Improving FRET Dynamic Range with Bright Green and Red Fluorescent Proteins

Lam, Amy; St-Pierre, François; Gong, Yiyang; Marshall, Jesse D.; McKeown, M.; Schnitzer, Mark J.; Tsien, Roger Y.; Lin, Michael Z.

doi:10.1016/j.bpj.2012.11.3773

Cited by 173 publications

(245 citation statements)

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“…Each FP also has a characteristic maturation period (i.e., the time it takes for the newly synthesized FP polypeptide to fold into its mature fluorescent conformation). Although rapid maturation is beneficial for most applications (the dLanYFP derivative mNeonGreen requires <1 min whereas GFP requires~25 min; Shaner et al 2013), slow-folding RFPs requiring several hours (Baird et al 2000) support lineage analysis of organelle inheritance over successive rounds of cell division (Grallert et al 2004;Lam et al 2012). Maximum projections from 21 z-plane images of cam1-gfp (left panels), cam1-yfp (center panels), and cam1-mCherry (right panels) cells.…”

Section: Fluorescent Proteinsmentioning

confidence: 99%

Live Cell Imaging in Fission Yeast

Mulvihill

2017

Cold Spring Harb Protoc

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Live cell imaging complements the array of biochemical and molecular genetic approaches to provide a comprehensive insight into functional dependencies and molecular interactions in fission yeast. Fluorescent proteins and vital dyes reveal dynamic changes in the spatial distribution of organelles and the proteome and how each alters in response to changes in environmental and genetic composition. This introduction discusses key issues and basic image analysis for live cell imaging of fission yeast.

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Section: Fluorescent Proteinsmentioning

confidence: 99%

Live Cell Imaging in Fission Yeast

Mulvihill

2017

Cold Spring Harb Protoc

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“…Also, many laboratories have utilized a GFP-red-FP (RFP) pair, which originates from Aequorea victoria (jellyfish) and Discosoma sp. (sea anemone), respectively, for retrovirus/lentivirus-mediated generation of stable cell lines (29). Another method is to utilize a transposon-mediated gene transfer system, e.g., the Piggybac system, in which the biosensor construct is designed to be sandwiched between transposon-specific inverted terminal repeat sequences (30,31).…”

Section: Generation Of Stable Cell Lines and Transgenic Mice Expressimentioning

confidence: 99%

Future Perspective of Single-Molecule FRET Biosensors and Intravital FRET Microscopy

Hirata

Kiyokawa

2016

Biophysical Journal

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Fö rster (or fluorescence) resonance energy transfer (FRET) is a nonradiative energy transfer process between two fluorophores located in close proximity to each other. To date, a variety of biosensors based on the principle of FRET have been developed to monitor the activity of kinases, proteases, GTPases or lipid concentration in living cells. In addition, generation of biosensors that can monitor physical stresses such as mechanical power, heat, or electric/magnetic fields is also expected based on recent discoveries on the effects of these stressors on cell behavior. These biosensors can now be stably expressed in cells and mice by transposon technologies. In addition, two-photon excitation microscopy can be used to detect the activities or concentrations of bioactive molecules in vivo. In the future, more sophisticated techniques for image acquisition and quantitative analysis will be needed to obtain more precise FRET signals in spatiotemporal dimensions. Improvement of tissue/organ position fixation methods for mouse imaging is the first step toward effective image acquisition. Progress in the development of fluorescent proteins that can be excited with longer wavelength should be applied to FRET biosensors to obtain deeper structures. The development of computational programs that can separately quantify signals from single cells embedded in complicated three-dimensional environments is also expected. Along with the progress in these methodologies, two-photon excitation intravital FRET microscopy will be a powerful and valuable tool for the comprehensive understanding of biomedical phenomena.Since the discovery of green fluorescent protein (GFP) (1), scientists have been widely employing this gene-encoded fluorescent protein (FP) to investigate the spatiotemporal dynamics of molecules with subcellular resolution. One of the applications of FP engineering is the development of biosensors based on the principle of Förster (or fluorescence) resonance energy transfer (FRET) (2). FRET is a nonradiative energy transfer process between two fluorophores located in close proximity to each other, and its efficiency is strongly dependent on the distance between the fluorophores. More precisely, the energy transfer rate (k t ) from a donor to an acceptor fluorophore is calculated in the following Förster's equation;where J is the overlap of donor emission and acceptor excitation spectra, k 2 is an orientation factor of transition moment (a factor determined by the relative orientation of the two fluorophores), n is a refractive index, R is the distance between the donor and acceptor fluorophores, and k f is the donor fluorescence emission speed. Therefore, FRET efficiency is determined by the distance and orientation of the two fluorophores. To measure FRET efficiency, there are roughly two methods; ratiometric analysis and lifetime analysis. Ratiometric analysis utilizes fluorescence intensity of an acceptor (e.g., yellow FP (YFP)) and a donor (e.g., cyan FP (CFP)) upon the donor excitation. Because accept...

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“…Generally, the excitation and emission filters should have the largest overlap with each spectrum of the fluorophore to acquire a bright image while the dichroic mirror blocks any excitation light transmitted through the emission filter. The selection of filters and dichroic mirrors for the FRET pair 11 based GEVI recording follows the same principle except that it necessitates a second filter cube in the image splitter for concurrent observation of donor and acceptor fluorescence. The first filter cube placed in the microscope filter box needs to have an excitation filter (475 nm/23) for Clover.…”

Section: Equipment Setupmentioning

confidence: 99%

“…The first class of GEVI uses the voltage-sensing domain from the voltage-sensing phosphatase with either a single fluorescent protein (FP) [5][6][7][8][9] or a Förster resonance energy transfer (FRET) pair [10][11][12] . The second class of sensors uses microbial rhodopsin as a fluorescent indicator directly [13][14][15] or via electrochromic FRET 16,17 .…”

Section: Introductionmentioning

confidence: 99%

Imaging Membrane Potential with Two Types of Genetically Encoded Fluorescent Voltage Sensors

Lee

Piao

Sepheri-Rad

et al. 2016

JoVE

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Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate goal is to image neuronal activity in vivo, one must be able to image activity of a single cell to ensure successful in vivo preparations. This procedure will describe how to image membrane potential in a single cell to provide a foundation to eventually image in vivo. Here we describe methods for imaging GEVIs consisting of a voltage-sensing domain fused to either a single fluorescent protein (FP) or two fluorescent proteins capable of Förster resonance energy transfer (FRET) in vitro. Using an image splitter enables the projection of images created by two different wavelengths onto the same charge-coupled device (CCD) camera simultaneously. The image splitter positions a second filter cube in the light path. This second filter cube consists of a dichroic and two emission filters to separate the donor and acceptor fluorescent wavelengths depending on the FPs of the GEVI. This setup enables the simultaneous recording of both the acceptor and donor fluorescent partners while the membrane potential is manipulated via whole cell patch clamp configuration. When using a GEVI consisting of a single FP, the second filter cube can be removed allowing the mirrors in the image splitter to project a single image onto the CCD camera. Video LinkThe video component of this article can be found at

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Improving FRET Dynamic Range with Bright Green and Red Fluorescent Proteins

Cited by 173 publications

References 0 publications

Live Cell Imaging in Fission Yeast

Live Cell Imaging in Fission Yeast

Future Perspective of Single-Molecule FRET Biosensors and Intravital FRET Microscopy

Imaging Membrane Potential with Two Types of Genetically Encoded Fluorescent Voltage Sensors

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