Advanced Fluorescence Microscopy Techniques—FRAP, FLIP, FLAP, FRET and FLIM

Abstract

Fluorescence microscopy provides an efficient and unique approach to study fixed and living cells because of its versatility, specificity, and high sensitivity. Fluorescence microscopes can both detect the fluorescence emitted from labeled molecules in biological samples as images or photometric data from which intensities and emission spectra can be deduced. By exploiting the characteristics of fluorescence, various techniques have been developed that enable the visualization and analysis of complex dynamic events in cells, organelles, and sub-organelle components within the biological specimen. The techniques described here are fluorescence recovery after photobleaching (FRAP), the related fluorescence loss in photobleaching (FLIP), fluorescence localization after photobleaching (FLAP), Förster or fluorescence resonance energy transfer (FRET) and the different ways how to measure FRET, such as acceptor bleaching, sensitized emission, polarization anisotropy, and fluorescence lifetime imaging microscopy (FLIM). First, a brief introduction into the mechanisms underlying fluorescence as a physical phenomenon and fluorescence, confocal, and multiphoton microscopy is given. Subsequently, these advanced microscopy techniques are introduced in more detail, with a description of how these techniques are performed, what needs to be considered, and what practical advantages they can bring to cell biological research.

Non-enzymatic assay for glucose by using immobilized whole-cells of E. coli containing glucose binding protein fused to fluorescent proteins

“…Subsequently, the binding of glucose to GBP induces a conformational change which may apparently bring closer both these two fluorescent proteins and therefore there is an increase in FRET efficiency. Several research workers have reported that FRET is apparently a very complex process when free and immobilized whole-cells or cell-free extracts are involved [19][20][21][22]. Regarding the differences in FRET efficiencies between the free and immobilized nanosensors, there are a number of factors affecting immobilized and soluble proteins such as restricted mobility on immobilization, chemical modification due to the immobilization method used, nature of the microenvironment and diffusion limitation [20].…”

High-Contrast Fluorescence Imaging Based on the Polarization Dependence of the Fluorescence Enhancement Using an Optical Interference Mirror Slide

“…1 Chip-based biomolecular analysis methods, such as DNA chips and protein chips, are also based on fluorescence imaging. The quality of fluorescence imaging and the sensitivity of fluorescence detection are generally evaluated by the signal-to-noise (S/N) ratio, which indicates the ratio between the fluorescence signal from a fluorophore and the background light.…”

Cell shape recognition and segmentation in fluorescence microscopy images.

“…The rapid progress of modern life science depends to a large extent on the use of advanced imaging techniques [1,2]. In particular, the design of fluorescent markers that can be genetically encoded and expressed in vivo have enormously contributed to our understanding of subcellular processes.…”