Abstract
Over the past decades, the discovery and development of genetically encoded fluorescent proteins (FPs) has brought a revolution into our ability to study biologic phenomena directly within living matter. First, FPs enabled fluorescence-labeling of a variety of molecules of interest to study their localization, interactions and dynamic behavior at various scales—from cells to whole organisms/animals. Then, rationally engineered FP-based sensors facilitated the measurement of physicochemical parameters of living matter—especially at the intracellular level, such as ion concentration, temperature, viscosity, pressure, etc. In addition, FPs were exploited as inert tracers of the intracellular environment in which they are expressed. This oft-neglected role is made possible by two distinctive features of FPs: (i) the quite null, unspecific interactions of their characteristic β-barrel structure with the molecular components of the cellular environment; and (ii) their compatibility with the use of time-resolved fluorescence-based optical microscopy techniques. This review seeks to highlight the potential of such unique combinations of properties and report on the most significative and original applications (and related advancements of knowledge) produced to date. It is envisioned that the use of FPs as inert tracers of living matter structural organization holds a potential for several lines of further development in the next future, discussed in the last section of the review, which in turn can lead to new breakthroughs in bioimaging.
Highlights
Progress in human science is often marked by improvements in the imaging technologies, i.e., fundamental steps forward in our capability to decipher the world at smaller and smaller spatial scales
fluorescent proteins (FPs) enabled fluorescence-labeling of a variety of molecules of interest to study their localization, interactions and dynamic behavior at various scales—from cells to whole organisms/animals
This oft-neglected role is made possible by two distinctive features of FPs: (i) the quite null, unspecific interactions of their characteristic β-barrel structure with the molecular components of the cellular environment; and (ii) their compatibility with the use of time-resolved fluorescence-based optical microscopy techniques
Summary
Progress in human science (and knowledge) is often marked by improvements in the imaging technologies, i.e., fundamental steps forward in our capability to decipher the world at smaller and smaller spatial scales. Di Rienzo and collaborators, fused together the concept of raster image correlation spectroscopy (RICS, [44,45]) with its potential application to variable time scales [46], and used the imaging-derived mean squared displacement (iMSD, [47]) as quantitative readout to probe GFP diffusion properties in the 3D-cellular environment with no a priori assumptions on the biologic system [48] This approach allowed pushing the temporal resolution of image correlation spectroscopy down to 1 microsecond, being able to measure GFP average displacements well below the diffraction limit [48]. The FRET pair of CFP and the glycine-inserted FP yielded a crowding sensor able to detect the changes in protein concentrations during the cell division or upon cell swelling/shrinking [54]
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