Coumarin 6, Hypericin, Resorufins, and Flavins: Suitable Chromophores for Fluorescence Correlation Spectroscopy of Biological Molecules

In this work we show that the dyes coumarin 6, hypericin, 7-O-ethylresorufin and resorufin are suitable for fluorescence correlation spectroscopy (FCS) and demonstrate the use of these dyes in physiologically relevant protein studies. Since coumarins are metabolised by cytochromes P450, the binding of coumarin 6 to cytochrome P450 3A4 was investigated by FCS. Coumarin 6 appears to be a very bright non-covalent cytochrome P450 label. When titrating cytochrome P450 3A4 with coumarin 6, the diffusion time of the coumarin 6/ cytochrome P450 3A4 complex increases roughly two-fold at protein concentrations higher than 1 μmol l-1, indicating the formation of cytochrome aggregates. FCS of the flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) shows that both endogenous dyes undergo photobleaching. Moreover, FAD appears to be present to great extent, as a non-fluorescent intramolecular complex. Analysis of the FCS data of the flavoprotein NADPH-cytochrome P450 oxidoreductase (molecular weight 76 500) yielded two components. While the slow component corresponds to a globular protein with the molecular weight about 75 000, the fast component appears to be due to free diffusing FMN and FAD molecules. The amount of free FMN and FAD increases with increasing laser power. At high laser power a complete photodissociation of FMN and FAD occurs.

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Diffusion of nanoparticles within a semidilute polyelectrolyte solution

Soft Matter ◽

10.1039/c9sm01313j ◽

2019 ◽

Vol 15 (38) ◽

pp. 7616-7622 ◽

Cited By ~ 2

Author(s):

Kavindya K. Senanayake ◽

Namita Shokeen ◽

Ehsan Akbari Fakhrabadi ◽

Matthew W. Liberatore ◽

Ashis Mukhopadhyay

Keyword(s):

Molecular Weight ◽

Gold Nanoparticles ◽

High Molecular Weight ◽

Polyacrylic Acid ◽

Fluorescence Correlation Spectroscopy ◽

Correlation Spectroscopy ◽

Polyelectrolyte Solution ◽

Fluorescence Correlation ◽

Semidilute Solution

We studied the diffusion of charged gold nanoparticles within a semidilute solution of weakly charged polyelectrolyte, polyacrylic acid (PAA) of high molecular weight (Mw = 106 g mol−1) by using fluorescence correlation spectroscopy (FCS).

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Fluorescence Correlation Spectroscopy Combined with Multiphoton Laser Scanning Microscopy—A Practical Guideline

Applied Sciences ◽

10.3390/app11052122 ◽

2021 ◽

Vol 11 (5) ◽

pp. 2122

Author(s):

Jeemol James ◽

Jonas Enger ◽

Marica B. Ericson

Keyword(s):

Biomedical Research ◽

Fluorescence Correlation Spectroscopy ◽

Laser Power ◽

Laser Scanning ◽

Correlation Spectroscopy ◽

Operating Conditions ◽

Laser Scanning Microscopy ◽

Scanning Microscopy ◽

Fluorescence Correlation ◽

Practical Guideline

Multiphoton laser scanning microscopy (MPM) has opened up an optical window into biological tissues; however, imaging is primarily qualitative. Cell morphology and tissue architectures can be clearly visualized but quantitative analysis of actual concentration and fluorophore distribution is indecisive. Fluorescence correlation spectroscopy (FCS) is a highly sensitive photophysical methodology employed to study molecular parameters such as diffusion characteristics on the single molecule level. In combination with laser scanning microscopy, and MPM in particular, FCS has been referred to as a standard and highly useful tool in biomedical research to study diffusion and molecular interaction with subcellular precision. Despite several proof-of-concept reports on the topic, the implementation of MPM-FCS is far from straightforward. This practical guideline aims to clarify the conceptual principles and define experimental operating conditions when implementing MPM-FCS. Validation experiments in Rhodamine solutions were performed on an experimental MPM-FCS platform investigating the effects of objective lens, fluorophore concentration and laser power. An approach based on analysis of time-correlated single photon counting data is presented. It is shown that the requirement of high numerical aperture (NA) objective lenses is a primary limitation that restricts field of view, working distance and concentration range. Within these restrictions the data follows the predicted theory of Poisson distribution. The observed dependence on laser power is understood in the context of perturbation on the effective focal volume. In addition, a novel interpretation of the effect on measured diffusion time is presented. Overall, the challenges and limitations observed in this study reduce the versatility of MPM-FCS targeting biomedical research in complex and deep tissue—being the general strength of MPM in general. However, based on the systematic investigations and fundamental insights this report can serve as a practical guide and inspire future research, potentially overcoming the technical limitations and ultimately allowing MPM-FCS to become a highly useful tool in biomedical research.

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EGF Receptor Stalls upon Activation as Evidenced by Complementary Fluorescence Correlation Spectroscopy and Fluorescence Recovery after Photobleaching Measurements

International Journal of Molecular Sciences ◽

10.3390/ijms20133370 ◽

2019 ◽

Vol 20 (13) ◽

pp. 3370 ◽

Cited By ~ 4

Author(s):

György Vámosi ◽

Elza Friedländer-Brock ◽

Shehu M. Ibrahim ◽

Roland Brock ◽

János Szöllősi ◽

...

Keyword(s):

Time Scales ◽

Fluorescence Correlation Spectroscopy ◽

Time Course ◽

Fluorescence Recovery After Photobleaching ◽

Slow Component ◽

Chinese Hamster ◽

Correlation Spectroscopy ◽

Fluorescence Recovery ◽

Brownian Diffusion ◽

Fluorescence Correlation

To elucidate the molecular details of the activation-associated clustering of epidermal growth factor receptors (EGFRs), the time course of the mobility and aggregation states of eGFP tagged EGFR in the membranes of Chinese hamster ovary (CHO) cells was assessed by in situ mobility assays. Fluorescence correlation spectroscopy (FCS) was used to probe molecular movements of small ensembles of molecules over short distances and time scales, and to report on the state of aggregation. The diffusion of larger ensembles of molecules over longer distances (and time scales) was investigated by fluorescence recovery after photobleaching (FRAP). Autocorrelation functions could be best fitted by a two-component diffusion model corrected for triplet formation and blinking. The slow, 100–1000 ms component was attributed to membrane localized receptors moving with free Brownian diffusion, whereas the fast, ms component was assigned to cytosolic receptors or their fragments. Upon stimulation with 50 nM EGF, a significant decrease from 0.11 to 0.07 μm2/s in the diffusion coefficient of membrane-localized receptors was observed, followed by recovery to the original value in ~20 min. In contrast, the apparent brightness of diffusing species remained the same. Stripe FRAP experiments yielded a decrease in long-range molecular mobility directly after stimulation, evidenced by an increase in the recovery time of the slow component from 13 to 21.9 s. Our observations are best explained by the transient attachment of ligand-bound EGFRs to immobile or slowly moving structures such as the cytoskeleton or large, previously photobleached receptor aggregates.

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