540:) or a single photon avalanche photo diode (SPAD)) with respect to the excitation laser pulse. The recordings are repeated for multiple laser pulses and after enough recorded events, one is able to build a histogram of the number of events across all of these recorded time points. This histogram can then be fit to an exponential function that contains the exponential lifetime decay function of interest, and the lifetime parameter can accordingly be extracted. Multi-channel PMT systems with 16 to 64 elements have been commercially available, whereas the recently demonstrated CMOS single-photon avalanche diode (SPAD)-TCSPC FLIM systems can offer even higher number of detection channels and additional low-cost options.
566:. The fluorescence is (a.) demodulated and (b.) phase shifted; both quantities are related to the characteristic decay times of the fluorophore. Also, y-components to the excitation and fluorescence sine waves will be modulated, and lifetime can be determined from the modulation ratio of these y-components. Hence, 2 values for the lifetime can be determined from the phase-modulation method. The lifetimes are determined through a fitting procedures of these experimental parameters. An advantage of PMT-based or camera-based frequency domain FLIM is its fast lifetime image acquisition making it suitable for applications such as live cell research.
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allows for detection for the fraction of time when it is open after the delay. Thus, with an adjustable delay generator, one is able to collect fluorescence emission after multiple delay times encompassing the time range of the fluorescence decay of the sample. In recent years integrated intensified CCD cameras entered the market. These cameras consist of an image intensifier, CCD sensor and an integrated delay generator. ICCD cameras with shortest gating times of down to 200ps and delay steps of 10ps allow sub-nanosecond resolution FLIM. In combination with an endoscope this technique is used for intraoperative diagnosis of brain tumors.
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Laplace and
Fourier transformation along with Laguerre gauss expansion have been used to estimate the lifetime in transformed space. These approaches are faster than the deconvolution based methods but they suffer from truncation and sampling problems. Moreover, application of methods like Laguerre gauss expansion is mathematically complicated. In Fourier methods the lifetime of a single exponential decay curve is given by:
677:
technique is the least square iterative re-convolution which is based on the minimization of the weighted sum of the residuals. In this technique theoretical exponential decay curves are convoluted with the instrument response function, which is measured separately, and the best fit is found by iterative calculation of the residuals for different inputs until a minimum is found. For a set of observations
1622:{\displaystyle {\begin{matrix}{{A}_{n}}={\frac {\sum \limits _{t}{d(t)\sin(n\omega t)}}{\sum \limits _{t}{IRF(t)\sin(n\omega t)}}}={\frac {\omega \tau }{1+{{\omega }^{2}}{{\tau }^{2}}}},&{{B}_{n}}={\frac {\sum \limits _{t}{d(t)\cos(n\omega t)}}{\sum \limits _{t}{IRF\cos(n\omega t)}}}={\frac {1}{1+n{{\omega }^{2}}{{\tau }^{2}}}},&\omega ={\frac {2\pi }{T}}\\\end{matrix}}}
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a method to discriminate between the states/environments of the fluorophore. In contrast to intensity-based FRET measurements, the FLIM-based FRET measurements are also insensitive to the concentration of fluorophores and can thus filter out artifacts introduced by variations in the concentration and emission intensity across the sample.
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fitting methods are attractive because they offer a very fast solution to lifetime estimation. One of the major and straightforward techniques in this category is the rapid lifetime determination (RLD) method. RLD calculates the lifetimes and their amplitudes directly by dividing the decay curve into two parts of equal width
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Pulse excitation is still used in this method. Before the pulse reaches the sample, some of the light is reflected by a dichroic mirror and gets detected by a photodiode that activates a delay generator controlling a gated optical intensifier (GOI) that sits in front of the CCD detector. The GOI only
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Since the fluorescence lifetime of a fluorophore depends on both radiative (i.e. fluorescence) and non-radiative (i.e. quenching, FRET) processes, energy transfer from the donor molecule to the acceptor molecule will decrease the lifetime of the donor. Thus, FRET measurements using FLIM can provide
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For multi exponential decays this equation provides the average lifetime. This method can be extended to analyze bi-exponential decays. One major drawback of this method is that it cannot take into account the instrument response effect and for this reason the early part of the measured decay curves
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The instrumental response of the source, detector, and electronics can be measured, usually from scattered excitation light. Recovering the decay function (and corresponding lifetimes) poses additional challenges as division in the frequency domain tends to produce high noise when the denominator is
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signaling and trafficking. Time domain FLIM (tdFLIM) has also been used to show the interaction of both types of nuclear intermediate filament proteins lamins A and B1 in distinct homopolymers at the nuclear envelope, which further interact with each other in higher order structures. FLIM imaging
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One of the interesting features of the convolution theorem is that the integral of the convolution is the product of the factors that make up the integral. There are a few techniques which work in transformed space that exploit this property to recover the pure decay curve from the measured curve.
