Photoactivation in Fluorescence Microscopy

2009 ◽  
Vol 17 (4) ◽  
pp. 8-13 ◽  
Author(s):  
David W. Piston ◽  
Gert-Jan Kremers ◽  
Richard K.P. Benninger ◽  
Michael W. Davidson
Keyword(s):  
Cell Biology ◽  
Fluorescent Proteins ◽  
Photophysical Properties ◽  
Live Cells ◽  
General Description ◽  
Caged Compounds ◽  
Introductory Overview ◽  
Photoactivatable Gfp ◽  
Genetic Labeling ◽  
New Applications

The use of photoactive compounds in microscopy has a long history. Caged compounds have been used for almost forty years, not only to elicit chemical reactions in cells, but also to mark specific cells or regions within cells by photoactivation of fluorescence. During the last seven years, the advent of photoactivatable GFP (PA-GFP) and its successors has opened up a myriad of new applications. All of this work has, of course, been greatly facilitated in live cells through the possibility of genetic labeling that is given by the fluorescent proteins. However, even as more photo-activatable and photo-switchable proteins are discovered, they are still limited in terms of wavelength ranges and photophysical properties. Thus, there has been a resurgence of interest in small organic photoactive molecules for cell biology experiments. In this short introductory overview, we will present the basic concepts of photoactivation and discuss many of the strengths and limitations of various approaches. We will also provide a general description of the kinds of applications for which these probes can be used.

scholarly journals GABA transporter function, oligomerization state, and anchoring: correlates with subcellularly resolved FRET

2009 ◽  
Vol 134 (6) ◽  
pp. 489-521 ◽  
Author(s):  
Fraser J. Moss ◽  
P.I. Imoukhuede ◽  
Kimberly Scott ◽  
Jia Hu ◽  
Joanna L. Jankowsky ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Fluorescent Proteins ◽  
Resonance Energy ◽  
High Order ◽  
Live Cells ◽  
Gaba Uptake ◽  
Cell Periphery ◽  
Wild Type ◽  
Gaba Transporter ◽  
Amplitude Component

The mouse γ-aminobutyric acid (GABA) transporter mGAT1 was expressed in neuroblastoma 2a cells. 19 mGAT1 designs incorporating fluorescent proteins were functionally characterized by [3H]GABA uptake in assays that responded to several experimental variables, including the mutations and pharmacological manipulation of the cytoskeleton. Oligomerization and subsequent trafficking of mGAT1 were studied in several subcellular regions of live cells using localized fluorescence, acceptor photobleach Förster resonance energy transfer (FRET), and pixel-by-pixel analysis of normalized FRET (NFRET) images. Nine constructs were functionally indistinguishable from wild-type mGAT1 and provided information about normal mGAT1 assembly and trafficking. The remainder had compromised [3H]GABA uptake due to observable oligomerization and/or trafficking deficits; the data help to determine regions of mGAT1 sequence involved in these processes. Acceptor photobleach FRET detected mGAT1 oligomerization, but richer information was obtained from analyzing the distribution of all-pixel NFRET amplitudes. We also analyzed such distributions restricted to cellular subregions. Distributions were fit to either two or three Gaussian components. Two of the components, present for all mGAT1 constructs that oligomerized, may represent dimers and high-order oligomers (probably tetramers), respectively. Only wild-type functioning constructs displayed three components; the additional component apparently had the highest mean NFRET amplitude. Near the cell periphery, wild-type functioning constructs displayed the highest NFRET. In this subregion, the highest NFRET component represented ∼30% of all pixels, similar to the percentage of mGAT1 from the acutely recycling pool resident in the plasma membrane in the basal state. Blocking the mGAT1 C terminus postsynaptic density 95/discs large/zona occludens 1 (PDZ)-interacting domain abolished the highest amplitude component from the NFRET distributions. Disrupting the actin cytoskeleton in cells expressing wild-type functioning transporters moved the highest amplitude component from the cell periphery to perinuclear regions. Thus, pixel-by-pixel NFRET analysis resolved three distinct forms of GAT1: dimers, high-order oligomers, and transporters associated via PDZ-mediated interactions with the actin cytoskeleton and/or with the exocyst.

Illuminating subcellular structures and dynamics in plants: a fluorescent protein toolboxThis review is one of a selection of papers published in the Special Issue on Plant Cell Biology.

