Where Do We Stand with Super-Resolution Optical Microscopy?

Edited by Wilhelm JustThe recent development of fluorescence microscopy approaches overcoming the diffraction limit of light microscopy opened possibilities for studying small-scale cellular processes. The spatial resolution achieved by these novel techniques, together with the possibility to perform live-cell and multicolor imaging, make them ideally suited for visualization of native viruses and subviral structures within the complex environment of a host cell or organ, thus providing fundamentally new possibilities for investigating virus-cell interactions. Here, we review the use of super-resolution microscopy approaches to study virus-cell interactions, and discuss recent insights into human immunodeficiency virus biology obtained by exploiting these novel techniques.

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“… in this issue. Regarding technical developments in SRFM and CLEM, we refer the reader to several excellent recent reviews .…”

Section: Super‐resolution Fluorescence Microscopy: a Novel Tool In VImentioning

confidence: 99%

Super‐resolved insights into human immunodeficiency virus biology

et al. 2016

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“…In parallel, a new set of FPs termed phototransformable FPs (PTFPs) have become the focus of intensive research due to many potential applications in advanced microscopy and biotechnology [5,9]. The fluorescent state (on/off) or emission colour (e.g., green/red) of PTFPs can be precisely controlled by light, forming the core principle of several super resolution microscopy techniques such as PALM (“photo activated localization microscopy”) or RESOLFT (“reversible saturable optical fluorescence transitions”) [10]. In this context, mechanistic investigations of PTFPs have recently become the subject of focused investigation so as to rationally design variants with enhanced photophysical properties [7,11,12,13,14,15,16,17].…”

Section: Introductionmentioning

confidence: 99%

Deciphering Structural Photophysics of Fluorescent Proteins by Kinetic Crystallography

Bourgeois

2017

IJMS

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Because they enable labeling of biological samples in a genetically-encoded manner, Fluorescent Proteins (FPs) have revolutionized life sciences. Photo-transformable fluorescent proteins (PTFPs), in particular, recently attracted wide interest, as their fluorescence state can be actively modulated by light, a property central to the emergence of super-resolution microscopy. PTFPs, however, exhibit highly complex photophysical behaviours that are still poorly understood, hampering the rational engineering of variants with improved performances. We show that kinetic crystallography combined with in crystallo optical spectroscopy, modeling approaches and single-molecule measurements constitutes a powerful tool to decipher processes such as photoactivation, photoconversion, photoswitching, photoblinking and photobleaching. Besides potential applications for the design of enhanced PTFPs, these investigations provide fundamental insight into photoactivated protein dynamics.

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“…Briefly, the proteins are labeled with corresponding antibodies with different excitation spectra, allowing them to be visualized in different colors, and colocalization is assessed by the overlap between the different colored signals in the registered composite image (Zinchuk and Grossenbacher-Zinchuk, 2014). However, diffraction-limited imaging techniques are capable of achieving only 200- to 300-nm spatial lateral resolution and 500–700 nm in the z -axis (Nienhaus and Nienhaus, 2016). Thus what is assessed as colocalization using such techniques could reflect a range of possibilities, from proteins directly interacting via chemical bonds to proteins located within 300 nm of each other (Figure 1; MacDonald et al.…”

Section: Introductionmentioning

confidence: 99%