Fluorescence has long been recognized as a powerful tool for probing biological structure and function. Nearly sixty-five years ago, Coons and colleagues introduced the first fluorescent-antibody techniques as a means of detecting pneumococcal antigens in mouse tissue.1 In the years that followed, studies have been conducted in hundreds of cellular contexts using immunofluorescence and related techniques such as fluorescence in situ hybridization.2 Together, these studies have provided cell biologists with a unique glimpse into the molecular organization of cells and tissues. However, because such approaches typically require fixation prior to detection, they provide only a snapshot of the highly dynamic cellular processes that govern cell physiology at the molecular level. To add a crucial temporal component to the study of cell biology, researchers have developed a diverse set of genetically-encodable biosensors designed to probe dynamic cellular events in living cells with high spatial and temporal resolution. These sensors, which generally involve the incorporation of a fluorescent tag into the primary amino acid sequence of a protein or a selected protein domain, have enabled researchers to track various components of intracellular signaling networks in real time within the native cellular environment. In this review, we will first explore the molecular basis of several genetically-targetable fluorescent tags amenable to live cell imaging. We will then turn our attention to the development and use of fluorescent biosensors for studying dynamic signaling processes in living cells.

/pdf/genetically-encodable-fluorescent-biosensors-for-tracking-o92nh672yk.pdf

Genetically-encodable Fluorescent Biosensors for Tracking Signaling Dynamics in Living Cells

Analysis of single molecules in living cells has provided quantitative insights into the kinetics of fundamental biological processes; however, the dynamics of messenger RNA (mRNA) translation have yet to be addressed. We have developed a fluorescence microscopy technique that reports on the first translation events of individual mRNA molecules. This allowed us to examine the spatiotemporal regulation of translation during normal growth and stress and during Drosophila oocyte development. We have shown that mRNAs are not translated in the nucleus but translate within minutes after export, that sequestration within P-bodies regulates translation, and that oskar mRNA is not translated until it reaches the posterior pole of the oocyte. This methodology provides a framework for studying initiation of protein synthesis on single mRNAs in living cells.

https://science.sciencemag.org/content/sci/347/6228/1367.full.pdf

An RNA biosensor for imaging the first round of translation from single cells to living animals

Despite its short history, diffraction-unlimited fluorescence microscopy techniques have already made a substantial imprint in the biological sciences. In this review, we describe how stimulated emission depletion (STED) imaging originally evolved, how it compares to other optical super-resolution imaging techniques, and what advantages it provides compared to previous golden-standards for biological microscopy, such as diffraction-limited optical microscopy and electron microscopy. We outline the prerequisites for successful STED imaging experiments, emphasizing the equally critical roles of instrumentation, sample preparation, and photophysics, and describe major evolving strategies for how to push the borders of STED imaging even further in life science. Finally, we provide examples of how STED nanoscopy can be applied, within three different fields with particular potential for STED imaging experiments: neuroscience, plasma membrane biophysics, and subcellular clinical diagnostics. In these areas, and...

Stimulated Emission Depletion Microscopy

http://www.einstein.yu.edu/labs/vlad-verkhusha/publications/Morozova%20et%20al%20BJ%202010.pdf

Far-Red Fluorescent Protein Excitable with Red Lasers for Flow Cytometry and Superresolution STED Nanoscopy

For about a decade, superresolution fluorescence microscopy has been advancing steadily, maturing from the proof-of-principle stage to routine application. Of the various techniques, STED (stimulated emission depletion) microscopy was the first to break the diffraction barrier. Today, it is a prominent and versatile form of superresolution light microscopy. STED microscopy has shed a sharper light on numerous topics in cell biology, but also in material sciences. Both disciplines extend into the nanometer range, making detailed studies of structural and functional relationships difficult or even impossible to achieve using diffraction-limited microscopy. With recent advancements like spectral multiplexing or live-cell imaging, STED microscopy makes nanoscale materials and components of the cell accessible for fluorescence-based investigations. With multicolor superresolution imaging, even the interactions between biological and engineered nanostructures can be studied in detail. This review gives an introduction into the working principle of STED microscopy, provides a detailed overview of recent advancements and new techniques implemented for use with STED microscopy and shows how these have been applied in the life sciences and nanotechnologies.

STED microscopy and its applications: new insights into cellular processes on the nanoscale.

STED Nanoscopy in Living Cells Using Fluorogen Activating Proteins

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STED Nanoscopy in Living Cells Using Fluorogen Activating Proteins