2006 ◽  
Vol 84 (4) ◽  
pp. 515-522 ◽  
Author(s):  
Preetinder K. Dhanoa ◽  
Alison M. Sinclair ◽  
Robert T. Mullen ◽  
Jaideep Mathur
Keyword(s):  
Plant Cell ◽  
Cell Biology ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Live Imaging ◽  
Living Cells ◽  
Special Issue ◽  
Exciting Possibility ◽  
Selection Of

The discovery and development of multicoloured fluorescent proteins has led to the exciting possibility of observing a remarkable array of subcellular structures and dynamics in living cells. This minireview highlights a number of the more common fluorescent protein probes in plants and is a testimonial to the fact that the plant cell has not lagged behind during the live-imaging revolution and is ready for even more in-depth exploration.

scholarly journals tdLanYFP, a yellow, bright, photostable and pH insensitive fluorescent protein for live cell imaging and FRET-based sensing strategies

2021 ◽  
Author(s):  
Y. Bousmah ◽  
H. Valenta ◽  
G. Bertolin ◽  
U. Singh ◽  
V. Nicolas ◽  
...  
Keyword(s):  
Single Molecule ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Resonance Energy ◽  
Live Cell ◽  
Live Cells

AbstractYellow fluorescent proteins (YFP) are widely used as optical reporters in Förster Resonance Energy Transfer (FRET) based biosensors. Although great improvements have been done, the sensitivity of the biosensors is still limited by the low photostability and the poor fluorescence performances of YFPs at acidic pHs. In fact, today, there is no yellow variant derived from the EYFP with a pK1/2 below ∼5.5. Here, we characterize a new yellow fluorescent protein, tdLanYFP, derived from the tetrameric protein from the cephalochordate B. lanceolatum, LanYFP. With a quantum yield of 0.92 and an extinction coefficient of 133 000 mol−1.L.cm−1, it is, to our knowledge, the brightest dimeric fluorescent protein available, and brighter than most of the monomeric YFPs. Contrasting with EYFP and its derivatives, tdLanYFP has a very high photostability in vitro and preserves this property in live cells. As a consequence, tdLanYFP allows the imaging of cellular structures with sub-diffraction resolution with STED nanoscopy. We also demonstrate that the combination of high brightness and strong photostability is compatible with the use of spectro-microscopies in single molecule regimes. Its very low pK1/2 of 3.9 makes tdLanYFP an excellent tag even at acidic pHs. Finally, we show that tdLanYFP can be a FRET partner either as donor or acceptor in different biosensing modalities. Altogether, these assets make tdLanYFPa very attractive yellow fluorescent protein for long-term or single-molecule live-cell imaging that is also suitable for FRET experiment including at acidic pH.

scholarly journals Poly(catecholamine) Coating and Biofunctionalization of CsPbBr3 Microcrystals

2021 ◽  
Author(s):  
Sangyeon Cho ◽  
Seok-Hyun Yun
Keyword(s):  
Lipid Bilayers ◽  
Environmental Stability ◽  
Live Cells ◽  
Functional Coatings ◽  
Common Strategy ◽  
Novel Method ◽  
Halide Perovskites ◽  
The Stability ◽  
New Applications

<p>Lead halide perovskites (LHP) microcrystals are promising materials for various optoelectronic applications. Surface coating on particles is a common strategy to improve their functionality and environmental stability, but LHP is not amenable to most coating chemistries because of its intrinsic weakness against polar solvents. Here, we describe a novel method of synthesizing LHP microcrystals in a super-saturated polar solvent using sonochemistry and applying various functional coatings on individual microcrystals <i>in situ</i>. We synthesize cesium lead bromine perovskite (CsPbBr<sub>3</sub>) microparticles capped with organic poly-norepinephrine (pNE) layers. The catechol group of pNE coordinates to bromine-deficient lead atoms, forming a defect-passivating and diffusion-blocking shell. The pNE layer enhances the stability of CsPbBr<sub>3</sub> in water by 2,000-folds, enabling bright luminescence and lasing from single microcrystals in water. Furthermore, the pNE shell permits biofunctionalization with proteins, small molecules, and lipid bilayers. Luminescence from CsPbBr<sub>3</sub> microcrystals is sustained in water over 1 hour and observed in live cells. The functionalization method may enable new applications of LHP particles in water-rich environments.<b></b></p>

scholarly journals Differential equation methods for simulation of GFP kinetics in non–steady state experiments

2018 ◽  
Vol 29 (6) ◽  
pp. 763-771 ◽  
Author(s):  
Robert D. Phair
Keyword(s):  
Differential Equation ◽  
Steady State ◽  
Fluorescence Microscopy ◽  
Cell Biology ◽  
Fluorescent Proteins ◽  
Quantitative Information ◽  
Tracer Kinetics ◽  
Mathematical Framework ◽  
Experimental Protocols ◽  
Tracer Kinetic

