Abstract：
:Fluorescence contributions from immobile sources present a challenge for fluorescence fluctuation spectroscopy (FFS) because the absence of signal fluctuations from stationary fluorophores leads to a biased analysis. This is especially of concern for cellular FFS studies on proteins that interact with immobile structures. Here we present a method that correctly analyzes FFS experiments in the presence of immobile sources by exploiting selective photobleaching of immobile fluorophores. The fluorescence decay due to photobleaching of the immobile species is modeled taking into account the nonuniform illumination volume. The experimentally observed decay curve serves to separate the mobile and immobile fluorescence contribution, which is used to calculate the molecular brightness from the FFS data. We experimentally verify this approach in vitro using the fluorescent protein EGFP as our immobilized species and a diffusing dye of a different color as the mobile one. For this special case, we also use an alternative method of determining the brightness by spectrally resolving the two species. By conducting a dilution study, we show that the correct parameters are obtained using either technique for a wide range of mobile fractions. To demonstrate the application of our technique in living cells, we perform experiments using the histone core protein H2B fused with EGFP expressed in COS-1 cells. We successfully recovered the brightness of the mobile fraction of H2B-EGFP.
journal_name
Biophys Jjournal_title
Biophysical journalauthors
Skinner JP,Chen Y,Müller JDdoi
10.1529/biophysj.107.115642subject
Has Abstractpub_date
2008-03-15 00:00:00pages
2349-60issue
6eissn
0006-3495issn
1542-0086pii
S0006-3495(08)70579-4journal_volume
94pub_type
杂志文章abstract：:Myosin subfragment 1 (S1) can be specifically modified at Lys-553 with the fluorescent probe FHS (6-[fluorescein-5(and 6)-carboxamido]hexanoic acid succinimidyl ester) (Bertrand, R., J. Derancourt, and R. Kassab. 1995. Biochemistry. 34:9500-9507), and solvent quenching of FHS-S1 with iodide has been shown to be sensit...
journal_title：Biophysical journal
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journal_title：Biophysical journal
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journal_title：Biophysical journal
pub_type： 杂志文章
doi：10.1016/S0006-3495(94)80767-2
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journal_title：Biophysical journal
pub_type： 杂志文章
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更新日期：2000-12-01 00:00:00
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pub_type： 杂志文章
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更新日期：2020-07-07 00:00:00
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更新日期：2018-02-27 00:00:00
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更新日期：2020-10-20 00:00:00
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pub_type： 杂志文章
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更新日期：2005-04-01 00:00:00
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pub_type： 杂志文章
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更新日期：1994-07-01 00:00:00
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journal_title：Biophysical journal
pub_type： 杂志文章
doi：10.1016/j.bpj.2019.03.012
更新日期：2019-04-23 00:00:00
