Picosecond fluorescence studies of xanthene dyes

Fleming, G. R.; Knight, Andrew W.; Morris, John; Morrison, Richard J. S.; Robinson, G. W.

doi:10.1021/ja00455a017

Cited by 335 publications

(189 citation statements)

References 4 publications

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“…Many of the compounds currently used for immunofluorescent labeling such as fluorescein and rbodamine were chosen because of their high fluorescence quantum yields (4,f), and have comparatively low yields of singlet oxygen (tO2). Eosin, a brominated derivative of fluorescein, has a singlet oxygen quantum yield (4%~) approximately 19 times greater than fluorescein (Gandin et al, 1983), while still possessing moderate fluorescence (~20% as bright as fluorescein) (Fleming et al, 1977). We found that eosin conjugated via its 5-isothiocyanate derivative to secondary anti-immunoglobnlin antibodies or to streptavidin could be used for both high quality immunofluorescence imaging and for the efficient photooxiclarion of DAB for subsequent electron microscopic examination.…”

mentioning

confidence: 72%

“…A more likely way in which to improve this technique is to exploit the fact that the ~f and 6~o~ of most compounds are highly dependent on the solvent medium. For example, the ~l,f of eosin is threefold higher in ethanol than in water (Fleming et al, 1977) whereas the ~to2 is greater in water than ethanol (Gandin et al, 1983). We are currently working to determine the optimal medium for maximizing the fluorescence quantum yield of eosin as well as that for maximizing DAB photooxidation.…”

Section: Discussionmentioning

confidence: 99%

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Fluorescence photooxidation with eosin: a method for high resolution immunolocalization and in situ hybridization detection for light and electron microscopy.

Deerinck

Martone

Lev‐Ram

et al. 1994

The Journal of Cell Biology

191

194

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Abstract. A simple method is described for highresolution light and electron microscopic immunolocalization of proteins in cells and tissues by immunofluorescence and subsequent photooxidation of diaminobenzidine tetrahydrochloride into an insoluble osmiophilic polymer. By using eosin as the fluorescent marker, a substantial improvement in sensitivity is achieved in the photooxidation process over other conventional fluorescent compounds. The technique allows for precise correlative immunolocalization studies on the same sample using fluorescence, transmitted light and electron microscopy. Furthermore, because eosin is smaller in size than other conventional markers, this method results in improved penetration of labeling reagents compared to gold or enzyme based procedures. The improved penetration allows for three-dimensional immunolocalization using high voltage electron microscopy. Fluorescence photooxidation can also be used for high resolution light and electron microscopic localization of specific nucleic acid sequences by in situ hybridization utilizing biotinylated probes followed by an eosin-streptavidin conjugate.F LUORESCENT labeling techniques have become increasingly popular in many areas of cell biology, particularly due to the expanded use of laser scanning confocal microscopy. Immunofluorescent localization of proteins and fluorescence-based in situ hybridization detection of specific nucleic acid sequences in cells and tissues have several advantages over other labeling procedures including relative simplicity, shorter processing times, high sensitivity, and good spatial resolution. One of the major disadvantages of fluorescent labeling has been the inability to use this technique for correlated light and electron microscopic studies in cases requiring better than light microscopic resolution. Several methods have been introduced in an attempt to overcome this limitation. These include the use of anti-fluorophore antibodies, e.g., anti-fluorescein, conjugated to peroxidase (Peters et al., 1990) and the process of fluorescence photooxidation of diaminobenzidine (DAB)? This latter method, first described by Maranto (1982), involves the use of fluorescent dyes to oxidize DAB into an insoluble reaction product.Photooxidation represents a relatively simple method to render fluorescently labeled preparations suitable for elec- tron nficroscopic examination. Briefly, a fluorescent dye is excited in the presence of DAB and gradually an oxidized DAB reaction product forms. The reaction product is highly insoluble and easily visible with ordinary transmitted light microscopy. Since the DAB reaction product is osmiophilic, this technique can be used for correlative light and electron microscopic studies using a variety of fluorescent compounds including Lucifer yellow, DiI, Bodipy ceramide, fluorogold, and fluororuby (Pagano et al., 1989;Bentivoglio and Su, 1990;yon Bartheld et al., 1990;Balercia et al., 1992;Lubke, 1993;Papadopoulos and Dori, 1993;Schmued and Snavely, 1993;Takizawa et al., 1993). A poten...

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mentioning

confidence: 72%

Section: Discussionmentioning

confidence: 99%

Fluorescence photooxidation with eosin: a method for high resolution immunolocalization and in situ hybridization detection for light and electron microscopy.

Deerinck

Martone

Lev‐Ram

et al. 1994

The Journal of Cell Biology

191

194

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“…The fit of this model to both photohemolysis (Valenzeno and Pooler, 1982a) and nerve photomodification suggested that this assumption may have validity. Intersystem crossing rates have indeed been shown to be reduced in bulk phase solvents of polarity lower than water for four of the halogenated fluoresceins (Fleming et al, 1977). This effect has been attributed to shifts in electron energy levels with solvent.…”

Section: Characteristics Of Singlet Oxygen In Membranesmentioning

confidence: 92%

Photomodification of Biological Membranes With Emphasis on Singlet Oxygen Mechanisms

Valenzeno

1987

Photochem & Photobiology

263

118

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Abstract-This review discusses photomodification of biological membranes and model membrane systems. Current concepts of membrane structure are first reviewed briefly. The role of preillumination association of sensitizer with membranes as it relates to photomodification rate is discussed, as well as the role of singlet oxygen in membrane photomodification. Finally the characteristics of singlet oxygen generation in membranes are considered. The evidence clearly indicates that membrane photomodification cannot be understood based only on the properties of sensitizers and singlet oxygen in aqueous solution. Rather the properties of sensitizers in association with membranes are the determinants of membrane photomodification. These properties differ significantly in aqueous solution and in membranes.

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“…The radiative lifetimes r rad are determined by application of the Strickler-Berg formula [43,44] (for reported T RAD data of eosin Y see refs. [11,[45][46][47][48]). The fluorescence lifetime T s 1 is given by eq.…”

Section: Determination Of Steady-state Absorption and Fluorescence Spmentioning

confidence: 99%

Intersystem-crossing and excited-state absorption in eosin Y solutions determined by picosecond double pulse transient absorption measurements

Penzkofer

Beidoun

Daiber

1992

Journal of Luminescence

100

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Picosecond fluorescence studies of xanthene dyes

Cited by 335 publications

References 4 publications

Fluorescence photooxidation with eosin: a method for high resolution immunolocalization and in situ hybridization detection for light and electron microscopy.

Fluorescence photooxidation with eosin: a method for high resolution immunolocalization and in situ hybridization detection for light and electron microscopy.

Photomodification of Biological Membranes With Emphasis on Singlet Oxygen Mechanisms

Intersystem-crossing and excited-state absorption in eosin Y solutions determined by picosecond double pulse transient absorption measurements

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