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Non-invasive intravital imaging of cellular differentiation with a bright red-excitable fluorescent protein.

ABSTRACT: A method for non-invasive visualization of genetically labeled cells in animal disease models with micrometer-level resolution would greatly facilitate development of cell-based therapies. Imaging of fluorescent proteins (FPs) using red excitation light in the 'optical window' above 600 nm is one potential method for visualizing implanted cells. However, previous efforts to engineer FPs with peak excitation beyond 600 nm have resulted in undesirable reductions in brightness. Here we report three new red-excitable monomeric FPs obtained by structure-guided mutagenesis of mNeptune. Two of these, mNeptune2 and mNeptune2.5, demonstrate improved maturation and brighter fluorescence than mNeptune, whereas the third, mCardinal, has a red-shifted excitation spectrum without reduction in brightness. We show that mCardinal can be used to non-invasively and longitudinally visualize the differentiation of myoblasts into myocytes in living mice with high anatomical detail.

SUBMITTER: Chu J

PROVIDER: S-EPMC4008650 | biostudies-literature | 2014 May

REPOSITORIES: biostudies-literature

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Non-invasive intravital imaging of cellular differentiation with a bright red-excitable fluorescent protein.

Chu Jun J Haynes Russell D RD Corbel Stéphane Y SY Li Pengpeng P González-González Emilio E Burg John S JS Ataie Niloufar J NJ Lam Amy J AJ Cranfill Paula J PJ Baird Michelle A MA Davidson Michael W MW Ng Ho-Leung HL Garcia K Christopher KC Contag Christopher H CH Shen Kang K Blau Helen M HM Lin Michael Z MZ

Nature methods 20140316 5

A method for non-invasive visualization of genetically labeled cells in animal disease models with micrometer-level resolution would greatly facilitate development of cell-based therapies. Imaging of fluorescent proteins (FPs) using red excitation light in the 'optical window' above 600 nm is one potential method for visualizing implanted cells. However, previous efforts to engineer FPs with peak excitation beyond 600 nm have resulted in undesirable reductions in brightness. Here we report three ...[more]

PMID: 24633408

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Project description:Here, a simple one-step hydrothermal-assisted carbonization process was adopted for the preparation of nitrogen/phosphorous-doped carbon dots from a water-soluble polymer, poly 2-(methacryloyloxy)ethyl phosphorylcholine (PMPC). By the free-radical polymerization method, PMPC was synthesized using 2-(methacryloyloxy)ethyl phosphorylcholine (MPC) and 4,4'-azobis (4-cyanovaleric acid). The water-soluble polymers, PMPC, that have nitrogen/phosphorus moieties are used to prepare carbon dots (P-CDs). The resulting P-CDs were thoroughly characterized by various analytical techniques such as field emission-scanning electron microscopy (FESEM) with energy-dispersive X-ray spectroscopy (EDS), high-resolution transmittance electron microscopy (HRTEM), X-ray diffraction (XRD), Raman spectroscopy, attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, X-ray photoelectron spectroscopy (XPS), Ultraviolet-visible (UV-vis) spectroscopy and fluorescence spectroscopy to determine their structural and optical properties. The synthesized P-CDs displayed bright/durable fluorescence, were stable for long periods, and confirmed the enrichment of functionalities including oxygen, phosphorus, and nitrogen heteroatoms in the carbon matrix. Since the synthesized P-CDs showed bright fluorescence with excellent photostability, excitation-dependent fluorescence emission, and excellent quantum yield (23%), it has been explored as a fluorescent (security) ink for drawing and writing (anti-counterfeiting). Further, cytotoxicity study results advised for biocompatibility and thus were used for cellular multicolor imaging in nematodes. This work not only demonstrated the preparation of CDs from polymers that can be used as advanced fluorescence ink, a bioimaging agent for anti-counterfeiting, and cellular multicolor imaging candidate, but additionally prominently opened a new perspective on the bulk preparation of CDs simply and efficiently for various applications.

Dataset Information

Non-invasive intravital imaging of cellular differentiation with a bright red-excitable fluorescent protein.

Publications

Non-invasive intravital imaging of cellular differentiation with a bright red-excitable fluorescent protein.

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