Red Fluorescent Proteins: Advanced Imaging Applications and Future Design

Shcherbakova, Daria M.; Subach, Fedor V.; Verkhusha, Vladislav V.

doi:10.1002/anie.201200408

Cited by 150 publications

(167 citation statements)

References 129 publications

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“…14,15 In recent years, rsFPs, such as green Dronpa, 16 reversibly switchable enhanced green fluorescent protein, 17 and rsTagRFP, 18,19 have been applied to fluorescence imaging with enhanced spatial resolution in (F)PALM/ STORM, [20][21][22] RESOLFT, 5 and photochromic stochastic optical fluctuation imaging (pcSOFI). 23 They have also been used to enhance weak signals in optical lock-in detection imaging (OLID).…”

Section: Introductionmentioning

confidence: 99%

Reversibly switchable fluorescence microscopy with enhanced resolution and image contrast

Yao

Shcherbakova

et al. 2014

J. Biomed. Opt

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Abstract. Confocal microscopy with optical sectioning has revolutionized biological studies by providing sharper images than conventional optical microscopy. Here, we introduce a fluorescence imaging method with enhanced resolution and imaging contrast, which can be implemented using a commercial confocal microscope setup. This approach, called the reversibly switchable photo-imprint microscopy (rsPIM), is based on the switching dynamics of reversibly switchable fluorophores. When the fluorophores are switched from the bright (ON) state to the dark (OFF) state, their switching rate carries the information about the local excitation light intensity. In rsPIM, a polynomial function is used to fit the fluorescence signal decay during the transition. The extracted high-order coefficient highlights the signal contribution from the center of the excitation volume, and thus sharpens the resolution in all dimensions. In particular, out-of-focus signals are greatly blocked for large targets, and thus the image contrast is considerably enhanced. Notably, since the fluorophores can be cycled between the ON and OFF states, the whole imaging process can be repeated. RsPIM imaging with enhanced image contrast was demonstrated in both fixed and live cells using a reversibly switchable synthetic dye and a genetically encoded red fluorescent protein. Since rsPIM does not require the modification of commercial microscope systems, it may provide a simple and cost-effective solution for subdiffraction imaging of live cells.

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Section: Introductionmentioning

confidence: 99%

Reversibly switchable fluorescence microscopy with enhanced resolution and image contrast

Yao

Shcherbakova

et al. 2014

J. Biomed. Opt

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“…Studies reveal that the mechanism behind this phenomenon is fast excited-state proton transfer (eSPT) [51] through an internal proton wire [52]. eSPT is not common among FPs, but it can be exploited for engineering FPs that display very large apparent Stokes shifts [17,53,54].…”

Section: Chromophore Environment and Fluorescence Emissionmentioning

confidence: 99%

“…For these reasons, the optimal wavelength range for exciting and detecting the emission of fluorescent probes (often referred to as the "optical window") runs from approximately 650 to 1000 nm in biological tissues [13][14][15]. To better use the optical window for fluorescence imaging in vivo, much effort has been invested in engineering FPs with ever more red-shifted wavelengths and pushing both their excitation and emission wavelengths into the near-infrared [16][17][18][19]. an alternative approach is to use near-infrared 2-photon (2P) excitation to excite FPs that fluoresce at visible wavelengths [20,21].…”

Section: Fp Colorsmentioning

confidence: 99%

Fluorescent Proteins for Neuronal Imaging

Zhao

Campbell

2015

Biological and Medical Physics, Biomedical Engineering

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over the past two decades, the growing selection of engineered fluorescent proteins have helped drive a revolution in the ability of researchers to image protein localization and biochemical dynamics in live cells in real time. although the fluorescent proteins were long preceded by other fluorophores compatible with live cell imaging, the fact that fluorescent proteins are fully genetically encoded has enabled them to be applied in applications that would not otherwise be possible. in particular, fluorescent proteins have enabled the creation of transgenic animals in which specific neuronal cell types are uniquely and fluorescently labeled. Furthermore, through the use of highly engineered fluorescent proteins that change their fluorescence in response to a change in calcium ion concentration or membrane potential, fluorescent proteins have enabled high resolution minimally invasive imaging of neuronal activity in model organisms. in this chapter we will provide an overview of fluorescent protein technology and detail the technological developments that have made such experiments possible. Particular emphasis will be placed on the development of strategies for engineering ca 2+ and voltage indicators, and the latest breakthroughs in these directions will be highlighted.

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“…With these properties, it becomes valuable as a fluorescent tag in various cell biology applications. During the past decade many advanced red fluorescent proteins, including far-red proteins with emission wavelength larger than 620 nm have been developed (Shcherbakova et al 2012). …”

Section: Proteins Of Gfp Familymentioning

confidence: 99%

Molecular-Size Fluorescence Emitters

Demchenko

2015

Introduction to Fluorescence Sensing

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A variety of fluorescent and luminescent materials in the form of molecules, their complexes and nanoparticles are available for implementation as response units into sensing and imaging technologies and we discuss their general properties that are essential for these applications. Organic dyes were the first and remain to be the most popular emitters used with these purposes. Their labeling and responsive properties are overviewed. Natural fluorescent proteins and their engineered analogues incorporate organic fluorophores that are synthesized spontaneously from the polypeptide chain inside the living cells without the need of their intrusion, as the gene products. They resulted in revolutionary advancement in cell imaging and show good prospects in sensing and reporting on cellular processes. Organic heterocyclic compounds can incorporate metal ions generating long-living luminescence. Moreover, noble metal particles of a very small size exhibit unique light-absorbing and fluorescent emission properties. In this Chapter we focus on these types of fluorophores that possess subnanometer dimensions and display single type of absorption-emission cycle. The more complex nanostructures and nanocomposites will be overviewed in Chaps. 5 and 6 that follow. Fluorophores and Their Characteristics General Properties of Fluorescent DyesIn view of tremendous diversity of structures, chemical reactivities and spectroscopic properties, one needs certain practically useful criteria for the dye selection for the purpose of sensing and imaging. They can be formulated in the form of spectroscopic parameters that have to be optimized. It is the parameter describing the ability of molecule to absorb light at a particular wavelength. The absorbance E measured at any wavelength on spectrophotometer is proportional to the dye concentration c (in mol/l) and the light path length in a sample l (in cm). In this relation, ε plays the role of coefficient of proportionality, so that E = εcl. The value of molar absorbance is obtained by purifying and drying the dye, dissolving it at low concentration and measuring E on a spectrophotometer. The broadly used term extinction includes absorbance and light scattering. Absorbance is a unique characteristic of a molecule under certain environmental conditions. In general, the bigger the fluorophore size, the greater is the probability that the photon will be absorbed.The value of ε max at the maximum of absorption band is the common characteristic of the light-absorbing power of the dye. The dye 'brightness' that determines the absolute sensitivity of fluorescence detection (Wetzl et al. 2003) is the product of molar absorbance and quantum yield, Φ. The absorbance is related to optical cross-section and for organic dyes, ε max varies from ca. 10 3 l mol −1 cm −1 for small dyes, such as coumarins to ca. 10 5 l mol −1 cm −1 for larger fluorophores such as cyanines and phthalocyanines (Lavis and Raines 2008). It should be selected as high as possible, but the limiting factor is the fluorophore size. Us...

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Red Fluorescent Proteins: Advanced Imaging Applications and Future Design

Cited by 150 publications

References 129 publications

Reversibly switchable fluorescence microscopy with enhanced resolution and image contrast

Reversibly switchable fluorescence microscopy with enhanced resolution and image contrast

Fluorescent Proteins for Neuronal Imaging

Molecular-Size Fluorescence Emitters

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