ArticleChemphyschem : a European journal of chemical physics and physical chemistry2007
Circumvention of fluorophore photobleaching in fluorescence fluctuation experiments: a beam scanning approach.
Article in Chemphyschem : a European journal of chemical physics and physical chemistry, 2007. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 11 papers.
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Who cites it
11 citing papers in PubMed.
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- Confining Fluorescent Probes in Nanochannels to Construct Reusable Nanosensors for Ion Current and Fluorescence Dual Gating.Nanomaterials (Basel, Switzerland) · 2022Article
- Using FCS to accurately measure protein concentration in the presence of noise and photobleaching.Biophysical journal · 2021Article
- Scanning single-molecule counting system for Eprobe with highly simple and effective approach.PloS one · 2020Article
- N-acetyl ornithine deacetylase is a moonlighting protein and is involved in the adaptation of Entamoeba histolytica to nitrosative stress.Scientific reports · 2016Article
- Article
- The proapoptotic protein tBid forms both superficially bound and membrane-inserted oligomers.Biophysical journal · 2014Article
- Measuring the lamellarity of giant lipid vesicles with differential interference contrast microscopy.Biophysical journal · 2013Article
- Simultaneous pH and temperature measurements using pyranine as a molecular probe.Journal of fluorescence · 2011Article
- Recent developments in fluorescence correlation spectroscopy for diffusion measurements in planar lipid membranes.International journal of molecular sciences · 2010Review
- Accurate determination of membrane dynamics with line-scan FCS.Biophysical journal · 2009Article
Corrections and comments
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Authors and funding
4 authors.
Funding
Abstract
Photobleaching is a fluorophore-damaging process that commonly afflicts single-molecule fluorescence studies. It becomes an especially severe problem in fluorescence fluctuation experiments when studying slowly diffusing particles. One way to circumvent this problem is to use beam scanning to decrease the residence time of the fluorophores in the excitation volume. We report a systematic study of the effects of circular beam scanning on the photobleaching of fluorescent particles as observed in single-photon excitation fluorescence fluctuation experiments. We start by deriving a simple expression relating the average detected fluorescence to the photobleaching cross section of the fluorophores. We then perform numerical calculations of the spatial distribution of fluorescent particles in order to understand under which conditions beam scanning can prevent the formation of a photobleaching hole. To support these predictions, we show experimental results obtained for large unilamellar vesicles containing a small amount of the fluorescent lipophilic tracer DiD. We establish the required scanning radius and frequency range in order to obtain sufficient reduction of the photobleaching effect for that system. From the detected increase in fluorescence upon increase in scanning speed, we estimate the photobleaching cross section of DiD.
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