scholarly journals In vivo mouse and live cell STED microscopy of neuronal actin plasticity using far-red emitting fluorescent proteins

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Waja Wegner ◽  
Peter Ilgen ◽  
Carola Gregor ◽  
Joris van Dort ◽  
Alexander C. Mott ◽  
...  
Keyword(s):  
Fluorescent Proteins ◽  
Live Cell ◽  
Sted Microscopy

scholarly journals Multi-label in vivo STED microscopy by parallelized switching of reversibly switchable fluorescent proteins

Cell Reports ◽  
2021 ◽  
Vol 35 (9) ◽  
pp. 109192
Author(s):  
Katrin I. Willig ◽  
Waja Wegner ◽  
Antonia Müller ◽  
Valérie Calvet-Fournier ◽  
Heinz Steffens
Keyword(s):  
Fluorescent Proteins ◽  
Sted Microscopy

Fluorescent proteins for in vivo imaging, where's the biliverdin?

2020 ◽  
Vol 48 (6) ◽  
pp. 2657-2667
Author(s):  
Felipe Montecinos-Franjola ◽  
John Y. Lin ◽  
Erik A. Rodriguez
Keyword(s):  
Hydrogen Peroxide ◽  
In Vivo Imaging ◽  
Near Infrared ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Fluorescent Imaging ◽  
Infrared Light ◽  
Red Fluorescent Protein ◽  
Color Palette

Noninvasive fluorescent imaging requires far-red and near-infrared fluorescent proteins for deeper imaging. Near-infrared light penetrates biological tissue with blood vessels due to low absorbance, scattering, and reflection of light and has a greater signal-to-noise due to less autofluorescence. Far-red and near-infrared fluorescent proteins absorb light >600 nm to expand the color palette for imaging multiple biosensors and noninvasive in vivo imaging. The ideal fluorescent proteins are bright, photobleach minimally, express well in the desired cells, do not oligomerize, and generate or incorporate exogenous fluorophores efficiently. Coral-derived red fluorescent proteins require oxygen for fluorophore formation and release two hydrogen peroxide molecules. New fluorescent proteins based on phytochrome and phycobiliproteins use biliverdin IXα as fluorophores, do not require oxygen for maturation to image anaerobic organisms and tumor core, and do not generate hydrogen peroxide. The small Ultra-Red Fluorescent Protein (smURFP) was evolved from a cyanobacterial phycobiliprotein to covalently attach biliverdin as an exogenous fluorophore. The small Ultra-Red Fluorescent Protein is biophysically as bright as the enhanced green fluorescent protein, is exceptionally photostable, used for biosensor development, and visible in living mice. Novel applications of smURFP include in vitro protein diagnostics with attomolar (10−18 M) sensitivity, encapsulation in viral particles, and fluorescent protein nanoparticles. However, the availability of biliverdin limits the fluorescence of biliverdin-attaching fluorescent proteins; hence, extra biliverdin is needed to enhance brightness. New methods for improved biliverdin bioavailability are necessary to develop improved bright far-red and near-infrared fluorescent proteins for noninvasive imaging in vivo.

scholarly journals Real-time observation of tetrapyrrole binding to an engineered bacterial phytochrome

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Yusaku Hontani ◽  
Mikhail Baloban ◽  
Francisco Velazquez Escobar ◽  
Swetta A. Jansen ◽  
Daria M. Shcherbakova ◽  
...  
Keyword(s):  
Real Time ◽  
Rational Design ◽  
Near Infrared ◽  
Fluorescent Proteins ◽  
Covalent Bond ◽  
Binding Pocket ◽  
Tissue Imaging ◽  
Covalent Attachment ◽  
Time Resolved

AbstractNear-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes are widely used for structural and functional deep-tissue imaging in vivo. To fluoresce, NIR FPs covalently bind a chromophore, such as biliverdin IXa tetrapyrrole. The efficiency of biliverdin binding directly affects the fluorescence properties, rendering understanding of its molecular mechanism of major importance. miRFP proteins constitute a family of bright monomeric NIR FPs that comprise a Per-ARNT-Sim (PAS) and cGMP-specific phosphodiesterases - Adenylyl cyclases - FhlA (GAF) domain. Here, we structurally analyze biliverdin binding to miRFPs in real time using time-resolved stimulated Raman spectroscopy and quantum mechanics/molecular mechanics (QM/MM) calculations. Biliverdin undergoes isomerization, localization to its binding pocket, and pyrrolenine nitrogen protonation in <1 min, followed by hydrogen bond rearrangement in ~2 min. The covalent attachment to a cysteine in the GAF domain was detected in 4.3 min and 19 min in miRFP670 and its C20A mutant, respectively. In miRFP670, a second C–S covalent bond formation to a cysteine in the PAS domain occurred in 14 min, providing a rigid tetrapyrrole structure with high brightness. Our findings provide insights for the rational design of NIR FPs and a novel method to assess cofactor binding to light-sensitive proteins.

