Measurement of fluorescence decay time in living cells

Loeser, Ch.N.; Clark, Ellen; Maher, Marjorie; Tarkmeel, H.

doi:10.1016/0014-4827(72)90017-1

Cited by 9 publications

(6 citation statements)

References 12 publications

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“…Twenty years later other imaging fluorescence lifetime measurements in microscopes were reported (1) by comparing decay curves from an analog simulator with the fluorescence decay on an oscilloscope (Loeser and Clarck 1972;Loeser et al 1972), (2) using Ortec photon counting instrumentation (Arndt- Jovin et al 1979), and (3) with a homemade digital averager (Andreoni et al 1975(Andreoni et al , 1976Bottiroli et al 1979).…”

Section: A Short History Of the Development Of Lifetime-resolved Imagingmentioning

confidence: 98%

Fluorescence lifetime-resolved imaging

Chen

Clegg

2009

Photosynth Res

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This is a short account of fluorescence lifetime-resolved imaging, in order to acquaint readers who are not experts with the basic methods for measuring lifetime-resolved signals throughout an image. We present the early FLI (fluorescence lifetime imaging) history, review shortly the instrumentation and experimental design, discuss briefly the fundamentals of the measured fluorescence response, and introduce the basic measurement methodologies. We also emphasize the complex nature of the fluorescence response in FLI signals, and introduce certain analysis methods that are appropriate and informative for complex fluorescence decays. The advantages of model independent analyses are discussed and examples given.

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Section: A Short History Of the Development Of Lifetime-resolved Imagingmentioning

confidence: 98%

Fluorescence lifetime-resolved imaging

Chen

Clegg

2009

Photosynth Res

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“…These measurements were carried out by comparing decay curves from an analog simulator with the fluorescence decay on an oscilloscope 45,46 , with a homemade digital averager [47][48][49] , and by using Ortec photon counting instrumentation [50][51][52] . Because of low signal to noise, the measurement times were very long especially for photon counting methods when multi-exponential analyses were attempted.…”

Section: Fluorescence Lifetime-resolved Imagingmentioning

confidence: 99%

What is behind all those lifetimes anyway, and where do we go from here?

et al. 2009

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Fluorescence lifetime-resolved imaging microscopy (FLIM) has made tremendous strides in the last two decades. Exciting applications are being presented weekly, an extensive diversity of instrumentation and commercial devices have appeared and have improved dramatically, sophisticated algorithms for analysis and interpretation are now available, and FLIM is being coupled to other imaging modalities, such as spectral dispersion and anisotropy. In other words, FLIM has matured considerably, and is approaching a point where it can be routinely applied by researchers who are not involved with the instrumentation and analysis side of things. The number of interested users in FLIM has almost certainly surpassed that of the audience that previously employed single channel fluorescence lifetime measurements (in a cuvette). The reason is, of course, the imaging capability of FLIM and its exciting possibilities for biological applications, especially FRET. In this lecture I will attempt to give an overview of where we have come from together with a personal judgment on where we stand, propose possible areas of growth that will be of importance for the future of FLIM, and discuss some specific challenges that remain, especially considering the unique nature of the FLIM measurement and the complex biological and medical systems that require innovative and novel solutions from the FLIM community.

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“…Using 1 -cos α = 2 sin 2 (α/2) and sin(α) = 2 sin(α/2) cos(α/2) yields tan θ S − θ D = tan(Δ/2) (27) or…”

Section: Phase Suppression Imagingmentioning

confidence: 99%

“…Since the first lifetime measurements under a microscope were carried in the early 1970s, [27][28][29] little attention has been paid to time-resolved fluorescence microscopy (TRFM). The first techniques were developed using a flash lamp 27 or nitrogen laser, 28,29 and they used the pulse sampling method for time-resolved detection, Single-photon counting was used later. [30][31][32] Advances in high-repetition lasers for pulse excitation, TCSPC, and frequency-domain methods have permitted more reliable fluorescence lifetimes to be obtained from microscopically portioned sections.…”

Section: Introductionmentioning

confidence: 99%

[30] Fluorescence lifetime imaging microscopy: Homodyne technique using high-speed gated image intensifier

Szmacinski

Lakowicz²,

Johnson³

1994

Part B: Numerical Computer Methods

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In the previous sections we demonstrated imaging of intracellular Ca2+ using our approach to FLIM. What other analytes can be imaged using FLIM? We have now characterized the lifetime of a good number of ion indicators. Based on these studies we know that Cl- can be imaged using FLIM with probes such as SPQ or MQAE, pH can be imaged using resorufin and probes of the SNAFL and SNARF (Molecular Probes) series, and Mg2+ can be imaged using Magnesium Green, Mag-quin-2, or Mag-quin-1 (Molecular Probes). At present, the probe for K+, as PBFI, are just adequate as a lifetime probe, but it seems likely that newer probes for Na+ (Sodium Green) and K+ will be practical for effective imaging. Of course, imaging of oxygen is possible using a wide variety of fluorophores. It should be noted that a wide variety of substances and/or phenomena are known to alter decay times, acting as quenchers. These include the phenomena of resonance energy transfer, collisional quenching, temperature effects, and viscosity effects. Also, the FLIM method is not limited to microscopic objects but can be possibly used in remote imaging of any object. Hence, FLIM will allow the imaging of the chemical and physical properties of objects based on the effects of the local environment on the decay kinetics of fluorophores. The instrumentation for FLIM is presently complex and requires a moderately complex laser source, a gain-modulated image intensifier, and a slow-scan CCD camera. However, one can readily imagine the instrumentation becoming rather compact, and even all solid-state, owing to advances in laser and CCD technologies and, more importantly, advances in probe chemistry. To be specific, the dye laser shown in Fig. 1 may be replaced by a simpler UV laser, such as the 354 nm HeCd laser which has become available (Fig. 11). Intensity modulation of a continuous wave sources can be accomplished with acoustooptic modulators. The scientific slow-scan CCD cameras are presently rather expensive, but they are used in the present instrumentation because of their linearity and high dynamic range. However, the increasing use of CCD detectors suggest that even the scientific-grade CCD cameras will soon become less costly. Additionally, the frame rates of these detectors continue to increase in response to the need for faster imaging. Furthermore, the performance of the video CCD cameras is increasing, as seen by the introduction of 10-bit video analog-to-digital (A/D) converters.(ABSTRACT TRUNCATED AT 400 WORDS)

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Measurement of fluorescence decay time in living cells

Cited by 9 publications

References 12 publications

Fluorescence lifetime-resolved imaging

Fluorescence lifetime-resolved imaging

What is behind all those lifetimes anyway, and where do we go from here?

[30] Fluorescence lifetime imaging microscopy: Homodyne technique using high-speed gated image intensifier

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