Ammasi Periasamy

University of Virginia

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Peter T. C. So

Massachusetts Institute of Technology

Richard N. Day

Indiana University – Purdue University Indianapolis

Yuansheng Sun

Institute of Semitic Studies

Alberto Diaspro

University of Genoa

Cynthia F. Booker

University of Virginia Health System

Karsten Knig

J. N. Demas

University of Virginia

Robert M. Clegg

University of Illinois Urbana-Champaign

Mike Davidson

University of Manitoba

ZŠ

Zdeněk Švindrych

Dartmouth College

Cooperative Institutions

University of Pisa

Universidad Complutense de Madrid

Italian Institute of Technology

University of Wisconsin–Madison

University of Virginia

University of California, Irvine

University of Urbino

University of Gothenburg

University of Konstanz

IMDEA Nanoscience

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Fluorescence Microscopy Imaging in Biomedical Sciences

Biological and medical physics series (2012)

Yuansheng Sun Ammasi Periasamy

Fluorescence-lifetime imaging microscopy

Photoactivated localization microscopy

Two-photon excitation microscopy

10.1007/978-3-642-28391-8_3

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Citations (3)

Visible Fluorescent Proteins

Journal of Biomedical Optics (2008)

Ammasi Periasamy Alberto Diaspro

The Journal of Biomedical Optics (JBO) is a Gold Open Access journal that publishes peer-reviewed papers on the use of novel optical systems and techniques for improved health care and biomedical research.

10.1117/1.2939070

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Citations (2)

Multiphoton Microscopy in the Biomedical Sciences IV

Ammasi Periasamy Peter T. C. So

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Citations (5)

Characterization of an orange acceptor fluorescent protein for sensitized spectral fluorescence resonance energy transfer microscopy using a white-light laser

Journal of Biomedical Optics (2009)

Yuansheng Sun Cynthia F. Booker Sangeeta Kumari Richard N. Day Mike Davidson

Orange fluorescent proteins (FPs) are attractive candidates as Forster resonance energy transfer (FRET) partners, bridging the gap between green and red/far-red FPs, but they pose significant challenges using common fixed laser wavelengths. We investigated monomeric Kusabira orange 2 (mKO2) FP as a FRET acceptor for monomeric teal FP (mTFP) as donor on a FRET standard construct using a fixed-distance amino acid linker, expressed in live cells. We quantified the apparent FRET efficiency (E%) of this construct, using sensitized spectral FRET microscopy on the Leica TCS SP5 X imaging system equipped with a white-light laser that allows choosing any excitation wavelength from 470 to 670 nm in 1-nm increments. The E% obtained in sensitized spectral FRET microscopy was then confirmed with fluorescence lifetime measurements. Our results demonstrate that mKO2 and mTFP are good FRET partners given proper imaging setups. mTFP was optimally excited by the Argon 458 laser line, and the 540-nm wavelength excitation for mKO2 was chosen from the white-light laser. The white-light laser generally extends the usage of orange and red/far-red FPs in sensitized FRET microscopy assays by tailoring excitation and emission precisely to the needs of the FRET pair.

Acceptor

10.1117/1.3227036

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Citations (59)

Heterooligomerization between Vasotocin and Corticotropin-Releasing Hormone (CRH) Receptors Augments CRH-Stimulated 3′,5′-Cyclic Adenosine Monophosphate Production

Molecular Endocrinology (2007)

Marina V. Mikhailova Philip R. Mayeux Alexander Jurkevich Wayne J. Kuenzel Farrah N. Madison

In birds, ACTH release from the anterior pituitary gland during stress is controlled by CRH and arginine vasotocin (AVT). Using 5-wk-old male chicks, simultaneous iv injections of CRH and AVT were found to result in a greater than additive increase in plasma corticosterone levels compared with that obtained with individual administration of either peptide hormone. In order to investigate molecular mechanisms underlying this observation, the chicken CRH receptor (CRHR) and vasotocin VT2 receptor (VT2R) were fused to cyan and yellow fluorescent proteins and expressed in HeLa cells. The resulting CRHR and VT2R fusion proteins were expressed appropriately in the plasma membrane and were found to couple to downstream signal transduction pathways. Quantitative fluorescence resonance energy transfer (FRET) analysis was used to determine whether the CRHR and VT2R formed heterodimers. In the absence of CRH and AVT, the FRET efficiency was 15–18%, and the distance between receptors was 5–6 nm. Treatment of the cells that expressed both cyan fluorescent protein-CRHR and yellow fluorescent protein-VT2R with CRH or AVT alone did not lead to a significant change in the FRET efficiency. However, simultaneous addition of these hormones increased the efficiency of the FRET signal and decreased the distance between the two receptors. In HeLa cells expressing both CRHR and VT2R, treatment with CRH and AVT resulted in a significant increase in cAMP production over that with CRH alone, indicating that heterodimer formation may enhance the ability of the CRHR to activate downstream signal transduction.

Vasotocin

Cyclic adenosine monophosphate

10.1210/me.2007-0160

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Citations (51)

Series in Cellular and Clinical Imaging

Series in cellular and clinical imaging (2014)

Ammasi Periasamy

10.1201/crcsercelcli

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Citations (9)

Issue Information

Microscopy Research and Technique (2022)

Alberto Diaspro Ranieri Bizzarri M. L. Calvo Jenu V. Chacko Michelle A. Digman

US$16,406 (US), US$16,406 (Rest of World), €10,585 (Europe), £8375 (UK).Prices are exclusive of tax.Asia-Pacific GST,

10.1002/jemt.23826

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Citations (0)

Multiphoton Microscopy in the Biomedical Sciences VI

SPIE eBooks (2006)

Ammasi Periasamy Peter T. C. So

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Citations (4)

Imaging Protein Interactions in Living Cells Using the Fluorescent Proteins

Reviews in fluorescence (2009)

Richard N. Day Ammasi Periasamy Ignacio A. Demarco

Living cell

Fluorescent protein

Live cell imaging

Fluorescence-lifetime imaging microscopy

10.1007/978-1-4419-1260-2_14

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<title>Digital deconvolution FRET microscopy: 3D visualization of protein-protein interactions in a single living cell</title>

Proceedings of SPIE, the International Society for Optical Engineering/Proceedings of SPIE (1998)

Ammasi Periasamy

The spatiotemporal dynamics of dimerization of the transcription factor Pit-1 in the living cell nucleus have been visualized and monitored by expression of genetic vectors encoding green (GFP) and blue (BFP) fluorescent protein fusions and fluorescence resonance energy transfer (FRET) imaging techniques. However, in wide-field FRET imaging microscopy the FRET signals are a combination of signals from all focal planes and the fine image details are obscured by 'out-of-focus' light. Digital deconvolution FRET imaging microscopy is used here to remove the 'out-of-focus' light to improve the resolution of the protein localization in the optical axis. Cells expressing fluorescent Pit-1 fusion proteins were imaged with a high speed, high sensitivity CCD camera and a water immersion objective lens. The point spread function (PSF) of the system was used to deconvolve the donor and acceptor images which then were ratioed to obtain the FRET signal at different optical sections. These signals were used to create three-dimensional visualization of the distribution of Pit-1 protein dimers. Digitally deconvolved data with a water immersion lens has a better signal-to-noise ratio than data obtained with an oil immersion lens. The 3-D energy transfer imaging of Pit-1 protein in the nucleus of living cells offers the possibility of studying domains within the nucleus.

Yellow fluorescent protein

Point spread function

Fluorescence-lifetime imaging microscopy

10.1117/12.307087

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Citations (7)