A Quantitative and Reliable Calibration Standard for Dual‐Color Fluorescence Cross‐Correlation Spectroscopy

Werner, Stefan; Ebenhan, Jan; Haupt, Caroline; Bacia, Kirsten

doi:10.1002/cphc.201800576

Cited by 6 publications

(6 citation statements)

References 30 publications

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“…To determine the overlap volume correction factor ( V crct .=74.6%), we performed a controlled experiment using a mixture of single-labelled and double-labelled PEGylated liposomes (Figure S1). A related calibration approach has been recently described by Werner et al 60 Besides determining V crct this experiment validates the suitability of FCCS to determine colocalization of particles of ca. 100 nm, comparable to LPNPs.…”

Section: Resultssupporting

confidence: 68%

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Stability Criterion for the Assembly of Hybrid Lipid-Polymer-Nucleic Acid Nanoparticles

Paris

Gaspar

Coelho

et al. 2022

Preprint

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Hybrid lipid-polymer-nucleic acid nanoparticles (LPNPs) provide a range of delivery strategies for nonviral gene therapy. However, due to several pairwise interactions between their components, these formulations are difficult to predict and characterize. Here, we employed, for the first time, a novel methodology based on fluorescence cross correlation spectroscopy (FCCS) to directly quantify the extent of association between polycation-DNA cores (polyplexes) and cationic liposome shells. This leads to a rapid and easy evaluation of LPNP formation. As a result, we were able to unveil two critical insights for predicting LPNP assembly. Firstly, the interaction between the polycation (polylysine) and DNA is robust, with the polycation not being displaced by liposomes. Hence, the polyplex cores and liposome shells have to be oppositely charged to associate. Secondly, and most importantly, the liposome:poliplex number ratio (ρN), as opposed to the more common charge ratio, is a critical parameter to predict stable LPNP formation. We find that ρN≥1 is required to ensure that every polyplex is enveloped by a liposome, avoiding the coexistence of oppositely charged species, thus inhibiting aggregation. Since the chosen LPNP components are common, these observations should be applicable to many LPNP systems. Furthermore, this FCCS-based methodology is relevant to other self-assembly composite materials, for applications beyond gene delivery.

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Section: Resultssupporting

confidence: 68%

“…The overlap volume correction factor (𝑉 ? @?A = 74.6%), is determined in a controlled experiment using mixtures of single-labelled and double-labelled PEGylated liposomes (section S3 and Figure S3) 47 . After correcting the crosstalk 46 , the 𝐴 ; used in Eqs.…”

Section: S2-s3mentioning

confidence: 99%

Stability Criterion for the Assembly of Hybrid Lipid-Polymer-Nucleic Acid Nanoparticles

Paris

Gaspar

Coelho

et al. 2022

Preprint

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“…Only heterocomplexes containing both Alexa 488-KKETPV 14 and Cy5-PDZ 14 contributed to cross-correlation curves of dcFCCS measurements (Fig. 1B), whose relaxation times correlated with residence times of heterocomplexes within the detection volume (37). Therefore, large complexes have longer relaxation times than small ones ( Fig.…”

Section: Results Dcfccs Assay To Capture and To Quantify Biomolecularmentioning

confidence: 93%

“…According to published procedure (37), correlation factors Cr 488 and Cr 640 were determined to be 0.58 ± 0.05 and 0.76 ± 0.02, respectively, using a doubly labeled dsDNA containing both Alexa 488 and Cy5.…”

Section: Methodsmentioning

confidence: 99%

Phase separation at the nanoscale quantified by dcFCCS

Peng

Yao

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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Liquid–liquid phase separation, driven by multivalent macromolecular interactions, causes formation of membraneless compartments, which are biomolecular condensates containing concentrated macromolecules. These condensates are essential in diverse cellular processes. Formation and dynamics of micrometer-scale phase-separated condensates are examined routinely. However, limited by commonly used methods which cannot capture small-sized free-diffusing condensates, the transition process from miscible individual molecules to micrometer-scale condensates is mostly unknown. Herein, with a dual-color fluorescence cross-correlation spectroscopy (dcFCCS) method, we captured formation of nanoscale condensates beyond the detection limit of conventional fluorescence microscopy. In addition, dcFCCS is able to quantify size and growth rate of condensates as well as molecular stoichiometry and binding affinity of client molecules within condensates. The critical concentration to form nanoscale condensates, identified by our experimental measurements and Monte Carlo simulations, is at least several fold lower than the detection limit of conventional fluorescence microscopy. Our results emphasize that, in addition to micrometer-scale condensates, nanoscale condensates are likely to play important roles in various cellular processes and dcFCCS is a simple and powerful quantitative tool to examine them in detail.

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“…Cr 640 is calculated via A x / A 640 , which are extracted from data of AF488-dsDNA. Correction factor Cd 488 is calculated via \begin{document}$ {\tau }_{488}/{\tau }_{x} $\end{document} , which are extracted from data of AF488-dsDNA (Werner et al 2018 ).…”

Section: Methodsmentioning

confidence: 99%

Quantifying phase separation at the nanoscale by dual-color fluorescence cross-correlation spectroscopy (dcFCCS)

Yao¹,

Wang²,

Chen³

2022

Biophysics Reports

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Liquid–liquid phase separation (LLPS) causes the formation of membraneless condensates, which play important roles in diverse cellular processes. Currently, optical microscopy is the most commonly used method to visualize micron-scale phase-separated condensates. Because the optical spatial resolution is restricted by the diffraction limit (~200 nm), dynamic formation processes from individual biomolecules to micron-scale condensates are still mostly unknown. Herein, we provide a detailed protocol applying dual-color fluorescence cross-correlation spectroscopy (dcFCCS) to detect and quantify condensates at the nanoscale, including their size, growth rate, molecular stoichiometry, and the binding affinity of client molecules within condensates. We expect that the quantitative dcFCCS method can be widely applied to investigate many other important phase separation systems.

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A Quantitative and Reliable Calibration Standard for Dual‐Color Fluorescence Cross‐Correlation Spectroscopy

Cited by 6 publications

References 30 publications

Stability Criterion for the Assembly of Hybrid Lipid-Polymer-Nucleic Acid Nanoparticles

Stability Criterion for the Assembly of Hybrid Lipid-Polymer-Nucleic Acid Nanoparticles

Phase separation at the nanoscale quantified by dcFCCS

Quantifying phase separation at the nanoscale by dual-color fluorescence cross-correlation spectroscopy (dcFCCS)

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