squares (WNLLS) algorithm or an analytical method, such as the RLD algorithm The RLD algorithm is attractive for high-speed FLIM since the acquisition time is shorter (if only two time-gated images are used) and it is non-iterative. We discuss here several factors that can affect the accuracy of a lifetime as computed using this algorithm, including the width of the gates used to record each image and their separation [19]. We investigate the influence of the relative intensity of both time-gated images for the first time, to our knowledge, and we point out that this provides a means to further optimize high-speed FLIM
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