Fixation-resistant photoactivatable fluorescent proteins for CLEM

Segala, Maria G. Paez; Sun, Miao; Shtengel, Gleb; Viswanathan, Sarada; Baird, Michelle A.; Macklin, John J.; Patel, Ronak; Allen, John R.; Howe, Elizabeth S.; Piszczek, Grzegorz; Hess, Harald F.; Davidson, Michael W.; Wang, Yalin; Looger, Loren L.

doi:10.1038/nmeth.3225

Cited by 183 publications

(199 citation statements)

References 43 publications

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“…Most PAFPs lost fluorescence or the ability to photoactivation under conditions necessary for optimal ultrastructure fixation for EM. Recently published EosFP variants survive heavy OsO 4 fixation, enabling better correlative PALM and electron microscopy [61].…”

Section: Novel Tags and Approaches For Superresolution Fluorescence Mmentioning

confidence: 99%

Novel uses of fluorescent proteins

Mishin

Belousov

Solntsev

et al. 2015

Current Opinion in Chemical Biology

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The field of genetically encoded fluorescent probes is developing rapidly. New chromophore structures were characterized in proteins of green fluorescent protein (GFP) family. A number of red fluorescent sensors, for example, for pH, Ca(2+) and H2O2, were engineered for multiparameter imaging. Progress in development of microscopy hardware and software together with specially designed FPs pushed superresolution fluorescence microscopy towards fast live-cell imaging. Deeper understanding of FPs structure and photophysics led to further development of imaging techniques. In addition to commonly used GFP-like proteins, unrelated types of FPs on the base of flavin-binding domains, bilirubin-binding domains or biliverdin-binding domains were designed. Their distinct biochemical and photophysical properties opened previously unexplored niches of FP uses such as labeling under anaerobic conditions, deep tissue imaging and even patients' blood analysis.

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Section: Novel Tags and Approaches For Superresolution Fluorescence Mmentioning

confidence: 99%

Novel uses of fluorescent proteins

Mishin

Belousov

Solntsev

et al. 2015

Current Opinion in Chemical Biology

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show abstract

“…For now, our approach has been driven by the need to understand in vacuo FP imaging on a 'systems' level, and the practical implications for integrated imaging using FPs as dual-modality probes. Indeed, it will be interesting in the future to examine the vacuum response of new iterations of FPs that withstand fixation, leading to improved electron contrast whilst maintaining FP activity [26].…”

Section: -25]mentioning

confidence: 99%

Standard fluorescent proteins as dual-modality probes for correlative experiments in an integrated light and electron microscope

et al. 2015

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Integrated light and electron microscopes (ILEMs) will enable a new generation of high-precision correlative imaging experiments. To fully exploit these systems, samples must contain dual-modality probes that highlight the position of macromolecules in the context of cell ultrastructure. We demonstrate that the fluorescent proteins (FPs) GFP (green), YFP (yellow) and mCherry can be used as dualmodality probes for ILEM when preserved using the inresin fluorescence (IRF) technique, which delivers stable active fluorophores in lightly stained, resin-embedded cells and tissues. However, we found that vacuum pressure in the ILEM affects the photophysics of FPs in IRF sections. Here, we show that reducing the vacuum pressure reduces fluorescence intensity of GFP and YFP, which is a consequence of water extraction from the sample and is reversible on recreation of partial pressure with water vapour (but not oxygen or nitrogen gas). We also find that, although fluorescence intensity is reduced at a partial pressure of 200 Pa (created using water vapour), the FP intensity is remarkably stable over time in vacuum and resistant to photobleaching during imaging. We are thus able to define imaging strategies for standard FPs acting as dual-modality probes in a single 'multi-colour' integrated microscope system.

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“…25,29). Most recently, preservation of the fluorescence and photoswitching properties of the FP mEos4 was achieved after 0.5-1% OsO 4 treatment and resin embedding 11 . Cryofixation prevents fluorescence quenching and has been exploited for super-resolution CLEM.…”

Section: Super-resolution Fluorescence Clemmentioning

confidence: 99%

“…Mammalian cells are also used in super-resolution FM followed by overlay with EM, for example with PALM 68 , including the use of the optimized photoswitchable Eos4 protein 11 . With optimized protocols for fluorescence retention in thin sections, including the aforementioned cryotechniques 6,7,10,12 , sample preparation resulting in deformation is no longer needed.…”

Section: Mammalian Cell Culturementioning

confidence: 99%

Correlated light and electron microscopy: ultrastructure lights up!

2015

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Microscopy has gone hand in hand with the study of living systems since van Leeuwenhoek observed living microorganisms and cells in 1674 using his light microscope. A spectrum of dyes and probes now enable the localization of molecules of interest within living cells by fluorescence microscopy. With electron microscopy (EM), cellular ultrastructure has been revealed. Bridging these two modalities, correlated light microscopy and EM (CLEM) opens new avenues. Studies of protein dynamics with fluorescent proteins (FPs), which leave the investigator 'in the dark' concerning cellular context, can be followed by EM examination. Rare events can be preselected at the light microscopy level before EM analysis. Ongoing development-including of dedicated probes, integrated microscopes, large-scale and three-dimensional EM and super-resolution fluorescence microscopy-now paves the way for broad CLEM implementation in biology.

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Fixation-resistant photoactivatable fluorescent proteins for CLEM

Cited by 183 publications

References 43 publications

Novel uses of fluorescent proteins

Novel uses of fluorescent proteins

Standard fluorescent proteins as dual-modality probes for correlative experiments in an integrated light and electron microscope

Correlated light and electron microscopy: ultrastructure lights up!

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