Fluorescent proteins for live-cell imaging with super-resolution

Abstract

Fluorescent proteins (FPs) from the GFP family have become indispensable as marker tools for imaging live cells, tissues and entire organisms. A wide variety of these proteins have been isolated from natural sources and engineered to optimize their properties as genetically encoded markers. Here we review recent developments in this field. A special focus is placed on photoactivatable FPs, for which the fluorescence emission can be controlled by light irradiation at specific wavelengths. They enable regional optical marking in pulse-chase experiments on live cells and tissues, and they are essential marker tools for live-cell optical imaging with super-resolution. Photoconvertible FPs, which can be activated irreversibly via a photo-induced chemical reaction that either turns on their emission or changes their emission wavelength, are excellent markers for localization-based super-resolution microscopy (e.g., PALM). Patterned illumination microscopy (e.g., RESOLFT), however, requires markers that can be reversibly photoactivated many times. Photoswitchable FPs can be toggled repeatedly between a fluorescent and a non-fluorescent state by means of a light-induced chromophore isomerization coupled to a protonation reaction. We discuss the mechanistic origins of the effect and illustrate how photoswitchable FPs are employed in RESOLFT imaging. For this purpose, special FP variants with low switching fatigue have been introduced in recent years. Despite nearly two decades of FP engineering by many laboratories, there is still room for further improvement of these important markers for live cell imaging.

Image detection of yeast Saccharomyces cerevisiae by light-addressable potentiometric sensors (LAPS)

“…Optical microscopy, especially fluorescence microscopy, is most widely performed in live-cell imaging for the study of the physiological state of cells, cellular transport or cell growth [1]. The development of super-resolved fluorescence microscopy has brought optical microscopy into the nanodimension bypassing Abbe's diffraction limit and has enabled the visualisation of the pathways of individual molecules such as proteins inside living cells with the help of fluorescent molecules [2,3].…”

Characterization and development of photoactivatable fluorescent proteins for single-molecule–based superresolution imaging

“…Iteration of this process allows numerous fluorescent labels to be localized and an image with sub-diffraction-limit resolution to be reconstructed from the fluorophore localizations. Fluorescent proteins that can be activated from dark to fluorescent or converted from one color to another are widely used for such imaging approaches (4,5). Although photoactivatable fluorescent proteins (PAFPs) are generally dimmer than photoswitchable dyes (6,7) and hence give lower image resolution, the ease and high specificity of labeling protein targets in living cells with fluorescent proteins makes PAFPs highly appealing probes for imaging the dynamics of cellular structures (4,8).…”

Reproducibility in biological models of the blood-brain barrier

“…Development of live-cell imaging techniques (19, 49) and fluorescent probes (50,51) has enabled the study of dynamic events in a time resolved manner. These live cell imaging techniques include laser scanning-and spinning disc confocal microscopy, widefield fluorescence microscopy, total internal reflection fluorescence microscopy (TIRFM) (for monitoring events that take place on or near the basolateral side of plasma membrane) (52-54), Förster resonance energy transfer (for the measurement of intracellular molecular interactions) (55), fluorescence life-time imaging microscopy (for visualizing the life-time of a molecule's excitation state) (55).…”