Fluorescence Lifetime Imaging (FLIM) based on Time-Correlated Single Photon Counting (TCSPC) is nowadays a well
established technique that is very often realised as an add-on for confocal laser scanning microscopes. However, the
standard laser scanning technique limits the maximum scan range in these setups to a few millimetre, making it therefore
unsuited for e.g. fluorescence multiplexing in multi well plate based assays or for macroscopic material science studies
on solar cells, wafers and similar material. In order to also realize larger scanning ranges, we have developed a sample
scanning approach based on a xy-cross stage equipped with piezo linear motors. Using online position monitoring, this
approach permits fast acceleration and scanning as well as precise positioning and features scan ranges from 100×100
microns up to 80×80 mm with submicron positioning accuracy. Standard upright and inverse microscope bodies can
easily be equipped with this scanning device. Along with the necessary excitation and detection components "largearea"
FLIM thus becomes possible. We will show new results obtained with a modified MicroTime 100 (PicoQuant
GmbH) illustrating the system capabilities for lifetime based imaging in macroscopic samples such as the improvement
of the fluorescence sensitivity in 2D gel electrophoresis or the possibility to perform lifetime based fluorescence
multiplexing in μ-well plate based assays. Even Two Photon Excitation (TPE) imaging is possible with this widerange
sample scanning approach and first FLIM results on cockroach salivary glands, loaded with a chloride sensitive dye
(MQAE) will be presented.
This paper describes a new platform for quantitative intact proteomics, entitled Cumulative Time-resolved Emission
2-Dimensional Gel Electrophoresis (CuTEDGE). The CuTEDGE technology utilizes differences in fluorescent lifetimes to
subtract the confounding background fluorescence during in-gel detection and quantification of proteins, resulting in a
drastic improvement in both sensitivity and dynamic range compared to existing technology. The platform is primarily
designed for image acquisition in 2-dimensional gel electrophoresis (2-DE), but is also applicable to 1-dimensional gel
electrophoresis (1-DE), and proteins electroblotted to membranes. In a set of proof-of-principle measurements, we have
evaluated the performance of the novel technology using the MicroTime 100 instrument (PicoQuant GmbH) in
conjunction with the CyDye minimal labeling fluorochromes (GE Healthcare, Uppsala, Sweden) to perform differential
gel electrophoresis (DIGE) analyses. The results indicate that the CuTEDGE technology provides an improvement in the
dynamic range and sensitivity of detection of 3 orders of magnitude as compared to current state-of-the-art image
acquisition instrumentation available for 2-DE (Typhoon 9410, GE Healthcare). Given the potential dynamic range of
7-8 orders of magnitude and sensitivities in the attomol range, the described invention represents a technological leap in
detection of low abundance cellular proteins, which is desperately needed in the field of biomarker discovery.
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