Fluorescence photobleaching recovery techniques for translational and slow rotational diffusion in solution and on cell surfaces

Koppel, Dennis E.

doi:10.1042/bst0140842

Cited by 9 publications

(5 citation statements)

References 14 publications

(18 reference statements)

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“…With a few notable exceptions where photobleaching provides valuable information (Peters et al ., 1974; Koppel, 1986; Phair & Misteli, 2000), the loss of fluorescence that accompanies bleaching inevitably decreases the signal‐to‐noise ratio and so reduces the quality of images obtained by fluorescence microscopy (Pawley, 1995; Stelzer, 1998). If signal loss is severe, it can prevent the acquisition of full data sets; for example, when collecting a succession of images through a sample, or over time.…”

Section: Introductionmentioning

confidence: 99%

Minimizing photobleaching during confocal microscopy of fluorescent probes bound to chromatin: role of anoxia and photon flux

Bernaś

Zarębski

Dobrucki

et al. 2004

Journal of Microscopy

114

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SummaryExposure to light can destroy the ability of a molecule to fluoresce. Such photobleaching limits the use of fluorescence and confocal microscopy in biological studies. Loss of fluorescence decreases the signal-to-noise ratio and so image resolution; it also prevents the acquisition of meaningful data late during repeated scanning (e.g. when collecting three-dimensional images). The aim of this work was to investigate the role of oxygen in the photobleaching of fluorophores bound to DNA in fixed cells, and to explore whether anoxia could minimize such bleaching. Anoxia significantly reduced bleaching rates and changed the order of reaction of both propidium iodide (an intercalator) and chromomycin A 3 (a minor-groove binder) bound to DNA; it afforded the greatest protection at low photon fluxes. However, it had no effect on the bleaching of the green fluorescent protein (GFP) covalently attached to a histone and so bound to DNA, probably because the protein shielded the chromophore from oxygen. Bleaching of all three fluorophores depended on photon flux. Practical ways of minimizing bleaching were examined, and examples of three-dimensional images of DNA marked by propidium and GFP (collected under standard and optimized conditions) are presented.

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Section: Introductionmentioning

confidence: 99%

Minimizing photobleaching during confocal microscopy of fluorescent probes bound to chromatin: role of anoxia and photon flux

Bernaś

Zarębski

Dobrucki

et al. 2004

Journal of Microscopy

114

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“…With the capability of probing transport over length scales as small as a few micrometers, fluorescence recovery after photobleaching (FRAP) is particularly well suited for measuring slow diffusion and diffusion in confined regions. This technique is therefore frequently applied to biological systems (e.g., Golan and Veatch, 1980;Jacobson and Wojcieszyn, 1984;Elson, 1986;Koppel, 1986), most commonly the cell membrane and cytoplasm. It also has been applied to Langmuir-Blodgett films (Weiss et al, 1982;Wright et al, 1988;Pachence et al, 1990), polymer films (Smith, 1982), and lipid bilayers (Smith et al, 1979) as well as to proteins adsorbed at the solid-liquid interface (Burghardt and Axelrod, 1981;Tilton et al, 1990a,b; Rabe and Tilton, 1993;Gaspers et al, 1994).…”

Section: Introductionmentioning

confidence: 99%

Effect of concentration quenching on fluorescence recovery after photobleaching measurements

Robeson¹,

Tilton²

1995

Biophysical Journal

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Standard analysis of fluorescence recovery after photobleaching (FRAP) data is valid only if the quantum yield of unphotobleached fluorophores is independent of concentration, yet close molecular packing in two-dimensional systems may lead to significant fluorescence concentration quenching. Using total internal reflection fluorescence, we quantified the surface concentration dependence of the relative quantum yield of fluorescein isothiocyanate-labeled proteins adsorbed to polymeric surfaces before performing measurements of fluorescence recovery after pattern photobleaching. Adsorbed layers of FITC-labeled ribonuclease A displayed significant concentration quenching, and thus the standard FRAP analysis method was unacceptable. We present an extended FRAP analysis procedure that accounts for the changing quantum yield of diffusing fluorophores in systems that are influenced by concentration quenching. The extended analysis shows that if concentration quenching conditions prevail, there may be significant error in the transport parameters obtained from FRAP measurements by using the standard procedures.

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“…It forms the basis of a variety of techniques designed to follow probe translational and rotational dynamics (e.g. Koppel, 1986). Moreover, the spatial heterogeneity of photobleaching rate constants in individual cells is in itself of interest (Benson et al, 1985).…”

Section: Introductionmentioning

confidence: 99%

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

Koppel

Carlson

Smilowitz

1989

Journal of Microscopy

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SUMMARY The method of cumulants has been applied to digital video fluorescence microscopy. The method is used to reconstruct the distribution of fluorescent molecules before the initiation of fluorescence photobleaching, and to characterize heterogeneous photobleaching by imaging one or more of the cumulants of the bleaching decay rate. Using the pipelined pixel processor of the image analysis system for the bulk of the calculations, rather than the general‐purpose host‐computer CPU, the video kinetics imaging can be performed in near real‐time. The method is applied to chick embryo myotubes labelled with fluorescein‐conjugated α‐bungarotoxin. The pre‐bleach fluorescence distribution is derived, and the image of fluorescein fluorescence is separated from glutaraldehyde‐induced autofluorescence on the basis of the spatially resolved average photobleaching decay rate.

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Fluorescence photobleaching recovery techniques for translational and slow rotational diffusion in solution and on cell surfaces

Cited by 9 publications

References 14 publications

Minimizing photobleaching during confocal microscopy of fluorescent probes bound to chromatin: role of anoxia and photon flux

Minimizing photobleaching during confocal microscopy of fluorescent probes bound to chromatin: role of anoxia and photon flux

Effect of concentration quenching on fluorescence recovery after photobleaching measurements

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

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