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Besides experimental difficulties, including the wavelength dependent instrument response function, mathematical treatment of the iterative de-convolution problem is not straightforward and it is a slow process which in the early days of FLIM made it impractical for a pixel-by-pixel analysis. Non
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lifetime (FLT) of the fluorophore, rather than its intensity, is used to create the image in FLIM. Fluorescence lifetime depends on the local micro-environment of the fluorophore, thus precluding any erroneous measurements in fluorescence intensity due to change in brightness of the light source,
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The goal of the analysis algorithm is to extract the pure decay curve from the measured decay and to estimate the lifetime(s). The latter is usually accomplished by fitting single or multi exponential functions. A variety of methods have been developed to solve this problem. The most widely used
524:) is usually employed because it compensates for variations in source intensity and single photon pulse amplitudes. Using commercial TCSPC equipment a fluorescence decay curve can be recorded with a time resolution down to 405 fs. The recorded fluorescence decay histogram obeys
431:
pulse of light, the time-resolved fluorescence will decay exponentially as described above. However, if the excitation pulse or detection response is wide, the measured fluorescence, d(t), will not be purely exponential. The instrumental response function, IRF(t) will be
57:
background light intensity or limited photo-bleaching. This technique also has the advantage of minimizing the effect of photon scattering in thick layers of sample. Being dependent on the micro-environment, lifetime measurements have been used as an indicator for
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is difficult. The technique was developed in the late 1980s and early 1990s (Gating method: Bugiel et al. 1989. König 1989, Phase modulation: Lakowicz at al. 1992,) before being more widely applied in the late 1990s. In cell culture, it has been used to study
415:, which allows one to view contrast between materials with different fluorescence decay rates (even if those materials fluoresce at exactly the same wavelength), and also produces images which show changes in other decay pathways, such as in
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Fluorescence lifetimes can be determined in the frequency domain by a phase-modulation method. The method uses a light source that is pulsed or modulated at high frequency (up to 500 MHz) such as an LED, diode laser or a
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is particularly useful in neurons, where light scattering by brain tissue is problematic for ratiometric imaging. In neurons, FLIM imaging using pulsed illumination has been used to study
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with a certain probability based on the decay rates through a number of different (radiative and/or nonradiative) decay pathways. To observe fluorescence, one of these pathways must be by
507:
214:
2133:
Elson, D. S.; Munro, I; Requejo-Isidro, J; McGinty, J; Dunsby, C; Galletly, N; Stamp, G W; Neil, M A A; Lever, M J; Kellett, P A; Dymoke-Bradshaw, A; Hares, J; French, P M W (2004).
165:
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715:
2572:
Yasuda, Ryohei (2006). "Imaging spatiotemporal dynamics of neuronal signaling using fluorescence resonance energy transfer and fluorescence lifetime imaging microscopy".
1661:, and Ran family proteins. FLIM has been used in clinical multiphoton tomography to detect intradermal cancer cells as well as pharmaceutical and cosmetic compounds.
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311:
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Verveer, Peter J.; Wouters, FS; Reynolds, AR; Bastiaens, PI (2000). "Quantitative
Imaging of Lateral ErbB1 Receptor Signal Propagation in the Plasma Membrane".
238:
1071:{\displaystyle {\begin{matrix}{{D}_{0}}=\sum \limits _{i=1}^{K/2}{{{I}_{i}}\delta t}&{{D}_{1}}=\sum \limits _{i=K/2}^{K}{{{I}_{i}}\delta t}\\\end{matrix}}}
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Fluorescence lifetimes can be determined in the time domain by using a pulsed source. When a population of fluorophores is excited by an ultrashort or
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is independent of the initial intensity and of the emitted light. This can be utilized for making non-intensity based measurements in chemical sensing.
