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

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Zdeněk Švindrych

Dartmouth College

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University of Pisa

Universidad Complutense de Madrid

Italian Institute of Technology

University of Wisconsin–Madison

University of Virginia

University of Urbino

University of California, Irvine

University of Gothenburg

University of Konstanz

IMDEA Nanoscience

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Fluorescence resonance energy transfer (FRET) microscopy imaging of live cell protein localizations

The Journal of Cell Biology (2003)

Rajesh B. Sekar Ammasi Periasamy

The current advances in fluorescence microscopy, coupled with the development of new fluorescent probes, make fluorescence resonance energy transfer (FRET) a powerful technique for studying molecular interactions inside living cells with improved spatial (angstrom) and temporal (nanosecond) resolution, distance range, and sensitivity and a broader range of biological applications.

Nanosecond

Fluorescence-lifetime imaging microscopy

Fluorescence cross-correlation spectroscopy

10.1083/jcb.200210140

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Citations (854)

Molecular Imaging, FRET Microscopy and Spectroscopy

Journal of Biomedical Optics (2006)

Ammasi Periasamy Richard N. Day Barry R. Masters

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.

10.1117/1.2388275

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Citations (167)

Guest editorial: Special section on visible fluorescent proteins

Ammasi Periasamy Alberto Diaspro

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Special section

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Characterization of spectral FRET imaging microscopy for monitoring nuclear protein interactions

Journal of Microscopy (2007)

Ye Chen Joshua P. Mauldin Richard N. Day Ammasi Periasamy

Summary The spectral processed Förster resonance energy transfer (psFRET) imaging method provides an effective and fast method for measuring protein–protein interactions in living specimens. The commercially available linear unmixing algorithms efficiently remove the contribution of donor spectral bleedthrough to the FRET signal. However, the acceptor contribution to spectral bleedthrough in the FRET image cannot be similarly removed, since the acceptor spectrum is identical to the FRET spectrum. Here, we describe the development of a computer algorithm that measures and removes the contaminating ASBT signal in the sFRET image. The new method is characterized in living cells that expressed FRET standards in which the donor and acceptor fluorescent proteins are tethered by amino acid linkers of specific lengths. The method is then used to detect the homo‐dimerization of a transcription factor in the nucleus of living cells, and then to measure the interactions of that protein with a second transcription factor.

Acceptor

Fluorescent protein

10.1111/j.1365-2818.2007.01838.x

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Citations (80)

Application of Nonlinear Microscopy in Life Sciences

Zdeněk Švindrych Ammasi Periasamy

As was the case with the introduction of confocal fluorescence microscopy in life sciences, the application of second harmonic and multiphoton imaging was another milestone in biological imaging, since it enabled researchers to move from cultured cells deep into tissues and whole organs, even in live animal models. A well-aligned confocal microscope provides faithful representation of the 3D distribution of the fluorophores without image-processing artifacts. Deep tissue imaging, however important in life sciences, is very difficult and faces many limitations and challenges. Methods based on x-ray absorption, nuclear magnetic resonance or emission from radioactive markers, as mere examples, do allow whole body imaging, but lack the resolution and specificity of light microscopy. The high power of incident light required for appreciable 2P absorption in fluorescently labeled samples necessitates the use of lasers as excitation sources. With these lasers, multiphoton imaging of some fluorescent proteins and second harmonic generation imaging are readily achievable.

10.1201/9781315206554-6

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Monitoring Protein Interactions in Living Cells with Fluorescence Lifetime Imaging Microscopy

Methods in enzymology on CD-ROM/Methods in enzymology (2012)

Yuansheng Sun Nicole M. Hays Ammasi Periasamy Michael W. Davidson Richard N. Day

Fluorescence-lifetime imaging microscopy

10.1016/b978-0-12-391857-4.00019-7

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Citations (66)

Multiphoton Microscopy in the Biomedical Sciences

Ammasi Periasamy Peter T. C. So

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FLIM Applications in the Biomedical Sciences

Chapman and Hall/CRC eBooks (2009)

Robert Clegg Ammasi Periasamy

10.1201/9781420078916.sec4

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The characterization of optimized fluorescent proteins for Förster resonance energy transfer microscopy

Proceedings of SPIE, the International Society for Optical Engineering/Proceedings of SPIE (2009)

Richard N. Day Yuansheng Sun Cynthia F. Booker Sangeeta Kumari Ammasi Periasamy

The genetically encoded fluorescent proteins (FP), used in combination with Förster resonance energy transfer (FRET) microscopy, provide the tools necessary for the direct visualization of protein interactions inside living cells. Currently, the FPs most commonly used for live-cell FRET studies are the Cerulean and Venus variants of the cyan and yellow FPs. However, there are problems associated with this donor-acceptor pair, and these might be overcome by exploiting the characteristics of some of the newer FPs. For example, earlier we showed that the monomeric teal FP (mTFP) has advantages over Cerulean as a FRET donor for Venus. Here, using mTFP as the common donor fluorophore, we characterize a variety of different yellow, orange and red FPs as potential acceptors of FRET. We employed a "FRET standard" genetic construct to minimize variability in the separation distance and positioning of the fused donor and acceptor FPs. Using spectral FRET imaging and fluorescence lifetime measurements from living cells expressing the fused proteins, we characterized both sensitized acceptor emission and the shortening of the donor lifetime resulting from quenching for each of the fused FP pairs. Surprisingly, we found disagreements between the spectral FRET and lifetime measurements for some of the different FP pairs. Our results appear to indicate that some of the orange and red FPs can quench the mTFP donor while yielding little sensitized emission. We are characterizing the basis for this observation.

Acceptor

10.1117/12.814918

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Multiphoton Microscopy in the Biomedical Sciences II

SPIE eBooks (2002)

Ammasi Periasamy Peter T. C. So

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Citations (7)