FLIM Publications

A novel lysosome‐to‐mitochondria signaling pathway disrupted by amyloid‐β oligomers
by  Andrés Norambuena, et al.

  • ABSTRACT: The mechanisms of mitochondrial dysfunction in Alzheimer's disease are incompletely understood. Using two‐photon fluorescence lifetime microscopy of the coenzymes, NADH and NADPH, and tracking brain oxygen metabolism with multi‐parametric photoacoustic microscopy, we show that activation of lysosomal mechanistic target of rapamycin complex 1 (mTORC1) by insulin or amino acids stimulates mitochondrial activity and regulates mitochondrial DNA synthesis in neurons...

The EMBO Journal 37: e100241, 2018.
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Segmented cell analyses to measure redox states of autofluorescent NAD(P)H, FAD & Trp in cancer cells by FLIM​
by Horst Wallrabe, et al.

  • ABSTRACT: Multiphoton FLIM microscopy offers many opportunities to investigate processes in live cells, tissue and animal model systems. For redox measurements, FLIM data is mostly published by cell mean values and intensity-based redox ratios. Our method is based entirely on FLIM parameters generated by 3-detector time domain microscopy capturing autofluorescent signals of NAD(P)H, FAD and novel FLIM-FRET application of Tryptophan and NAD(P)H-a2%/FAD-a1% redox ratio...

Sci. Rep. 8: 79 (2018)
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Investigation of Mitochondrial Metabolic Response to Doxorubicin in Prostate Cancer Cells: An NADH, FAD and Tryptophan FLIM Assay​
by Shagufta Rehman Alam, et al.

  • ABSTRACT: Prostate cancer (PCa) is one of the leading cancers in men in the USA. Lack of experimental tools that predict therapy response is one of the limitations of current therapeutic regimens. Mitochondrial dysfunctions including defective oxidative phosphorylation (OXPHOS) in cancer inhibit apoptosis by modulating ROS production and cellular signaling. Thus, correction of mitochondrial dysfunction and induction of apoptosis are promising strategies in cancer treatment...

Sci. Rep. 7: 10451 (2017)
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Localizing Protein–Protein Interactions in Living Cells Using Fluorescence Lifetime Imaging Microscopy
by Yuansheng Sun & Ammasi Periasamy

  • ABSTRACT: In the past decade, advances in fluorescence lifetime imaging have extensively applied in the life sciences, from fundamental biological investigations to advanced clinical diagnosis. Fluorescence lifetime imaging microscopy (FLIM) is now routinely used in the biological sciences to monitor dynamic signaling events inside living cells, e.g., Protein–Protein interactions...

Methods in Mol. Biol., Vol. 1251: 83-108 (2015)
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Monitoring Protein Interactions in Living Cells with Fluorescence Lifetime Imaging Microscopy​
by Yuansheng Sun, et al.

  • ABSTRACT: Fluorescence lifetime imaging microscopy (FLIM) is now routinely used for dynamic measurements of signaling events inside single living cells, such as monitoring changes in intracellular ions and detecting protein–protein interactions. Here, we describe the digital frequency domain FLIM data acquisition and analysis...

Meth. Enzymol., 504: 371-391​ (2012)

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Investigating protein-protein interactions in living cells using fluorescence lifetime imaging microscopy 
by Yuansheng Sun, Richard N Day & Ammasi Periasamy

  • ABSTRACT: Fluorescence lifetime imaging microscopy (FLIM) is now routinely used for dynamic measurements of signaling events inside living cells, including detection of protein-protein interactions. An understanding of the basic physics of fluorescence lifetime measurements is required to use this technique. In this protocol, we describe both the time-correlated single photon counting and the frequency-domain methods for FLIM data acquisition and analysis...

Nature Protocol Vol. 6 No. 9, 2011. 
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Characterization of two‐photon excitation fluorescence lifetime imaging microscopy for protein localization​
by Ye Chen & Ammasi Periasamy

  • ABSTRACT: Two‐photon excitation fluorescence resonance energy transfer (2P‐FRET) imaging microscopy can provide details of specific protein molecule interactions inside living cells. Fluorophore molecules used for 2P‐FRET imaging have characteristic absorption and emission spectra that introduce spectral cross‐talk (bleed‐through) in the FRET signal that should be removed in the 2P‐FRET images, to establish that FRET has actually occurred and to have a basis for distance estimations.​..

Microscopy Research and Techniques. 63:72-80​ (2004)

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Protein interactions in cells and tissues using FLIM and FRET​​
by Ye Chen, James D. Mills & Ammasi Periasamy

  • ABSTRACT: Interacting proteins assemble into molecular machines that control cellular homeostasis in living cells. While the in vitro screening methods have the advantage of providing direct access to the genetic information encoding unknown protein partners, they do not allow direct access to interactions of these protein partners in their natural environment inside the living cell. Using wide-field, confocal, or two-photon (2p) fluorescence resonance energy transfer (FRET) microscopy, this information can be obtained from living cells and tissues with nanometer resolution.​..

