FRET: Signals hidden within the noise

Leavesley, Silas J.; Rich, Thomas C.

doi:10.1002/cyto.a.22568

Cited by 4 publications

(8 citation statements)

References 13 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Among the several existing ratiometric techniques, Forster resonance energy transfer (FRET) 8 – 10 and fluorescence polarization (FP)/fluorescence anisotropy (FA) 11 , 12 are probably among the most widespread. Ratiometric determination of the efficiency of fluorescence (FRET) is typically used to analyze protein clustering and conformation.…”

Section: Introductionmentioning

confidence: 99%

“…This effect is particularly relevant in all those cases where the fluorescence imaging process is characterized by a low efficiency and can be only partially compensated by longer integration times. As a consequence, the ratio formed in the presence of weak signals can be highly inaccurate due to fluctuations in the ratio, which result in compounding error propagation 9 , 10 and wild ratio estimates. Temporal or spatial filtering could be effective in mitigating these effects but at the expenses of the temporal or spatial resolutions, 13 particularly when dealing with dynamic imaging.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Extended dynamic range imaging for noise mitigation in fluorescence anisotropy imaging

2020

View full text Add to dashboard Cite

Significance: Fluorescence polarization (FP) and fluorescence anisotropy (FA) microscopy are powerful imaging techniques that allow to translate the common FP assay capabilities into the in vitro and in vivo cellular domain. As a result, they have found potential for mapping drugprotein or protein-protein interactions. Unfortunately, these imaging modalities are ratiometric in nature and as such they suffer from excessive noise even under regular imaging conditions, preventing accurate image-feature analysis of fluorescent molecules behaviors. Aim: We present a high dynamic range (HDR)-based FA imaging modality for improving image quality in FA microscopy. Approach: The method exploits ad hoc acquisition schemes to extend the dynamic range of individual FP channels, allowing to obtain FA images with increased signal-to-noise ratio. Results: A direct comparison between FA images obtained with our method and the standard, clearly indicates how an HDR-based FA imaging approach allows to obtain high-quality images, with the ability to correctly resolve image features at different values of FA and over a substantially higher range of fluorescence intensities. Conclusion: The method presented is shown to outperform standard FA imaging microscopy narrowing the spread of the propagated error and yielding higher quality images. The method can be effectively and routinely used on any commercial imaging system and could be also translated to other microscopy ratiometric imaging modalities.

show abstract

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Extended dynamic range imaging for noise mitigation in fluorescence anisotropy imaging

2020

View full text Add to dashboard Cite

show abstract

“…The accuracy of the calculation of FRET efficiency based on intensity ratios could be increased computationally even in samples having low signal to noise ratios by applying maximum likelihood estimation (MLE) (6,7). The accuracy of the calculation of FRET efficiency based on intensity ratios could be increased computationally even in samples having low signal to noise ratios by applying maximum likelihood estimation (MLE) (6,7).…”

mentioning

confidence: 99%

“…This enormous increase in the application of FRET in biological sciences is partially due to the increase in the number of methods for evaluating FRET quantitatively, including improvements in intensity-and lifetime-based FRET methods. The accuracy of the calculation of FRET efficiency based on intensity ratios could be increased computationally even in samples having low signal to noise ratios by applying maximum likelihood estimation (MLE) (6,7). The improvement occurs because the MLE method takes the Poisson statistics inherent to photon-based detection into consideration (6,7).…”

mentioning

confidence: 99%

“…The accuracy of the calculation of FRET efficiency based on intensity ratios could be increased computationally even in samples having low signal to noise ratios by applying maximum likelihood estimation (MLE) (6,7). The improvement occurs because the MLE method takes the Poisson statistics inherent to photon-based detection into consideration (6,7). Not only intensity ratios but changes in the fluorescence lifetime can be used to estimate the FRET efficiency.…”

mentioning

confidence: 99%

See 1 more Smart Citation

The flow of events: How the sequence of molecular interactions is seen by the latest, user‐friendly high throughput flow cytometric FRET

2016

View full text Add to dashboard Cite

F€ orster (or less precisely Fluorescence) Resonance Energy Transfer (FRET) is widely used by biologists to study the clustering of biomolecules tagged appropriately with chromophores in order to decipher the inner workings of the cell. FRET is a collision-free interchromophoric relaxation process that transfers energy nonradiatively from an initially excited donor to a ground-state acceptor in a distance-dependent way. FRET occurs over interatomic distances due to resonance-based dipole-dipole interaction of chromophores without transmission of photons from donor to acceptor species. Therefore, it is inaccurate to use fluorescence with the acronym FRET, since fluorescence involves emission of photons. At distances below 1 nm, collisions between the donor and acceptor would prevail, whereas at distances higher than 10 nm, photon emission by the donor would dominate, so FRET occurs only in the near field, i.e., in the range of 1-10 nm (1-3). Measuring the efficiency of FRET can serve as a "spectroscopic ruler" within this range, and provide absolute quantitative data describing interand intramolecular interactions (4).The application of FRET in biological sciences has seen an incredible expansion lately with almost 9000 hits retrieved by PubMed using FRET as a unique keyword. While the number of published papers applying FRET fluctuated between zero and one annually before 1985, >1000 publications could be found in the PubMed database in the year of 2015. This stark contrast clearly demonstrates the astonishing expansion rate of FRET applications. Partly because of this tremendous increase in the number of FRET studies, two international discussion meetings were devoted solely to FRET in life sciences. The FRET1 meeting was held from the 27th till the 30th of March in 2011 (http://www.fret.uni-duesseldorf.de/cms/fret_1.html), and the FRET 2 conference took place between the 3rd and the 6th of April in 2016 (http://www.fret.uni-duesseldorf.de/cms), both of them in G€ ottingen. Both FRET meetings were very successful, and the participants of the first meeting compiled an excellent book about "FRET from theory to applications" (5).This enormous increase in the application of FRET in biological sciences is partially due to the increase in the number of methods for evaluating FRET quantitatively, including improvements in intensity-and lifetime-based FRET methods. The accuracy of the calculation of FRET efficiency based on intensity ratios could be increased computationally even in samples having low signal to noise ratios by applying maximum likelihood estimation (MLE) (6,7). The improvement occurs because the MLE method takes the Poisson statistics inherent to photon-based detection into consideration (6,7). Not only intensity ratios but changes in the fluorescence lifetime can be used to estimate the FRET efficiency. Fluorescence

show abstract

Generation and flow cytometric quality control of clinical‐scale TCRαβ/CD19‐depleted grafts

Bremm

Cappel

Erben

et al. 2015

Cytometry Part B Clinical

View full text Add to dashboard Cite

show abstract

FRET: Signals hidden within the noise

Cited by 4 publications

References 13 publications

Extended dynamic range imaging for noise mitigation in fluorescence anisotropy imaging

Extended dynamic range imaging for noise mitigation in fluorescence anisotropy imaging

The flow of events: How the sequence of molecular interactions is seen by the latest, user‐friendly high throughput flow cytometric FRET

Generation and flow cytometric quality control of clinical‐scale TCRαβ/CD19‐depleted grafts

Contact Info

Product

Resources

About