An Efficient Aequorea victoria Green Fluorescent Protein for Stimulated Emission Depletion Super-Resolution Microscopy

Storti, Barbara; Carlotti, Benedetta; Chiellini, Grazia; Ruglioni, Martina; Salvadori, Tiziano; Scotto, Marco; Elisei, Fausto; Diaspro, Alberto; Bianchini, Paolo; Bizzarri, Ranieri

doi:10.3390/ijms23052482

Cited by 2 publications

(2 citation statements)

References 52 publications

(76 reference statements)

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“…Raster-scanning the sample allows the sequential registration of fluorescence from only those dyes that are effectively excited at each spatial location [ 26 ]. STED may reach a lateral resolution of 30–50 nm, albeit the strong laser intensity requires specific fluorophores [ 27 ]. Lower depletion intensities can be obtained by exploiting fluorophore lifetime [ 28 ] or through a modified version called reversible saturable optical fluorescence transitions (RESOLFT), which requires reversibly photoswitching fluorophores [ 29 ].…”

Section: Super-resolution Microscopy (Srm)mentioning

confidence: 99%

“…While STED has been utilized to image live samples [ 33 , 34 , 35 ], it is usually applied to image fixed samples. This is mostly due to the complex genetic encoding of fluorophores which comply with the strong depletion intensities of STED (particularly in conventional continuous-wave STED) [ 27 ]. The need for specific fluorophores also reduces the spectral multiplexing of STED.…”

Section: Super-resolution Microscopy (Srm)mentioning

confidence: 99%

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On the Advent of Super-Resolution Microscopy in the Realm of Polycomb Proteins

Nepita

Piazza

Ruglioni

et al. 2023

Biology

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The genomes of metazoans are organized at multiple spatial scales, ranging from the double helix of DNA to whole chromosomes. The intermediate genomic scale of kilobases to megabases, which corresponds to the 50–300 nm spatial scale, is particularly interesting, as the 3D arrangement of chromatin is implicated in multiple regulatory mechanisms. In this context, polycomb group (PcG) proteins stand as major epigenetic modulators of chromatin function, acting prevalently as repressors of gene transcription by combining chemical modifications of target histones with physical crosslinking of distal genomic regions and phase separation. The recent development of super-resolution microscopy (SRM) has strongly contributed to improving our comprehension of several aspects of nano-/mesoscale (10–200 nm) chromatin domains. Here, we review the current state-of-the-art SRM applied to PcG proteins, showing that the application of SRM to PcG activity and organization is still quite limited and mainly focused on the 3D assembly of PcG-controlled genomic loci. In this context, SRM approaches have mostly been applied to multilabel fluorescence in situ hybridization (FISH). However, SRM data have complemented the maps obtained from chromosome capture experiments and have opened a new window to observe how 3D chromatin topology is modulated by PcGs.

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Section: Super-resolution Microscopy (Srm)mentioning

confidence: 99%

Section: Super-resolution Microscopy (Srm)mentioning

confidence: 99%

On the Advent of Super-Resolution Microscopy in the Realm of Polycomb Proteins

Nepita

Piazza

Ruglioni

et al. 2023

Biology

Self Cite

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Reversibly switchable fluorescent proteins: “the fair switch project”

Nifosì,

Storti,

Bizzarri

2024

Riv. Nuovo Cim.

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Fluorescent proteins (FPs) have transformed cell biology through their use in fluorescence microscopy, enabling precise labeling of proteins via genetic fusion. A key advancement is altering primary sequences to customize their photophysical properties for specific imaging needs. A particularly notable family of engineered mutants is constituted by Reversible Switching Fluorescent Proteins (RSFPs), i.e. variant whose optical properties can be toggled between a bright and a dark state, thereby adding a further dimension to microscopy imaging. RSFPs have strongly contributed to the super-resolution (nanoscopy) revolution of optical imaging that has occurred in the last 20 years and afforded new knowledge of cell biochemistry at the nanoscale. Beyond high-resolution applications, the flexibility of RSFPs has been exploited to apply these proteins to other non-conventional imaging schemes such as photochromic fluorescence resonance energy transfer (FRET). In this work, we explore the origins and development of photochromic behaviors in FPs and examine the intricate relationships between structure and photoswitching ability. We also discuss a simple mathematical model that accounts for the observed photoswitching kinetics. Although we review most RSFPs developed over the past two decades, our main goal is to provide a clear understanding of key switching phenotypes and their molecular bases. Indeed, comprehension of photoswitching phenotypes is crucial for selecting the right protein for specific applications, or to further engineer the existing ones. To complete this picture, we highlight in some detail the exciting applications of RSFPs, particularly in the field of super-resolution microscopy.

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An Efficient Aequorea victoria Green Fluorescent Protein for Stimulated Emission Depletion Super-Resolution Microscopy

Cited by 2 publications

References 52 publications

On the Advent of Super-Resolution Microscopy in the Realm of Polycomb Proteins

On the Advent of Super-Resolution Microscopy in the Realm of Polycomb Proteins

Reversibly switchable fluorescent proteins: “the fair switch project”

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