Abstract
Fluorescence lifetime imaging (FLIM) is increasingly used to read out cellular autofluorescence originating from the coenzyme NADH in the context of investigating cell metabolic state. We present here an automated multiwell plate reading FLIM microscope optimized for UV illumination with the goal of extending high content fluorescence lifetime assays to readouts of metabolism. We demonstrate its application to automated cellular autofluorescence lifetime imaging and discuss the key practical issues associated with its implementation. In particular, we illustrate its capability to read out the NADH-lifetime response of cells to metabolic modulators, thereby illustrating the potential of the instrument for cytotoxicity studies, assays for drug discovery and stratified medicine.
Highlights
The benets of automated high content assays are well established for drug discovery and are increasingly recognized for systems biology and basic research in the life sciences
In contrast to multiphoton microscopy of NADH, which is inherently optically sectioned, wide- ̄eld single photon excitation measurements of NADH auto°uorescence lifetime are prone to background °uorescence, which might arise from UV excitation of °uorescence in the microscope optics, sample holders and culture media
We have reported the development and demonstration of what we believe to be therst automated multiwell plate reading FLIM microscope for application to auto°uorescence lifetime imaging under wide- ̄eld UV illumination
Summary
The benets of automated high content assays are well established for drug discovery and are increasingly recognized for systems biology and basic research in the life sciences. The capacity to increase the number of cells that can practically be imaged and the number of \repeats" per condition in a single \experiment" mitigates for biological noise, with the ability to average readouts over large datasets enabling quantitative measurements of challenging assays such as protein–protein interactions to become statistically robust. To this end, high content analysis (HCA) has evolved from simple automated intensity-based imaging to utilize spectroscopic image-based readouts of sample arrays, harnessing the molecular specicity of °uorescence labeling. This paper concerns the emerging application of °uorescence lifetime imaging (FLIM) to HCA, which has previously been used to provide high content readouts of F€orster resonance energy transfer (FRET) and o®ers the potential to screen interactions of proteins labeled with exogenous, genetically encoded donor °uorescent fusion proteins.[1,2,3] FLIM can be used to gain insight into cellular processes via the auto°uorescence lifetimes of endogenous cellular °uorophores and we report here the translation of such readouts to an automated FLIM plate reader
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