Unsupervised Fluorescence Lifetime Imaging Microscopy for High Content and High Throughput Screening

Abstract

Proteomics and cellomics clearly benefit from the molecular insights in cellular biochemical events that can be obtained by advanced quantitative microscopy techniques like fluorescence lifetime imaging microscopy and Fö rster resonance energy transfer imaging. The spectroscopic information detected at the molecular level can be combined with cellular morphological estimators, the analysis of cellular localization, and the identification of molecular or cellular subpopulations. This allows the creation of powerful assays to gain a detailed understanding of the molecular mechanisms underlying spatiotemporal cellular responses to chemical and physical stimuli. This work demonstrates that the high content offered by these techniques can be combined with the high throughput levels offered by automation of a fluorescence lifetime imaging microscope setup capable of unsupervised operation and image analysis. Systems and software dedicated to image cytometry for analysis and sorting represent important emerging tools for the field of proteomics, interactomics, and cellomics. These techniques could soon become readily available both to academia and the drug screening community by the application of new allsolid-state technologies that may results in cost-effective turnkey systems. Here the application of this screening technique to the investigation of intracellular ubiquitination levels of ␣-synuclein and its familial mutations that are causative for Parkinson disease is shown. The finding of statistically lower ubiquitination of the mutant ␣-synuclein forms supports a role for this modification in the mechanism of pathological protein aggregation. Molecular & Cellular Proteomics 6:1446 -1454, 2007.Above and beyond the isolation and identification of proteins, the field of proteomics faces the challenges of detecting protein cellular localization and quantifying molecular states such as protein conformations, protein-protein interactions, and post-translational modifications. In the past decade, Fö rster resonance energy transfer (FRET) 1 and fluorescence lifetime imaging microscopy (FLIM) have proven to be instrumental for the quantitative imaging of these biochemical states in single cells (1). Similarly the analysis of different cellular populations (cellomes) will also benefit from these imaging methods. Quantitative multiparametric microscopy is a very young field in which advances in liquid/sample handling robotics and information technology are gradually being integrated into automated microscopes (2, 3). These automated imaging systems merge the high content image information with the high throughput volumes provided by their automation and unsupervised operation.Screening techniques have now reached (ultra)high throughput levels, i.e. they are capable of performing more than 10 5 assays/day in microliter volumes. Such a high throughput is necessary for applications where (bio)chemical libraries are tested, e.g. for drug discovery and interactomics research (4). Although the advance in throughput scale is neces...

Beyond Range: Innovating Fluorescence Microscopy

“…However successful that development was, one key element in allowing the biomedical community to benefit from FLIM systems, to enable the screening technologies we were pioneering [1] and to get closer to the routine application of FLIM, was still missing: replacing the expensive, comparatively fragile, and not very user-friendly, multi-channel plates used in FLIM with better technologies. The basic idea was to find a silicon-based detector capable of demodulating fluorescence signals on a bi-dimensional array of pixels.…”

“…Automated microscopes are used to characterize complex biological processes with high throughput capabilities [1,37]. This enables the screening of large libraries of genes and molecules.…”

Advances in cellular, subcellular, and nanoscale imaging in vitro and in vivo

“…Our group has generated and applied many biosensors that are based on this detection method (19)(20)(21). However, for NIR FRET-based probes and sensors, it is more practical to exploit what happens with the red-shifted acceptor emission.…”

Towards high-throughput FLIM for protein-protein interaction screening of live cells and tissue microarrays

“…Fluorescence lifetime microscopy (FLIM) has been used for some time and systems are available commercially, however, to date, there has only been one example of an automated system that incorporates FLIM [5] and none that use time-domain FLIM via time-correlated single photon counting (TCSPC) [6]. The analysis of mixed populations of fluorophores, some experiencing FRET and some not, results in bi-exponential fluorescence decays making FLIM by single-frequency-domain methods troublesome.…”