Using FCS to accurately measure protein concentration in the presence of noise and photobleaching

Zhang, Lili; Perez-Romero, Carmina Angelica; Dostatni, Nathalie; Fradin, Cécile

doi:10.1016/j.bpj.2021.06.035

Cited by 4 publications

(6 citation statements)

References 49 publications

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“…This strategy would be suitable not only for fixed sample, but also for living cells. Photobleaching in live cells has been proposed, outside of the oligomerization context, to retrieve the molecular brightness and to transform the confocal images into concentration maps . However, this method cannot be used for cases in which the studied protein forms homo-oligomers, in contrast to the present work.…”

Section: Discussionmentioning

confidence: 99%

Combining Fluorescence Fluctuations and Photobleaching to Quantify Surface Density

et al. 2022

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We have established a self-calibrated method, called pbFFS for photobleaching fluctuation fluorescence spectroscopy, which aims to characterize molecules or particles labeled with an unknown distribution of fluorophores. Using photobleaching as a control parameter, pbFFS provides information on the distribution of fluorescent labels and a reliable estimation of the absolute density or concentration of these molecules. We present a complete theoretical derivation of the pbFFS approach and experimentally apply it to measure the surface density of a monolayer of fluorescently tagged streptavidin molecules, which can be used as a base platform for biomimetic systems. The surface density measured by pbFFS is consistent with the results of spectroscopic ellipsometry, a standard surface technique. However, pbFFS has two main advantages: it enables in situ characterization (no dedicated substrates are required) and can be applied to low masses of adsorbed molecules, which we demonstrate here by quantifying the density of biotin-Atto molecules that bind to the streptavidin layer. In addition to molecules immobilized on a surface, we also applied pbFFS to molecules diffusing in solution, to confirm the distribution of fluorescent labels found on a surface. Hence, pbFFS provides a set of tools for investigating the molecules labeled with a variable number of fluorophores, with the aim of quantifying either the number of molecules or the distribution of fluorescent labels, the latter case being especially relevant for oligomerization studies.

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Section: Discussionmentioning

confidence: 99%

Combining Fluorescence Fluctuations and Photobleaching to Quantify Surface Density

et al. 2022

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“…The photobleaching decay was taken into account by adding a dedicated term to the ACF (see section and Ref. (58)), which allowed extracting dynamic information from the very first curves obtained in each series, when the signal-to-noise ratio is the highest, and before photobleaching has started significantly affecting the brightness of eventual oligomers. From these fits, four characteristic times are obtained: the photophysics relaxation time τ T ≈ 20 μ s, the characteristic diffusion times of the fast and slow diffusing fractions τ Df ≈ 1 m s and τ Ds ≈ 10 - 100 m s, and the photobleaching relaxation time τ P ≈ 40 s. The measured values of these four parameters, remarkably constant over time, are shown for all three proteins in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The photo-bleaching contribution is given by (58): where t M is the FCS measurement time, τ P is the photo-bleaching characteristic time and r is the signal-to-noise at the beginning of the measurement (58).…”

Section: Discussionmentioning

confidence: 99%

“…Following the work done in (58), ACFs obtained as the result of nuclear FCS measurements were analyzed as the sum of two contributions: where G D (τ) accounted for the fluorophore dynamics (diffusion, transient immobilization, photophysics, etc…) and G P (τ) accounted for the gradual decrease in fluorescence due to continuous photobleaching in the small nuclear space.…”

Section: Discussionmentioning

confidence: 99%

“…where 𝑡 𝑀 is the FCS measurement time, 𝜏 𝑃 is the photobleaching characteristic time and 𝑟 is the signal-to-noise at the beginning of the measurement (58). Amplitude of the slow diffusive term and photobleaching.…”

Section: Fcsmentioning

confidence: 99%

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Both the transcriptional activator, Bcd, and transcriptional repressor, Cic, form small mobile oligomeric clusters in early fly embryo nuclei

Zhang,

Hodgins,

Sakib

et al. 2024

Preprint

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Transcription factors play an essential role in pattern formation during early embryo development, generating a strikingly fast and precise transcriptional response that results in sharp gene expression boundaries. To characterize the steps leading up to transcription, we performed a side-by-side comparison of the nuclear dynamics of two morphogens, a transcriptional activator, Bicoid (Bcd), and a transcriptional repressor, Capicua (Cic), both involved in body patterning along the anterior-posterior axis of the early Drosophila embryo. We used a combination of fluorescence recovery after photobleaching, fluorescence correlation spectroscopy, and single particle tracking to access a wide range of dynamical timescales. Despite their opposite effects on gene transcription, we find that Bcd and Cic have very similar nuclear dynamics, characterized by the co-existence of a freely diffusing monomer population with a number of oligomeric clusters, which range from low stoichiometry and high mobility clusters to larger, DNA-bound hubs. Our observations are consistent with the inclusion of both Bcd and Cic into transcriptional hubs or condensates, while putting constraints on the mechanism by which these form. These results fit in with the recent proposal that many transcription factors might share a common search strategy for target genes regulatory regions that makes use of their large unstructured regions, and may eventually help explain how the transcriptional response they elicit can be at the same time so fast and so precise.

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Both the transcriptional activator, Bcd, and repressor, Cic, form small mobile oligomeric clusters

Zhang,

Hodgins,

Sakib

et al. 2024

Biophysical Journal

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Using FCS to accurately measure protein concentration in the presence of noise and photobleaching

Cited by 4 publications

References 49 publications

Combining Fluorescence Fluctuations and Photobleaching to Quantify Surface Density

Combining Fluorescence Fluctuations and Photobleaching to Quantify Surface Density

Both the transcriptional activator, Bcd, and transcriptional repressor, Cic, form small mobile oligomeric clusters in early fly embryo nuclei

Both the transcriptional activator, Bcd, and repressor, Cic, form small mobile oligomeric clusters

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