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should be ignored in the analyses. This means that part of the signal is discarded and the accuracy for estimating short lifetimes goes down.
2273:"Fluorescence lifetime imaging microscopy (flimscopy). Methodology development and application to studies of endosome fusion in single cells"
1173:
2725:
Mueller-Harvey, Irene; Feucht, Walter; Polster, Juergen; Trnková, Lucie; Burgos, Pierre; Parker, Anthony W.; Botchway, Stanley W. (2012).
630:
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Sun, Yinghua; Hatami, Nisa; Yee, Matthew; Marcu, Jennifer; Elson, Daniel S.; Gorin, Fredric; Schrot, Rudolph J.; Phipps, Laura (2010).
2029:
Li, Day-Uei; Arlt, Jochen; Richardson, Justin; Walker, Richard; Buts, Alex; Stoppa, David; Charbon, Edoardo; Henderson, Robert (2010).
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FLIM has primarily been used in biology as a method to detect photosensitizers in cells and tumors as well as FRET in instances where
612:
2135:"Real-time time-domain fluorescence lifetime imaging including single-shot acquisition with a segmented optical image intensifier"
722:
2956:
416:
2031:"Real-time fluorescence lifetime imaging system with a 32 Ă— 32 0.13ÎĽm CMOS low dark-count single-photon avalanche diode array"
2530:"The truncated prelamin A in Hutchinson–Gilford progeria syndrome alters segregation of A-type and B-type lamin homopolymers"
2255:
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records times at which individual photons are detected by a fast single-photon detector (typically a photo-multiplier tube (
1960:
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Delbarre, Erwan; Tramier, Marc; Coppey-Moisan, Maïté; Gaillard, Claire; Courvalin, Jean-Claude; Buendia, Brigitte (2006).
1779:
Levitt, James A.; Kuimova, Marina K.; Yahioglu, Gokhan; Chung, Pei-Hua; Suhling, Klaus; Phillips, David (9 July 2009).
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Ruedas-Rama, Maria J.; Orte, Angel; Hall, Elizabeth A. H.; Alvarez-Pez, Jose M.; Talavera, Eva M. (20 February 2012).
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1999:
1726:"Application of fluorescence lifetime imaging of enhanced green fluorescent protein to intracellular pH measurements"
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Ii is the recorded signal in the i-th channel and K is the number of channels. The lifetime can be estimated using:
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1952:
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2727:"Two-photon excitation with pico-second fluorescence lifetime imaging to detect nuclear association of flavanols"
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2831:"Optimization of FLIM imaging, fitting and analysis for auto-fluorescent NAD(P)H and FAD in cells and tissues"
2678:"The design of Förster (fluorescence) resonance energy transfer (FRET)-based molecular sensors for Ran GTPase"
107:
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Lakowicz, Joseph R.; Szmacinski, Henryk; Nowaczyk, Kazimierz; Berndt, Klaus W.; Johnson, Michael (1992).
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of the fluorescence signal in time bin i, the lifetime estimation is carried out by minimization of:
2616:
1781:"Membrane-Bound Molecular Rotors Measure Viscosity in Live Cells via Fluorescence Lifetime Imaging"
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are the rates for each decay pathway, at least one of which must be the fluorescence decay rate
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1838:"A chloride ion nanosensor for time-resolved fluorimetry and fluorescence lifetime imaging"
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Fluorescence-lifetime imaging yields images with the intensity of each pixel determined by
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Nakabayashi, Takakazu; Wang, Hui-Ping; Kinjo, Masataka; Ohta, Nobuhiro (4 June 2008).
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t. The analysis is performed by integrating the decay curve in equal time intervals
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Datta, Rupsa; Alfonso-GarcĂa, Alba; Cinco, Rachel; Gratton, Enrico (2015-05-20).
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1948:
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2621:"The Spread of Ras Activity Triggered by Activation of a Single Dendritic Spine"
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Cao, Ruofan; Wallrabe, Horst; Siller, Karsten; Periasamy, Ammasi (2020-02-05).