Genetically encoded fluorescent proteins, combined with fluorescence microscopy, are widely used in cell biology to collect kinetic data on intracellular trafficking. Methods for extraction of quantitative information from these data are based on the mathematics of diffusion and tracer kinetics. Current methods, although useful and powerful, depend on the assumption that the cellular system being studied is in a steady state, that is, the assumption that all the molecular concentrations and fluxes are constant for the duration of the experiment. Here, we derive new tracer kinetic analytical methods for non–steady state biological systems by constructing mechanistic nonlinear differential equation models of the underlying cell biological processes and linking them to a separate set of differential equations governing the kinetics of the fluorescent tracer. Linking the two sets of equations is based on a new application of the fundamental tracer principle of indistinguishability and, unlike current methods, supports correct dependence of tracer kinetics on cellular dynamics. This approach thus provides a general mathematical framework for applications of GFP fluorescence microscopy (including photobleaching [FRAP, FLIP] and photoactivation to frequently encountered experimental protocols involving physiological or pharmacological perturbations (e.g., growth factors, neurotransmitters, acute knockouts, inhibitors, hormones, cytokines, and metabolites) that initiate mechanistically informative intracellular transients. When a new steady state is achieved, these methods automatically reduce to classical steady state tracer kinetic analysis.

scholarly journals Using Fluorescent Proteins to Monitor Glucagon Granules in Live Cells

2020 ◽  
Vol 118 (3) ◽  
pp. 402a
Author(s):  
Alessandro Ustione ◽  
Priya Mathur ◽  
David W. Piston
Keyword(s):  
Fluorescent Proteins ◽  
Live Cells

scholarly journals Intracellular Imaging with Genetically Encoded RNA-based Molecular Sensors

Nanomaterials ◽  
2019 ◽  
Vol 9 (2) ◽  
pp. 233 ◽  
Author(s):  
Zhining Sun ◽  
Tony Nguyen ◽  
Kathleen McAuliffe ◽  
Mingxu You
Keyword(s):  
Cell Biology ◽  
Design Principle ◽  
Fluorescent Proteins ◽  
Rna Recognition ◽  
Molecular Sensors ◽  
Intracellular Imaging ◽  
Biological Targets ◽  
Target Analytes ◽  
Wide Range ◽  
Reporting Systems

Genetically encodable sensors have been widely used in the detection of intracellular molecules ranging from metal ions and metabolites to nucleic acids and proteins. These biosensors are capable of monitoring in real-time the cellular levels, locations, and cell-to-cell variations of the target compounds in living systems. Traditionally, the majority of these sensors have been developed based on fluorescent proteins. As an exciting alternative, genetically encoded RNA-based molecular sensors (GERMS) have emerged over the past few years for the intracellular imaging and detection of various biological targets. In view of their ability for the general detection of a wide range of target analytes, and the modular and simple design principle, GERMS are becoming a popular choice for intracellular analysis. In this review, we summarize different design principles of GERMS based on various RNA recognition modules, transducer modules, and reporting systems. Some recent advances in the application of GERMS for intracellular imaging are also discussed. With further improvement in biostability, sensitivity, and robustness, GERMS can potentially be widely used in cell biology and biotechnology.

scholarly journals Dissecting pigment architecture of individual photosynthetic antenna complexes in solution

2015 ◽  
Vol 112 (45) ◽  
pp. 13880-13885 ◽  
Author(s):  
Quan Wang ◽  
W. E. Moerner
Keyword(s):  
Single Molecule ◽  
Light Harvesting ◽  
Fluorescent Proteins ◽  
Protein Complexes ◽  
Photophysical Properties ◽  
Critical Role ◽  
Photosynthetic Pigment ◽  
Emergent Properties ◽  
Aqueous Environment ◽  
Harvesting Systems

Oligomerization plays a critical role in shaping the light-harvesting properties of many photosynthetic pigment−protein complexes, but a detailed understanding of this process at the level of individual pigments is still lacking. To study the effects of oligomerization, we designed a single-molecule approach to probe the photophysical properties of individual pigment sites as a function of protein assembly state. Our method, based on the principles of anti-Brownian electrokinetic trapping of single fluorescent proteins, step-wise photobleaching, and multiparameter spectroscopy, allows pigment-specific spectroscopic information on single multipigment antennae to be recorded in a nonperturbative aqueous environment with unprecedented detail. We focus on the monomer-to-trimer transformation of allophycocyanin (APC), an important antenna protein in cyanobacteria. Our data reveal that the two chemically identical pigments in APC have different roles. One (α) is the functional pigment that red-shifts its spectral properties upon trimer formation, whereas the other (β) is a “protective” pigment that persistently quenches the excited state of α in the prefunctional, monomer state of the protein. These results show how subtleties in pigment organization give rise to functionally important aspects of energy transfer and photoprotection in antenna complexes. The method developed here should find immediate application in understanding the emergent properties of other natural and artificial light-harvesting systems.

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