Differentiation. 71:528-541​ (2003)

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Protein localization in living cells and tissues using FRET and FLIM 
by Ye Chen, James D. Mills & Ammasi Periasamy

  • ABSTRACT: Interacting proteins assemble into molecular machines that control cellular homeostasis in living cells. While the in vitro screening methods have the advantage of providing direct access to the genetic information encoding unknown protein partners, they do not allow direct access to interactions of these protein partners in their natural environment inside the living cell. Using wide-.eld, confocal, or two-photon (2p) .uorescence resonance energy transfer (FRET) microscopy, ..........

Differentiation (2003) 71:528?41 
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Dynamic Imaging Using Fluorescence Resonance Energy Transfer 
by Masilamani Elangovan, Richard N. Day & Ammasi Periasamy 

  • ABSTRACT: Recently, fluorescnce resonance energy transfer (FRET) and fluorescnce lifetime imaging microscopy (FLIM) have been the buzzwords for monitoring protein-protein interactions in living cells....

Biotechniques Vol. 32 No. 6 (2002) 
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Nanosecond Fluorescence Resonance Energy Transfer-Fluorescence Lifetime Imaging Microscopy To Localize The Protein Interactions In A Single Living Cell 
by Masilamani Elangovan, Richard N. Day & Ammasi Periasamy 

  • ABSTRACT: Visualizing and quantifying protein-protein interactions is a recent trend in biomedical imaging. The current advances in fluorescnce microscopy, coupled with the development of new fluorescent probes such as green fluorescent proteins, allow fluorescent resonance energy transfer (FRET) to be used to study protein interactions in living spectrums....

Journal Of Microscopy, Vol. 205, Pt 1 January 2002, Pp. 3-14 
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Fluorescence Lifetime Imaging (FLIM) Of Green Fluorescent Fusion Proteins In Living Cells 
by Ammasi Periasamy, et al.

  • ABSTRACT: Fluorescence microscopy is an exception al tool for looling inside cells and tissues. Recent advances in fluorescence microscopy, including improved optics, sensitive fluorescent dyes, and high-sensitivity cameras, coupled with technological advances in computes and sophisticated software now permit quantitative measurement and noninvasive acquisition of spectroscopic information from a single living cell....

Methods In Molecular Biology, Vol. 183: Green Fluorescent Protein: Applications And Protocals (2002) 
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Error Analysis Of The Rapid Lifetime Determination Method For Double-Exponential Decays And New Windowing Schemes 
by Kristin K. Sharman, et al.

  • ABSTRACT: The rapid lifetime determination method (RLD) is a mathematical technique for extremely rapid evaluations of lifetimes in exponential decays. It has been applied in luminescence microscopy and single-molecule lifetime evaluation....

Anal. Chem.1999, 71,947-952 
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Time-Resolved Fluorescence Lifetime Imaging Microscopy Using A Picosecond Pulsed Tunable Dye Laser System 
by Ammasi Periasamy, et al.

  • ABSTRACT: The design and implementation of a time-resolved fluorescence lifetime imaging microscope (TRFLIM) for the biomedical sciences are described. The measurement of fluorescence lifetimes offers many benefits, among which is that they are independent of local signal intensity and concentration of the fluorophore and they provide visualization of the molecular environment in a single living cell.

Rev. Sci. Instrum. 67 (10), October 1996 
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Time-Gated Fluorescence Microscopy For Clinical Imaging 
by Ammasi Periasamy, Majid Siadat-Pajouh & Pawel Wodnicki

  • ABSTRACT: Fluorescence microscopy has been an essential tool for generations of biologists, allowing the examination of details of individual living or fixed cells. The development of a large number of fluorescnece dyes and the ability to attach them to specific molecular cell constituents allows real-time observation and quantification of the activities of these constituents in cells and tissues....

USA Microscopy And Analyses, March 1995 19 
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Fluorescence Lifetime Imaging Microscopy (FLIM): Instrumentation and Applications​ 
by Xue Feng Wang, et al.

  • ABSTRACT: The new and novel techniques of fluorescence lifetime imaging (FLI)∗∗ and fluorescence lifetime imaging microscopy (FLIM) provide the investigator with the capacity to quantitate two-dimensional fluorescence intensity distributions and lifetimes. The concept, theory, and instrumentation of FLI and FLIM are reviewed in this paper. The implementation of FLIM instrumentation with conventional and confocal microscopic systems is discussed...

Critical Rev. Analyt. Chem., 23(5): 369-395 (1992)
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