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Proceedings of the
National Academy of Sciences of the United States of America
2186:"Fluorescence lifetime imaging microscopy for brain tumor image-guided surgery"
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2438:"Fluorescence lifetime imaging of receptor tyrosine kinase activity in cells"
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Agronskaia, Alexandra V.; Tertoolen, L.; Gerritsen, Hans C. (November 2004).
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2774:"Fluorescence lifetime imaging of endogenous biomarker of oxidative stress"
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and n is the harmonic number and T is the total time range of detection.
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Multi-photon FLIM is increasingly used to detect auto-fluorescence from
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description, the fluorescence emitted will decay with time according to
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2007:
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Lakowicz, Joseph R.; Szmacinski, H; Nowaczyk, K; Johnson, ML (1992).
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1229:{\displaystyle \tau ={\frac {1}{n\omega }}{\frac {{A}_{n}}{{B}_{n}}}}
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1893:"Fast fluorescence lifetime imaging of calcium in living cells"
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2379:"Fluorescence Lifetime Imaging of Free and Protein-Bound NADH"
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Phasor approach to fluorescence lifetime and spectral imaging
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863:{\displaystyle {{\chi }^{2}}=\sum \limits _{i}{{\left}^{2}}}
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2006:. Becker & Hickl GmbH. April 26, 2017. Archived from
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from a sample. It can be used as an imaging technique in
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is an imaging technique based on the differences in the
1154:{\displaystyle \tau =\delta t/\ln({{D}_{0}}/{{D}_{1}})}
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source combined with an electro-optic modulator or an
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2894:"Lifetime Imaging Techniques for Optical Microscopy"
2436:Wouters, Fred S.; Bastiaens, Philippe I.H. (1999).
604:. Unsourced material may be challenged and removed.
2092:. Methods in Cell Biology. Vol. 81. pp.
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2917:Lifetime and spectral analysis tools in ImageJ:
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1687:as markers for changes in mammalian metabolism.
1664:More recently FLIM has also been used to detect
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2615:Harvey, Christopher D.; Yasuda, R; Zhong, H;
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502:{\displaystyle {d}(t)={IRF}(t)\otimes {F}(t)}
209:{\displaystyle {\frac {1}{\tau }}=\sum k_{i}}
1730:Photochemical & Photobiological Sciences
2913:Fluorescence Excited-State Lifetime Imaging
19:"FLIM" redirects here. For other uses, see
2085:"Fluorescence Lifetime Imaging Microscopy"
613:"Fluorescence-lifetime imaging microscopy"
436:or blended with the decay function, F(t).
2892:Becker, Wolfgang; Bergmann, Axel (2003).
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1980:. Becker & Hickl GmbH. April 26, 2017
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664:Learn how and when to remove this message
72:
2931:Fluorescence Lifetime Imaging Microscopy
2835:Methods and Applications in Fluorescence
520:Time-correlated single-photon counting (
27:Fluorescence-lifetime imaging microscopy
2244:Gadella, Theodorus W. J. (2011-07-29).
2243:
1949:Principles of Fluorescence Spectroscopy
16:Imaging technique based on fluorescence
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422:
160:{\displaystyle I(t)=I_{0}e^{-t/\tau }}
2271:Oida, T.; Sako, Y; Kusumi, A (1993).
2676:Kaláb, Petr; Soderholm, Jon (2010).
602:adding citations to reliable sources
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532:during fitting. More specifically,
1785:The Journal of Physical Chemistry C
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528:which is considered in determining
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367:. More importantly, the lifetime,
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37:rate of the photon emission of a
1974:"SPC-150NX, Product description"
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47:two-photon excitation microscopy
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2574:Current Opinion in Neurobiology
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2330:"Fluorescence lifetime imaging"
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2090:Digital Microscopy, 3rd Edition
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589:needs additional citations for
287:is the initial fluorescence at
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260:is the fluorescence lifetime,
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49:, and multiphoton tomography.
