Caveat fluorophore: an insiders’ guide to small-molecule fluorescent labels

Nature Methods, Journal Year: 2021, Volume and Issue: 19(2), P. 149 - 158

Published: Dec. 23, 2021

Language: Английский

---

Molecular Fluorophores for Deep-Tissue Bioimaging

ACS Central Science, Journal Year: 2020, Volume and Issue: 6(8), P. 1302 - 1316

Published: July 23, 2020

Fluorescence imaging has made tremendous inroads toward understanding the complexity of biological systems, but in vivo deep-tissue remains a great challenge due to optical opacity tissue. Recent improvements laser and detector manufacturing have allowed expansion nonlinear linear fluorescence underexplored "tissue-transparent" second near-infrared (NIR-II; 1000–1700 nm) window, opening up new opportunities for access deep inside opaque Molecular fluorophores historically played major role bioimaging. It is increasingly important design molecular fully unlock potential NIR-II techniques. In this outlook, we give an overview novel developed bioimaging past five years discuss their pros cons applications. Guidelines designing with desirable properties are also provided.

Language: Английский

---

Pushing the frontiers: tools for monitoring neurotransmitters and neuromodulators

Nature reviews. Neuroscience, Journal Year: 2022, Volume and Issue: 23(5), P. 257 - 274

Published: March 31, 2022

Language: Английский

---

Engineered HaloTag variants for fluorescence lifetime multiplexing

Nature Methods, Journal Year: 2021, Volume and Issue: 19(1), P. 65 - 70

Published: Dec. 16, 2021

Abstract Self-labeling protein tags such as HaloTag are powerful tools that can label fusion proteins with synthetic fluorophores for use in fluorescence microscopy. Here we introduce variants either increased or decreased brightness and lifetime compared HaloTag7 when labeled rhodamines. Combining these enabled live-cell multiplexing of three cellular targets one spectral channel using a single fluorophore the generation lifetime-based biosensor. Additionally, brightest variant showed up to 40% higher imaging applications.

Language: Английский

---

Kinetic and Structural Characterization of the Self-Labeling Protein Tags HaloTag7, SNAP-tag, and CLIP-tag

Biochemistry, Journal Year: 2021, Volume and Issue: 60(33), P. 2560 - 2575

Published: Aug. 2, 2021

The self-labeling protein tags (SLPs) HaloTag7, SNAP-tag, and CLIP-tag allow the covalent labeling of fusion proteins with synthetic molecules for applications in bioimaging biotechnology. To guide selection an SLP–substrate pair provide guidelines design substrates, we report a systematic comparative study kinetics substrate specificities CLIP-tag. HaloTag7 reaches almost diffusion-limited rate constants certain rhodamine which are more than 2 orders magnitude higher those SNAP-tag corresponding substrates. constants, however, less affected by structure label vary over 6 commonly employed Determining crystal structures labeled fluorescent substrates allowed us to rationalize their preferences. We also demonstrate how these insights can be exploited improved kinetics.

Language: Английский

---

A general method for the development of multicolor biosensors with large dynamic ranges

Nature Chemical Biology, Journal Year: 2023, Volume and Issue: 19(9), P. 1147 - 1157

Published: June 8, 2023

Fluorescent biosensors enable the study of cell physiology with spatiotemporal resolution; yet, most suffer from relatively low dynamic ranges. Here, we introduce a family designed Förster resonance energy transfer (FRET) pairs near-quantitative FRET efficiencies based on reversible interaction fluorescent proteins fluorescently labeled HaloTag. These enabled straightforward design for calcium, ATP and NAD+ unprecedented The color each these can be readily tuned by changing either protein or synthetic fluorophore, which enables simultaneous monitoring free in different subcellular compartments following genotoxic stress. Minimal modifications furthermore allow their readout to switched fluorescence intensity, lifetime bioluminescence. thus establish new concept development highly sensitive tunable biosensors.

Language: Английский

---

Fast and sensitive GCaMP calcium indicators for neuronal imaging

The Journal of Physiology, Journal Year: 2023, Volume and Issue: 602(8), P. 1595 - 1604

Published: Feb. 22, 2023

Abstract We review the principles of development and deployment genetically encoded calcium indicators (GECIs) for detection neural activity. Our focus is on popular GCaMP family green GECIs, culminating in recent release jGCaMP8 sensors, with dramatically improved kinetics relative to previous generations. summarize properties GECIs multiple colour channels (blue, cyan, green, yellow, red, far‐red) highlight areas further improvement. With their low‐millisecond rise‐times, allow new classes experiments following activity time frames approaching underlying computations. image

Language: Английский

---

Hybrid Small-Molecule/Protein Fluorescent Probes

Chemical Reviews, Journal Year: 2024, Volume and Issue: 124(10), P. 6198 - 6270

Published: May 8, 2024

Hybrid small-molecule/protein fluorescent probes are powerful tools for visualizing protein localization and function in living cells. These hybrid constructed by diverse site-specific chemical labeling approaches through reactions to exogenous peptide/small tags, enzymatic post-translational modifications, bioorthogonal genetically incorporated unnatural amino acids, ligand-directed reactions. The employed imaging trafficking, conformational changes, bioanalytes surrounding proteins. In addition, facilitate visualization of dynamics at the single-molecule level defined structure with super-resolution imaging. this review, we discuss development bioimaging applications based on hybrids.

Language: Английский

---

Fluorescent and Bioluminescent Calcium Indicators with Tuneable Colors and Affinities

Journal of the American Chemical Society, Journal Year: 2022, Volume and Issue: 144(15), P. 6928 - 6935

Published: April 5, 2022

We introduce a family of bright, rhodamine-based calcium indicators with tuneable affinities and colors. The can be specifically localized to different cellular compartments are compatible both fluorescence bioluminescence readouts through conjugation HaloTag fusion proteins. Importantly, their increase in upon localization enables no-wash live-cell imaging, which greatly facilitates use biological assays. Applications as fluorescent rat hippocampal neurons include the detection single action potentials fluxes endoplasmic reticulum. bioluminescent recording pharmacological modulation nuclear high-throughput versatility remarkable ease these make them powerful tools for bioimaging bioassays.

Language: Английский

---

HaloTag‐Based Reporters for Fluorescence Imaging and Biosensing

ChemBioChem, Journal Year: 2023, Volume and Issue: 24(12)

Published: Feb. 23, 2023

Abstract Visualizing the structure and dynamics of biomolecules is critical to understand biological function, requires methods fluorescently label targets interest in their cellular context. Self‐labelling proteins, which combine a genetically encoded tag with small‐molecule fluorophore, have attracted considerable attention for this purpose, as they can overcome limitations fluorescent proteins. Among them, HaloTag protein most broadly used, showing fast specific labelling small, easy functionalize cell‐permeant ligand. Synthetic chemistry engineering provided portfolio powerful imaging tools exploiting HaloTag, along general optimize adapt them applications. Here, we provide an overview reporters based on biosensing, highlighting strategies

Language: Английский

---