scholarly journals The Positive Switching RSFP Padron2 Enables Live-Cell RESOLFT Nanoscopy Without Sequential Irradiation Steps

2020 ◽  
Author(s):  
Timo Konen ◽  
Tim Grotjohann ◽  
Isabelle Jansen ◽  
Nickels Jensen ◽  
Stefan W. Hell ◽  
...  
Keyword(s):  
Fluorescent Proteins ◽  
Contrast Ratio ◽  
Light Response ◽  
Live Cell ◽  
Switching Behavior ◽  
Excitation Wavelength ◽  
Beam Scanning ◽  
Fluorescence Excitation ◽  
Green Fluorescent ◽  
Sequential Switching

AbstractReversibly switchable fluorescent proteins (RSFPs) can be repeatedly transferred between a fluorescent on- and a non-fluorescent off-state in response to irradiation with light of different wavelengths. Negative switching RSFPs are switched from the on- to the off-state with the same wavelength which also excites fluorescence. Positive switching RSFPs have a reversed light response where the fluorescence excitation wavelength induces the transition from the off- to the on-state. Reversible saturable optical linear (fluorescence) transitions (RESOLFT) nanoscopy utilizes these switching states to achieve diffraction-unlimited resolution, but so far has primarily relied on negative switching RSFPs by using time sequential switching schemes.Based on the green fluorescent RSFP Padron, we engineered the positive switching RSFP Padron2. Compared to its predecessor, it can undergo 50-fold more switching cycles while displaying a contrast ratio between the on- and the off-state of more than 100:1. Because of its robust switching behavior, Padron2 supports a RESOLFT imaging scheme that entirely refrains from sequential switching as it only requires beam scanning of two spatially overlaid light distributions. Using Padron2, we demonstrate live-cell RESOLFT nanoscopy without sequential irradiation steps.

Structural Evidence of Photoisomerization Pathways in Fluorescent Proteins

2019 ◽  
Author(s):  
Jeffrey Chang ◽  
Matthew Romei ◽  
Steven Boxer
Keyword(s):  
Rational Design ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Super Resolution ◽  
Unit Cell Size ◽  
Chlorine Substituent ◽  
Gfp Chromophore ◽  
The One ◽  
Green Fluorescent ◽  
Super Resolution Microscopy

<p>Double-bond photoisomerization in molecules such as the green fluorescent protein (GFP) chromophore can occur either via a volume-demanding one-bond-flip pathway or via a volume-conserving hula-twist pathway. Understanding the factors that determine the pathway of photoisomerization would inform the rational design of photoswitchable GFPs as improved tools for super-resolution microscopy. In this communication, we reveal the photoisomerization pathway of a photoswitchable GFP, rsEGFP2, by solving crystal structures of <i>cis</i> and <i>trans</i> rsEGFP2 containing a monochlorinated chromophore. The position of the chlorine substituent in the <i>trans</i> state breaks the symmetry of the phenolate ring of the chromophore and allows us to distinguish the two pathways. Surprisingly, we find that the pathway depends on the arrangement of protein monomers within the crystal lattice: in a looser packing, the one-bond-flip occurs, whereas in a tighter packing (7% smaller unit cell size), the hula-twist occurs.</p><p> </p><p> </p><p> </p><p> </p><p> </p><p> </p> <p> </p>

scholarly journals Coherent Dynamics of Photoexcited Green Fluorescent Proteins

2001 ◽  
Vol 86 (15) ◽  
pp. 3439-3442 ◽  
Author(s):  
Riccardo A. G. Cinelli ◽  
Valentina Tozzini ◽  
Vittorio Pellegrini ◽  
Fabio Beltram ◽  
Giulio Cerullo ◽  
...  
Keyword(s):  
Fluorescent Proteins ◽  
Green Fluorescent Proteins ◽  
Coherent Dynamics ◽  
Green Fluorescent

scholarly journals Red-Shifted Aminated Derivatives of GFP Chromophore for Live-Cell Protein Labeling with Lipocalins

2018 ◽  
Vol 19 (12) ◽  
pp. 3778 ◽  
Author(s):  
Nina Bozhanova ◽  
Mikhail Baranov ◽  
Nadezhda Baleeva ◽  
Alexey Gavrikov ◽  
Alexander Mishin
Keyword(s):  
Green Fluorescent Protein ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Fluorescent Protein ◽  
Live Cell ◽  
Protein Labeling ◽  
Cell Protein ◽  
Gfp Chromophore ◽  
Green Fluorescent ◽  
Derivatives Of

Fluorogens are an attractive type of dye for imaging applications, eliminating time-consuming washout steps from staining protocols. With just a handful of reported fluorogen-protein pairs, mostly in the green region of spectra, there is a need for the expansion of their spectral range. Still, the origins of solvatochromic and fluorogenic properties of the chromophores suitable for live-cell imaging are poorly understood. Here we report on the synthesis and labeling applications of novel red-shifted fluorogenic cell-permeable green fluorescent protein (GFP) chromophore analogs.

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