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2957:Optical microscopy techniques
2507:10.1126/science.290.5496.1567
2455:10.1016/S0960-9822(99)80484-9
2297:10.1016/S0006-3495(93)81427-9
2102:10.1016/S0091-679X(06)81024-1
2000:"PML-16, Product description"
1711:
2346:10.1016/0003-2697(92)90112-K
2193:Journal of Biomedical Optics
1897:Journal of Biomedical Optics
710:{\displaystyle d({{t}_{i}})}
7:
2694:10.1016/j.ymeth.2010.01.022
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2586:10.1016/j.conb.2006.08.012
1672:Autofluorescent coenzymes
18:
2743:10.1016/j.aca.2011.12.068
2160:10.1088/1367-2630/6/1/180
2855:10.1088/2050-6120/ab6f25
2537:Human Molecular Genetics
2247:FRET and FLIM Techniques
515:
2952:Fluorescence techniques
2938:(Becker&Hickl GmbH)
2936:Principle of TCSPC FLIM
2645:10.1126/science.1159675
2334:Analytical Biochemistry
906:{\displaystyle \delta }
886:{\displaystyle \delta }
564:acousto-optic modulator
2731:Analytica Chimica Acta
2404:10.1073/pnas.89.4.1271
2199:(5): 056022–056022–5.
2139:New Journal of Physics
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408:{\displaystyle \tau }
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97:of a photon. In the
65:and chemical species
2056:10.1364/OE.18.010257
1947:Joseph R. Lakowicz.
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2919:http://spechron.com
2847:2020MApFl...8b4001C
2637:2008Sci...321..136H
2499:2000Sci...290.1567V
2395:1992PNAS...89.1271L
2289:1993BpJ....64..676O
2277:Biophysical Journal
2205:2010JBO....15e6022S
2151:2004NJPh....6..180E
2078:Chang, CW; Sud, D;
2047:2010OExpr..1810257L
1909:2004JBO.....9.1230A
1854:2012Ana...137.1500R
1791:(27): 11634–11642.
1641:ratiometric imaging
423:Pulsed illumination
306:{\displaystyle t=0}
43:confocal microscopy
2924:2013-03-11 at the
2778:Scientific Reports
2550:10.1093/hmg/ddl026
2004:Becker & Hickl
1978:Becker & Hickl
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2010:on March 3, 2018
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1848:(6): 1500–1508.
1833:
1827:
1826:
1808:
1776:
1770:
1769:
1742:10.1039/B800391B
1721:
1668:in plant cells.
1628:
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708:
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658:
655:
649:
647:
606:
582:
574:
553:Phase modulation
508:
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2926:Wayback Machine
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2609:
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2483:
2479:
2448:(19): 1127–30.
2442:Current Biology
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2371:
2326:
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2269:
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2258:
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691:
690:
682:
679:
678:
670:
659:
653:
650:
607:
605:
595:
583:
572:
560:continuous wave
555:
546:
530:goodness of fit
518:
512:close to zero.
485:
462:
445:
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439:
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372:
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75:
24:
17:
12:
11:
5:
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2907:External links
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2110:
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2035:Optics Express
2021:
1991:
1965:
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1828:
1771:
1736:(6): 668–670.
1715:
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2888:
2880:
2876:
2872:
2868:
2864:
2860:
2856:
2852:
2848:
2844:
2841:(2): 024001.
2840:
2836:
2832:
2825:
2817:
2813:
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2803:
2799:
2795:
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2688:(2): 220–32.
2687:
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2638:
2634:
2630:
2626:
2622:
2618:
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2587:
2583:
2580:(5): 551–61.
2579:
2575:
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2542:
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2524:
2516:
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2389:(4): 1271–5.
2388:
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2356:
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2347:
2343:
2340:(2): 316–30.
2339:
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2331:
2324:
2316:
2312:
2307:
2302:
2298:
2294:
2290:
2286:
2283:(3): 676–85.
2282:
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2111:9780123740250
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1951:3rd edition.
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1806:10044/1/15590
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1303:
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1276:
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1259:
1254:
1239:
1236:
1219:
1214:
1207:
1202:
1192:
1189:
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1180:
1177:
1169:
1165:
1161:
1142:
1137:
1130:
1123:
1118:
1109:
1106:
1102:
1098:
1095:
1092:
1089:
1081:
1078:
1060:
1057:
1051:
1046:
1037:
1032:
1028:
1024:
1021:
1018:
1010:
1004:
999:
990:
987:
981:
976:
967:
963:
959:
954:
951:
948:
940:
934:
929:
914:
900:
880:
870:
854:
848:
841:
838:
835:
832:
826:
821:
809:
806:
801:
795:
786:
781:
769:
764:
757:
748:
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734:
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718:
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639:
636:
632:
629:
625:
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618:
615: –
614:
610:
609:Find sources:
603:
599:
593:
592:
587:This article
585:
581:
576:
575:
567:
565:
561:
550:
544:Gating method
541:
539:
535:
531:
527:
523:
513:
509:
493:
486:
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476:
469:
466:
463:
459:
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402:
388:
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352:
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325:
321:
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272:
268:
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201:
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182:
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171:
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127:
123:
117:
111:
104:
103:
102:
100:
96:
92:
88:
84:
80:
70:
68:
67:concentration
64:
60:
55:
50:
48:
44:
40:
36:
32:
28:
22:
2899:. p. 4.
