Dual-color fluorescence imaging of tumor/host interaction with green and red fluorescent proteins

2004 ◽  
Author(s):  
Meng Yang ◽  
Yasuyuki Amoh ◽  
Lingna Li ◽  
Eugene Baranov ◽  
Jin Wei Wang ◽  
...  
Keyword(s):  
Fluorescence Imaging ◽  
Fluorescent Proteins ◽  
Host Interaction ◽  
Dual Color ◽  
Red Fluorescent Proteins

scholarly journals LSSmScarlet, dCyRFP2s, dCyOFP2s and CRISPRed2s, Genetically Encoded Red Fluorescent Proteins with a Large Stokes Shift

2021 ◽  
Vol 22 (23) ◽  
pp. 12887
Author(s):  
Oksana M. Subach ◽  
Anna V. Vlaskina ◽  
Yuliya K. Agapova ◽  
Pavel V. Dorovatovskii ◽  
Alena Y. Nikolaeva ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Rational Design ◽  
Fluorescent Proteins ◽  
Stokes Shift ◽  
Confocal Imaging ◽  
Large Stokes Shift ◽  
Two Photon ◽  
Dual Color ◽  
Red Fluorescent Proteins

Genetically encoded red fluorescent proteins with a large Stokes shift (LSSRFPs) can be efficiently co-excited with common green FPs both under single- and two-photon microscopy, thus enabling dual-color imaging using a single laser. Recent progress in protein development resulted in a great variety of novel LSSRFPs; however, the selection of the right LSSRFP for a given application is hampered by the lack of a side-by-side comparison of the LSSRFPs’ performance. In this study, we employed rational design and random mutagenesis to convert conventional bright RFP mScarlet into LSSRFP, called LSSmScarlet, characterized by excitation/emission maxima at 470/598 nm. In addition, we utilized the previously reported LSSRFPs mCyRFP1, CyOFP1, and mCRISPRed as templates for directed molecular evolution to develop their optimized versions, called dCyRFP2s, dCyOFP2s and CRISPRed2s. We performed a quantitative assessment of the developed LSSRFPs and their precursors in vitro on purified proteins and compared their brightness at 488 nm excitation in the mammalian cells. The monomeric LSSmScarlet protein was successfully utilized for the confocal imaging of the structural proteins in live mammalian cells and multicolor confocal imaging in conjugation with other FPs. LSSmScarlet was successfully applied for dual-color two-photon imaging in live mammalian cells. We also solved the X-ray structure of the LSSmScarlet protein at the resolution of 1.4 Å that revealed a hydrogen bond network supporting excited-state proton transfer (ESPT). Quantum mechanics/molecular mechanics molecular dynamic simulations confirmed the ESPT mechanism of a large Stokes shift. Structure-guided mutagenesis revealed the role of R198 residue in ESPT that allowed us to generate a variant with improved pH stability. Finally, we showed that LSSmScarlet protein is not appropriate for STED microscopy as a consequence of LSSRed-to-Red photoconversion with high-power 775 nm depletion light.

Semiconductor Quantum Dots for Multicolor Fluorescence Imaging and Spectroscopy of Single Cancer Cells

2003 ◽  
Vol 773 ◽  
Author(s):  
Xiaohu Gao ◽  
Shuming Nie ◽  
Wallace H. Coulter
Keyword(s):  
Quantum Dots ◽  
Cancer Cells ◽  
Fluorescence Imaging ◽  
Organic Dyes ◽  
Fluorescent Proteins ◽  
Single Cells ◽  
Quantitative Imaging ◽  
Semiconductor Quantum Dots ◽  
Single Cancer ◽  
Imaging And Spectroscopy

AbstractLuminescent quantum dots (QDs) are emerging as a new class of biological labels with unique properties and applications that are not available from traditional organic dyes and fluorescent proteins. Here we report new developments in using semiconductor quantum dots for quantitative imaging and spectroscopy of single cancer cells. We show that both live and fixed cells can be labeled with multicolor QDs, and that single cells can be analyzed by fluorescence imaging and wavelength-resolved spectroscopy. These results raise new possibilities in cancer imaging, molecular profiling, and disease staging.

