Multi-dimensional time-correlated single photon counting (TCSPC) fluorescence lifetime imaging microscopy (FLIM) to detect FRET in cells

Journal of Microscopy
R R DuncanM J Shipston

Abstract

We present a novel, multi-dimensional, time-correlated single photon counting (TCSPC) technique to perform fluorescence lifetime imaging with a laser-scanning microscope operated at a pixel dwell-time in the microsecond range. The unsurpassed temporal accuracy of this approach combined with a high detection efficiency was applied to measure the fluorescent lifetimes of enhanced cyan fluorescent protein (ECFP) in isolation and in tandem with EYFP (enhanced yellow fluorescent protein). This technique enables multi-exponential decay analysis in a scanning microscope with high intrinsic time resolution, accuracy and counting efficiency, particularly at the low excitation levels required to maintain cell viability and avoid photobleaching. Using a construct encoding the two fluorescent proteins separated by a fixed-distance amino acid spacer, we were able to measure the fluorescence resonance energy transfer (FRET) efficiency determined by the interchromophore distance. These data revealed that ECFP exhibits complex exponential fluorescence decays under both FRET and non-FRET conditions, as previously reported. Two approaches to calculate the distance between donor and acceptor from the lifetime delivered values within a 10% error r...Continue Reading

Citations

Apr 2, 2010·Chemical Reviews·Mikhail Y Berezin, Samuel Achilefu
Sep 15, 2009·The Journal of Biological Chemistry·Colin Rickman, Rory R Duncan
Feb 1, 2007·The Journal of Biological Chemistry·Colin RickmanRory R Duncan
Feb 23, 2012·Journal of Biomedical Optics·Ryan A ColyerXavier Michalet
Apr 28, 2005·BMC Cell Biology·Christopher D StubbsAnthony W Parker
Oct 4, 2011·Journal of Nanobiotechnology·Peter N YaronKris Noel Dahl
Apr 17, 2012·PloS One·Daniel R MatthewsSimon M Ameer-Beg
Nov 3, 2012·PloS One·John SaegerViola Vogel
Jul 31, 2013·PloS One·Atul A BhardeDonna J Arndt-Jovin
Nov 28, 2006·Frontiers in Bioscience : a Journal and Virtual Library·Peter G BushAndrew C Hall
Jan 17, 2012·Cellular and Molecular Bioengineering·Tristan TabouillotPeter J Butler
Aug 26, 2009·The Journal of Cell Biology·Jian-Ping XiongM Amin Arnaout
Oct 6, 2010·The Journal of Cell Biology·Markus PoschJason R Swedlow
Dec 20, 2014·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·Jakub NedbalSimon M Ameer-Beg
Jan 20, 2016·ACS Applied Materials & Interfaces·Patrick BoyerKris Noel Dahl
Oct 1, 2015·Molecular Microbiology·David J MothersoleC Neil Hunter
Dec 17, 2008·Microscopy Research and Technique·Aymeric LerayLaurent Héliot
May 25, 2012·Journal of Microscopy·W Becker
Jul 11, 2013·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·Iwona ZiomkiewiczDonna J Arndt-Jovin
Jan 9, 2010·Journal of Microscopy·D R MatthewsS M Ameer-Beg
Nov 21, 2008·IEEE Journal of Solid-state Circuits·David Eric SchwartzKenneth L Shepard
Dec 24, 2014·The FEBS Journal·Haik ChosrowjanFumio Tanaka
Jun 10, 2015·BMC Research Notes·Daniel SewellPing Ma
Nov 17, 2007·Biochimica Et Biophysica Acta·Kira V GromovaGregory S Harms
May 15, 2012·Biochimica Et Biophysica Acta·Dita StrachotováZdena Palková
Dec 22, 2007·Biophysical Journal·Michelle A DigmanEnrico Gratton
Jul 31, 2013·Proceedings of the National Academy of Sciences of the United States of America·Nicholas Stephane CaronRay Truant
Jan 9, 2013·Journal of Diabetes Science and Technology·Jithesh V VeetilKaiming Ye
Mar 30, 2007·Microscopy Research and Technique·V CaorsiA Diaspro
Mar 31, 2007·Microscopy Research and Technique·Claire N MedineRory R Duncan
Mar 30, 2007·Microscopy Research and Technique·Wolfgang BeckerChristoph Biskup
Jul 1, 2010·Optics Express·Gilbert O FruhwirthFrederic Festy
Jun 8, 2007·The Journal of Biological Chemistry·Mari ValkonenRory R Duncan
Nov 13, 2019·Methods and Applications in Fluorescence·Herbert Schneckenburger
Dec 4, 2019·Methods and Applications in Fluorescence·Julien Godet, Yves Mely
May 19, 2020·Methods and Applications in Fluorescence·Deborah S BarkauskasMichael S Roberts

❮ Previous
Next ❯

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.

Related Papers

Microscopy Research and Technique
Wolfgang BeckerChristoph Biskup
Nature Biotechnology
Elizabeth A Jares-Erijman, Thomas M Jovin
© 2022 Meta ULC. All rights reserved