scholarly journals Measuring fast gene dynamics in single cells with time-lapse luminescence microscopy

2014 ◽  
Vol 25 (22) ◽  
pp. 3699-3708 ◽  
Author(s):  
Anyimilehidi Mazo-Vargas ◽  
Heungwon Park ◽  
Mert Aydin ◽  
Nicolas E. Buchler
Keyword(s):  
Gene Expression ◽  
Fluorescent Proteins ◽  
Single Cells ◽  
Time Lapse ◽  
Photon Flux ◽  
Luminescence Microscopy ◽  
Cell Cycle Genes ◽  
Light Excitation ◽  
Better Than

Time-lapse fluorescence microscopy is an important tool for measuring in vivo gene dynamics in single cells. However, fluorescent proteins are limited by slow chromophore maturation times and the cellular autofluorescence or phototoxicity that arises from light excitation. An alternative is luciferase, an enzyme that emits photons and is active upon folding. The photon flux per luciferase is significantly lower than that for fluorescent proteins. Thus time-lapse luminescence microscopy has been successfully used to track gene dynamics only in larger organisms and for slower processes, for which more total photons can be collected in one exposure. Here we tested green, yellow, and red beetle luciferases and optimized substrate conditions for in vivo luminescence. By combining time-lapse luminescence microscopy with a microfluidic device, we tracked the dynamics of cell cycle genes in single yeast with subminute exposure times over many generations. Our method was faster and in cells with much smaller volumes than previous work. Fluorescence of an optimized reporter (Venus) lagged luminescence by 15–20 min, which is consistent with its known rate of chromophore maturation in yeast. Our work demonstrates that luciferases are better than fluorescent proteins at faithfully tracking the underlying gene expression.

Labeling cellular structures in vivo using confined primed conversion of photoconvertible fluorescent proteins

2016 ◽  
Vol 11 (12) ◽  
pp. 2419-2431 ◽  
Author(s):  
Manuel Alexander Mohr ◽  
Paul Argast ◽  
Periklis Pantazis
Keyword(s):  
Fluorescent Proteins ◽  
Cellular Structures

Dermal Fibroblasts from Chronic Wounds Exhibit Paradoxically Enhanced Proliferative and Migratory Activities that May be Related to the Non-Canonical Wnt Signaling Pathway

2021 ◽  
Vol 39 ◽  
Author(s):  
Marta Vinas ◽  
◽  
Xiaofeng Lin ◽  
Susan MacLauchlan ◽  
Polly Carson ◽  
...  
Keyword(s):  
Chronic Wounds ◽  
Chronic Wound ◽  
Wnt Signaling Pathway ◽  
High Energy ◽  
Dermal Fibroblasts ◽  
Live Cell ◽  
Mrna Levels ◽  
Venous Leg Ulcer ◽  
Recurrence Rates

It is generally thought that dermal fibroblasts from chronic wounds are in a state of senescence, which contributes to the failure to heal. This assumption, based on limited experimental evidence, has led to the widespread use of therapeutic approaches focused on delivering new fibroblasts and/or increasing resident fibroblast activity to promote healing. In this study, we decided to re-visit the evidence for the relative inactivity of resident chronic wound fibroblasts. We therefore evaluated the proliferative and migratory activities of matching, patient-derived dermal fibroblasts from a chronic wound (wound dermal fibroblasts, or WDF), ipsilateral thigh newly created acute wound dermal fibroblasts (ADF, Day-3 after wounding the normal thigh skin), and ipsilateral thigh normal dermal skin fibroblasts (NDF). This approach was used in each of 10 consecutive non-selected individual patients with a venous leg ulcer, and allowed us to determine whether WDF are intrinsically less active than NDF and AWD. Cell migration and proliferation were quantified by a live-cell analysis system and MTT assay, respectively, in low (0.5%) or high (10%) levels of fetal bovine serum (FBS). In addition, the ability of patient-derived fibroblasts to modulate wound re-epithelialization in vivo was analyzed by transplantation in a mouse tail full-thickness wound model. Wnt5a mRNA, its ROR1 co-receptors, and ROR2 mRNA levels were determined by qRT-PCR. We report that WDF had increased -SMA and increased levels of Wnt5a. Moreover, using live-cell imaging in a scratch assay monolayer model, WDF showed baseline migratory activity similar to those of NDF and ADF, and such activity was not stimulated by FBS. WDF showed the same capacity to increase wound re-epithelialization as NDF and ADF. Together, these results suggest that WDF are not actually less "active" than NDF and ADF. This enhanced activity of chronic wound fibroblasts may lead to high energy requirements that contribute to a failure to heal. The findings may represent a new paradigm for wound chronicity, impaired healing, and high recurrence rates.

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