Abstract
Fluorescent proteins are promising toolsfor studying intracellular signaling processes in lymphocytes. This brief review summarizes fluorescence-based imaging techniques developed in recent years and discusses new methodological advances, such as fluorescent photoswitches, fluorescence recovery after photobleaching (FRAP), fluorescent resonance energy transfer (FRET), fluorescence lifetime imaging microscopy (FLIM), photoactivated localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), stimulated emission depletion (STED), total internal reflection fluorescence (TIRF) and other techiques. This survey also highlights recent advances in vitro imaging of live tissues, novel applications of flow cytometry with genetically modifed fluorescent proteins, and future prospects for the development of new immunological test systems based on fluorescent protein technology.
Highlights
The immune cell activation is a complex and multistage process
This review discusses novel non-invasive research approaches based on fluorescent proteins that are applicable to the study of different stages of lymphocyte activation
Fluorescent proteins are well suited for studies of intermolecular interactions that occur in intact living cells and tissues that otherwise would have been destroyed during fixation and staining
Summary
The history of genetically encoded fluorescent proteins (FP) began with the discovery of green. The main disadvantage of methods based on synthetic dyes and quantum labels concerns the delivery of the label into the cell Since these probes are used as synthetic conjugates with specific antibodies or, for example, receptor proteins, they cannot be genetically encoded and their delive ry through the plasma membrane into the cytosol and other intracellular compartments of living cells is technically very difficult. The activation alarm reaches chromatin at the expense of transport of signaling molecules to the cell nucleus and provides persistent changes of gene expression of adhesion proteins, receptors, cytokines and chemokines. The methods using genetically encoded fluorescent proteins that allow multicolor labeling, minimal impact on the studied processes and the possibility of using in vivo, are promising to study the activation of lymphocytes [18,19,20,21,22]
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