Bright cyan fluorescent protein variants identified by fluorescence lifetime screening

Goedhart, Joachim; Weeren, Laura van; Hink, Mark A.; Vischer, Norbert O. E.; Jalink, Kees; Gadella, Theodorus W. J.

doi:10.1038/nmeth.1415

Cited by 256 publications

(259 citation statements)

References 18 publications

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“…This reduces calcium buffering caused by the indicator and potential residual biological activity of the sensing domain. Other improvements may arise from incorporating brighter fluorescent protein variants, such as mCerulean3 45 or mTurquoise 46 , to allow lower expression levels while maintaining the same signal-to-noise levels.…”

Section: Discussionmentioning

confidence: 99%

Biocompatibility of a genetically encoded calcium indicator in a transgenic mouse model

et al. 2012

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Engineering efforts of genetically encoded calcium indicators predominantly focused on enhancing fluorescence changes, but how indicator expression affects the physiology of host organisms is often overlooked. Here, we demonstrate biocompatibility and widespread functional expression of the genetically encoded calcium indicator Tn-XXL in a transgenic mouse model. To validate the model and characterize potential effects of indicator expression we assessed both indicator function and a variety of host parameters, such as anatomy, physiology, behaviour and gene expression profiles in these mice. We also demonstrate the usefulness of primary cells and organ explants prepared from these mice for imaging applications. Although we find mild signatures of indicator expression that may be further reduced in future sensor generations, the 'green' indicator mice generated provide a well-characterized resource of primary cells and tissues for in vitro and in vivo calcium imaging applications.

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Section: Discussionmentioning

confidence: 99%

Biocompatibility of a genetically encoded calcium indicator in a transgenic mouse model

et al. 2012

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“…Based on previous studies demonstrating that wavelengths near 800 nm could be used for relatively selective excitation of CFP over YFP 38,39 , we focused our evaluations of potential FRET acceptors on newer, improved variants of YFP. Based on these studies, we identified monomeric (m)Turquoise 35 and mVenus 40 as optimal FPs for TPLSM. We found that illumination at 810 nm efficiently excited mTurquoise with minimal direct excitation of mVenus 33 .…”

Section: Development Of the Protocolmentioning

confidence: 98%

“…Since the cyan FPs (CFP) are optimally excited close to the power maximum of the titanium-sapphire lasers used in most TPLSM systems, we focused our evaluations of potential FRET donors on the newer variants of the CFPs that have improved brightness and photostability, and no photo-switching behavior [34][35][36][37] . Based on previous studies demonstrating that wavelengths near 800 nm could be used for relatively selective excitation of CFP over YFP 38,39 , we focused our evaluations of potential FRET acceptors on newer, improved variants of YFP.…”

Section: Development Of the Protocolmentioning

confidence: 99%

“…Measurement of spectral crosstalk correction factors. The imaging protocol described here exploits the relatively selective 2PE of mTurquoise 35 over mVenus 40 using illumination at 810 nm, which enables the measurement of FRET ratios from single, two-channel images ( Figure 3). The accurate measurement of FRET efficiencies (EFRET), however, requires the application of correction factors for spectral crosstalk (Box 1).…”

Section: Overview Of the Proceduresmentioning

confidence: 99%

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A simple approach for measuring FRET in fluorescent biosensors using two-photon microscopy

2016

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“…Recently, CFP mutants with a single, long lifetime and increased brightness and photostability (e.g., mTurquoise or mCerulean3) have been developed that are suitable for donor FPs in FRET biosensors, with great potential for in vivo application (48,49). However, there is a paradoxical concern about the application of this pair to two-photon excitation intravital FRET microscopy, mainly due to the proximity of their excitation profiles at relatively short wavelength.…”

Section: Improvement Of Biosensors For Two-photon Intravital Imagingmentioning

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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Bright cyan fluorescent protein variants identified by fluorescence lifetime screening

Cited by 256 publications

References 18 publications

Biocompatibility of a genetically encoded calcium indicator in a transgenic mouse model

Biocompatibility of a genetically encoded calcium indicator in a transgenic mouse model

A simple approach for measuring FRET in fluorescent biosensors using two-photon microscopy

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

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