Genetically encodable fluorescent protein markers in advanced optical imaging

Abstract

Optical fluorescence microscopy plays a pivotal role in the exploration of biological structure and dynamics, especially on live specimens. Progress in the field relies, on the one hand, on technical advances in imaging and data processing and, on the other hand, on progress in fluorescent marker technologies. Among these, genetically encodable fluorescent proteins (FPs) are invaluable tools, as they allow facile labeling of live cells, tissues or organisms, as these produce the FP markers all by themselves after introduction of a suitable gene. Here we cover FP markers from the GFP family of proteins as well as tetrapyrrole-binding proteins, which further complement the FP toolbox in important ways. A broad range of FP variants have been endowed, by using protein engineering, with photophysical properties that are essential for specific fluorescence microscopy techniques, notably those offering nanoscale image resolution. We briefly introduce various advanced imaging methods and show how they utilize the distinct properties of the FP markers in exciting imaging applications, with the aim to guide researchers toward the design of powerful imaging experiments that are optimally suited to address their biological questions.

Fluorescent Probes and Quenchers in Studies of Protein Folding and Protein‐Lipid Interactions

“…Early experimental and theoretical studies of GFP have revealed a complex interplay between a relatively simple chromophore formed after specific biosynthesis, and the spatial and dynamic organization of its protein carrier (Scheme 4). [94,[98][99] Today, through mutation and selection, many GFP variants were designed that differ in protein and chromophore structure, intending to tune their absorbance and emission characteristics. Popular variants of more enhanced and stable GFP were produced, such as cyan, and yellow fluorescent proteins (ECFP, EYFP, etc.).…”

“…Early experimental and theoretical studies of GFP have revealed a complex interplay between a relatively simple chromophore formed after specific biosynthesis, and the spatial and dynamic organization of its protein carrier (Scheme 4). [94,98–99] …”

Effect of photoconversion conditions on the spectral and cytotoxic properties of photoconvertible fluorescent polymer markers

“…Other staining approaches include the use of genetically encodable fluorescent proteins. 8 Their signal is sufficiently bright and stable and allows tracking individual cells and molecular parts. However, the gene modification that is required for these proteins’ expression can lead to spontaneous mutation and changes in the properties of cell culture relative to the original, which calls into question the adequacy of data obtained from the transfected culture.…”

A Primer on Luminescence Sensing

“…The synthesis of luminescent nanoparticles [9] can be even more challenging and is driven by advancements in the materials sciences [10] . Fluorescent proteins represent another important type of luminescent reporters that are mainly used for imaging applications [11] …”

“…[10] Fluorescent proteins represent another important type of luminescent reporters that are mainly used for imaging applications. [11] Although nanoparticles are larger in size compared to molecular reporters such as fluorescent dyes or lanthanide complexes, they provide several advantages as luminescent labels and probes. For example, luminescent nanoparticles are typically much brighter and more photostable than fluorescent dyes, which are prone to photobleaching by singlet oxygen and thus not suitable for long-time applications under strong illumination.…”