Analysis of heterogeneous fluorescence photobleaching by video kinetics imaging: The method of cumulants

Koppel, Dennis E.; Carlson, Curt A.; Smilowitz, Henry M.

doi:10.1111/j.1365-2818.1989.tb02882.x

Cited by 22 publications

(17 citation statements)

References 7 publications

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“…Since the stretched exponential or Kohlrausch function can be considered as a superposition of mono-exponential decays, this result suggests a multi-step bleaching process. A similar behavior has been previously described for complex fluorescence lifetime and bleaching kinetics (Koppel et al, 1989;Lee et al, 2001). For a (pseudo)-unimolecular one-step excitation and bleaching process, there is a simple relation between the bleach rate constant, k B , and the quantum yield, Q F , first derived by Hirschfeld (1976) (Hirschfeld, 1976):…”

Section: Heterogeneous Photobleaching Of Bch2 In Fatty-acid Loaded Cellsmentioning

confidence: 70%

Quantitative assessment of sterol traffic in living cells by dual labeling with dehydroergosterol and BODIPY-cholesterol

Wüstner

Solanko

Sokol

et al. 2011

Chemistry and Physics of Lipids

105

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Section: Heterogeneous Photobleaching Of Bch2 In Fatty-acid Loaded Cellsmentioning

confidence: 70%

Quantitative assessment of sterol traffic in living cells by dual labeling with dehydroergosterol and BODIPY-cholesterol

Wüstner

Solanko

Sokol

et al. 2011

Chemistry and Physics of Lipids

105

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“…In some cases, the StrExp function can be used to discern a physical mechanism underlying the observed relaxation process [24]. More often, it is used as empirical fitting function to describe an experiment in quantitative terms [26-28,32]. Here, we use the StrExp function as empirical function to describe FLIP image sets on a pixel basis.…”

Section: Resultsmentioning

confidence: 99%

Quantitative fluorescence loss in photobleaching for analysis of protein transport and aggregation

et al. 2012

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BackgroundFluorescence loss in photobleaching (FLIP) is a widely used imaging technique, which provides information about protein dynamics in various cellular regions. In FLIP, a small cellular region is repeatedly illuminated by an intense laser pulse, while images are taken with reduced laser power with a time lag between the bleaches. Despite its popularity, tools are lacking for quantitative analysis of FLIP experiments. Typically, the user defines regions of interest (ROIs) for further analysis which is subjective and does not allow for comparing different cells and experimental settings.ResultsWe present two complementary methods to detect and quantify protein transport and aggregation in living cells from FLIP image series. In the first approach, a stretched exponential (StrExp) function is fitted to fluorescence loss (FL) inside and outside the bleached region. We show by reaction–diffusion simulations, that the StrExp function can describe both, binding/barrier–limited and diffusion-limited FL kinetics. By pixel-wise regression of that function to FL kinetics of enhanced green fluorescent protein (eGFP), we determined in a user-unbiased manner from which cellular regions eGFP can be replenished in the bleached area. Spatial variation in the parameters calculated from the StrExp function allow for detecting diffusion barriers for eGFP in the nucleus and cytoplasm of living cells. Polyglutamine (polyQ) disease proteins like mutant huntingtin (mtHtt) can form large aggregates called inclusion bodies (IB’s). The second method combines single particle tracking with multi-compartment modelling of FL kinetics in moving IB’s to determine exchange rates of eGFP-tagged mtHtt protein (eGFP-mtHtt) between aggregates and the cytoplasm. This method is self-calibrating since it relates the FL inside and outside the bleached regions. It makes it therefore possible to compare release kinetics of eGFP-mtHtt between different cells and experiments.ConclusionsWe present two complementary methods for quantitative analysis of FLIP experiments in living cells. They provide spatial maps of exchange dynamics and absolute binding parameters of fluorescent molecules to moving intracellular entities, respectively. Our methods should be of great value for quantitative studies of intracellular transport.

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“…Counterstaining tissue sections with solutions derived from flow cytometry such as crystal violet 27 or pontamine sky blue 28 to eliminate background autofluorescence may be impractical as aqueous processing can produce postexperimental drug diffusion. Time-resolved fluorometry, which utilizes the different decay times between autofluorescent molecules and fluorescent tags, has also been used to compensate for autofluorescence, 29,30 but utilizes a sophisticated fluorescence microscopy system and lengthy excitation times. In addition, this technique may require special fluorescent tags such as lanthanide chelates, 31 which may not be readily available individually or conjugated to compounds of interest.…”

Section: Discussionmentioning

confidence: 99%

Measurement of drug distribution in vascular tissue using quantitative fluorescence microscopy

Wan

Lovich

Hwang

et al. 1999

Journal of Pharmaceutical Sciences

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Quantitative tools to assess vascular macromolecular distributions have been limited by low signal-to-noise ratios, reduced spatial resolution, postexperimental motion artifact, and the inability to provide multidimensional drug distribution profiles. Fluorescence microscopy offers the potential of identifying exogenous compounds within intact tissue by reducing autofluorescence, the process by which endogenous compounds emit energy at the same wavelength as fluorescent labels. A new technique combining fluorescence microscopy with digital postprocessing has been developed to address these limitations and is now described in detail. As a demonstration, histologic cross-sections of calf carotid arteries that had been loaded endovascularly with FITC-Dextran (20 kD) ex vivo were imaged at two different locations of the electromagnetic spectrum, one exciting only autofluorescent structures and the other exciting both autofluorescent elements and exogenous fluorescent labels. The former image was used to estimate the autofluorescence in the latter. Subtraction of the estimated autofluorescence resulted in an autofluorescence-corrected image. A standard curve, constructed from arteries that were incubated until equilibrium in different bulk phase concentrations of FITC-Dextran, was used to convert fluorescent intensities to tissue concentrations. This resulted in a concentration map with spatial resolution superior to many of the previous methods used to quantify macromolecular distributions. The transvascular concentration profiles measured by quantitative fluorescence microscopy compared favorably with those generated from the proven en face serial sectioning technique, validating the former. In addition, the fluorescence method demonstrated markedly increased spatial resolution. This new technique may well prove to be a valuable tool for elucidating the mechanisms of macromolecular transport, and for the rational design of drug delivery systems.

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Analysis of heterogeneous fluorescence photobleaching by video kinetics imaging: The method of cumulants

Cited by 22 publications

References 7 publications

Quantitative assessment of sterol traffic in living cells by dual labeling with dehydroergosterol and BODIPY-cholesterol

Quantitative assessment of sterol traffic in living cells by dual labeling with dehydroergosterol and BODIPY-cholesterol

Quantitative fluorescence loss in photobleaching for analysis of protein transport and aggregation

Measurement of drug distribution in vascular tissue using quantitative fluorescence microscopy

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