POSSIBLE APPLICATION OF METAL SENSITIVE RED FLUORESCENT PROTEINS IN ENVIRONMENTAL MONITORING

2012 ◽  
Vol 11 (1) ◽  
pp. 193-198 ◽  
Author(s):  
Laszlo Szilagyi ◽  
Maria Szabo (Palfi) ◽  
Judit Petres ◽  
Ildiko Miklossy ◽  
Beata Abraham ◽  
...  
Keyword(s):  
Environmental Monitoring ◽  
Fluorescent Proteins ◽  
Red Fluorescent Proteins

scholarly journals Split-wrmScarlet and split-sfGFP: Tools for faster, easier fluorescent l abeling of endogenous proteins in Caenorhabditis elegans

Genetics ◽  
2021 ◽  
Author(s):  
Jérôme Goudeau ◽  
Catherine S Sharp ◽  
Jonathan Paw ◽  
Laura Savy ◽  
Manuel D Leonetti ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Fluorescent Labeling ◽  
Large Fragment ◽  
C Elegans ◽  
High Affinity ◽  
Red Fluorescent Proteins ◽  
Invaluable Tool ◽  
Endogenous Proteins ◽  
Fluorescent Tags

Abstract We create and share a new red fluorophore, along with a set of strains, reagents and protocols, to make it faster and easier to label endogenous C. elegans proteins with fluorescent tags. CRISPR-mediated fluorescent labeling of C. elegans proteins is an invaluable tool, but it is much more difficult to insert fluorophore-size DNA segments than it is to make small gene edits. In principle, high-affinity asymmetrically split fluorescent proteins solve this problem in C. elegans: the small fragment can quickly and easily be fused to almost any protein of interest, and can be detected wherever the large fragment is expressed and complemented. However, there is currently only one available strain stably expressing the large fragment of a split fluorescent protein, restricting this solution to a single tissue (the germline) in the highly autofluorescent green channel. No available C. elegans lines express unbound large fragments of split red fluorescent proteins, and even state-of-the-art split red fluorescent proteins are dim compared to the canonical split-sfGFP protein. In this study, we engineer a bright, high-affinity new split red fluorophore, split-wrmScarlet. We generate transgenic C. elegans lines to allow easy single-color labeling in muscle or germline cells and dual-color labeling in somatic cells. We also describe a novel expression strategy for the germline, where traditional expression strategies struggle. We validate these strains by targeting split-wrmScarlet to several genes whose products label distinct organelles, and we provide a protocol for easy, cloning-free CRISPR/Cas9 editing. As the collection of split-FP strains for labeling in different tissues or organelles expands, we will post updates at doi.org/10.5281/zenodo.3993663

scholarly journals Dual-Color Fluorescence Imaging of EpCAM and EGFR in Breast Cancer Cells with a Bull’s Eye-Type Plasmonic Chip

Sensors ◽  
2017 ◽  
Vol 17 (12) ◽  
pp. 2942 ◽  
Author(s):  
Shota Izumi ◽  
Shohei Yamamura ◽  
Naoko Hayashi ◽  
Mana Toma ◽  
Keiko Tawa
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Fluorescence Imaging ◽  
Breast Cancer Cells ◽  
Dual Color ◽  
Bull's Eye

Generation and Characterization of a Rat Monoclonal Antibody Specific for Multiple Red Fluorescent Proteins

Hybridoma ◽  
2008 ◽  
Vol 27 (5) ◽  
pp. 337-343 ◽  
Author(s):  
Andrea Rottach ◽  
Elisabeth Kremmer ◽  
Danny Nowak ◽  
Heinrich Leonhardt ◽  
M. Cristina Cardoso
Keyword(s):  
Monoclonal Antibody ◽  
Fluorescent Proteins ◽  
Red Fluorescent Proteins

scholarly journals Monomerization of far-red fluorescent proteins

2018 ◽  
Vol 115 (48) ◽  
pp. E11294-E11301 ◽  
Author(s):  
Timothy M. Wannier ◽  
Sarah K. Gillespie ◽  
Nicholas Hutchins ◽  
R. Scott McIsaac ◽  
Sheng-Yi Wu ◽  
...  
Keyword(s):  
Molecular Markers ◽  
Biological Markers ◽  
Fluorescent Proteins ◽  
Biological Tissues ◽  
Biological Applications ◽  
Emission Maximum ◽  
Naturally Occurring ◽  
Red Fluorescent Proteins ◽  
Animal Imaging ◽  
Structural Perturbations

Anthozoa-class red fluorescent proteins (RFPs) are frequently used as biological markers, with far-red (λem ∼ 600–700 nm) emitting variants sought for whole-animal imaging because biological tissues are more permeable to light in this range. A barrier to the use of naturally occurring RFP variants as molecular markers is that all are tetrameric, which is not ideal for cell biological applications. Efforts to engineer monomeric RFPs have typically produced dimmer and blue-shifted variants because the chromophore is sensitive to small structural perturbations. In fact, despite much effort, only four native RFPs have been successfully monomerized, leaving the majority of RFP biodiversity untapped in biomarker development. Here we report the generation of monomeric variants of HcRed and mCardinal, both far-red dimers, and describe a comprehensive methodology for the monomerization of red-shifted oligomeric RFPs. Among the resultant variants is mKelly1 (emission maximum, λem = 656 nm), which, along with the recently reported mGarnet2 [Matela G, et al. (2017) Chem Commun (Camb) 53:979–982], forms a class of bright, monomeric, far-red FPs.

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