2887:
2838:
2834:
2824:
2781:
2777:
2767:
2734:
2730:
2720:
2685:
2681:
2671:
2628:
2624:
2610:
2577:
2573:
2567:
2540:
2536:
2523:
2490:
2486:
2480:
2445:
2441:
2431:
2386:
2382:
2372:
2337:
2333:
2323:
2280:
2276:
2266:
2250:. Elsevier.
2246:
2239:
2196:
2192:
2179:
2142:
2138:
2128:
2089:
2073:
2038:
2034:
2024:
2012:. Retrieved
2008:the original
2003:
1994:
1982:. Retrieved
1977:
1968:
1943:
1900:
1896:
1886:
1845:
1841:
1831:
1788:
1784:
1774:
1733:
1729:
1719:
1694:
1691:FRET imaging
1682:
1663:
1646:EGF receptor
1638:
1635:Applications
1630:
1240:
1237:
1170:
1166:
1162:
1082:
1079:
915:
871:
719:
675:
660:
651:
641:
634:
627:
620:
608:
596:Please help
591:verification
588:
556:
547:
519:
510:
438:
426:
417:FRET imaging
394:
219:
169:
91:ground state
76:
54:fluorescence
51:
30:
26:
25:
2784:(1): 9848.
2170:10044/1/578
391:Measurement
79:fluorophore
39:fluorophore
2946:Categories
2617:Svoboda, K
2145:(1): 180.
1712:References
624:newspapers
2879:210883495
2863:2050-6120
2798:2045-2322
2737:: 68–75.
2080:Mycek, MA
2014:April 26,
1984:April 26,
1927:1083-3668
1870:1364-5528
1815:1932-7447
1750:1474-9092
1685:coenzymes
1666:flavanols
1607:π
1595:ω
1576:τ
1562:ω
1528:ω
1519:
1497:∑
1484:ω
1475:
1450:∑
1410:τ
1396:ω
1382:τ
1379:ω
1360:ω
1351:
1320:∑
1307:ω
1298:
1273:∑
1193:ω
1178:τ
1110:
1096:δ
1090:τ
1058:δ
1015:∑
988:δ
945:∑
901:δ
881:δ
842:τ
796:−
745:∑
730:χ
483:⊗
434:convolved
403:τ
375:τ
248:τ
240:is time,
194:∑
186:τ
153:τ
142:−
81:which is
63:viscosity
2922:Archived
2871:31972557
2816:25993434
2759:24094780
2751:22340533
2712:20096786
2663:18556515
2619:(2008).
2602:54398436
2594:16971112
2559:16481358
2515:11090353
2464:10531012
2231:21054116
2120:17519182
2082:(2007).
2065:20588879
1955:(2006).
1953:Springer
1935:15568944
1878:22324050
1823:96097931
1766:42881416
1758:18528549
1700:See also
654:May 2014
570:Analysis
99:ensemble
2843:Bibcode
2807:4438616
2703:2884063
2682:Methods
2654:2745709
2633:Bibcode
2625:Science
2495:Bibcode
2487:Science
2472:7640970
2423:1741380
2391:Bibcode
2364:1519759
2355:6986422
2315:8471720
2306:1262380
2285:Bibcode
2222:2966493
2201:Bibcode
2147:Bibcode
2094:495–524
2043:Bibcode
1905:Bibcode
1850:Bibcode
1842:Analyst
1674:NAD(P)H
1238:Where:
638:scholar
83:excited
2877:
2869:
2861:
2814:
2804:
2796:
2757:
2749:
2710:
2700:
2661:
2651:
2600:
2592:
2557:
2513:
2470:
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