Quantitative FRET measurement based on spectral unmixing of donor, acceptor and spontaneous excitation‐emission spectra

Du, Mingde; Zhang, Chenshuang; Chen, Tongsheng

doi:10.1002/jbio.201800314

Cited by 7 publications

(8 citation statements)

References 32 publications

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“…To accurately analyze the signal amplification, FRET is also coupled with spectral unmixing technique to separate the signal contributions from the donor and the acceptor 62 , 63 . Although spectral unmixing has been rarely explored for CRET, we speculated that spectral unmixing could be feasible for our DAS-CRET.…”

Section: Resultsmentioning

confidence: 99%

Turn-on chemiluminescence probes and dual-amplification of signal for detection of amyloid beta species in vivo

et al. 2020

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Turn-on fluorescence imaging is routinely studied; however, turn-on chemiluminescence has been rarely explored for in vivo imaging. Herein, we report the design and validation of chemiluminescence probe ADLumin-1 as a turn-on probe for amyloid beta (Aβ) species. Two-photon imaging indicates that ADLumin-1 can efficiently cross the blood–brain barrier and provides excellent contrast for Aβ plaques and cerebral amyloid angiopathy. In vivo brain imaging shows that the chemiluminescence signal of ADLumin-1 from 5-month-old transgenic 5xFAD mice is 1.80-fold higher than that from the age-matched wild-type mice. Moreover, we demonstrate that it is feasible to further d ually- a mplify s ignal via c hemiluminescence r esonance e nergy t ransfer (DAS-CRET) using two non-conjugated smart probes (ADLumin-1 and CRANAD-3) in solutions, brain homogenates, and in vivo whole brain imaging. Our results show that DAS-CRET can provide a 2.25-fold margin between 5-month-old 5xFAD mice and wild type mice. We believe that our strategy could be extended to other aggregating-prone proteins.

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Section: Resultsmentioning

confidence: 99%

Turn-on chemiluminescence probes and dual-amplification of signal for detection of amyloid beta species in vivo

et al. 2020

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“…where W D , W S , and W A are the contributions of the S D , S S , and S A in S DA , respectively. We also obtain (Du et al, 2016;Zhang et al, 2018;Su et al, 2019;Zhang et al, 2019)…”

Section: Theorymentioning

confidence: 83%

“…ExEm-spFRET with as few as two excitation wavelengths makes it easy to perform microscopic ExEm-spFRET imaging for single living cells. Our recent studies have demonstrated the strong robustness of ExEm-spFRET imaging (Lin et al, 2018;Su et al, 2019). We recently also performed an ExEm-unmixing-based FRET two-hybrid assay in living cells to analyze the stoichiometric information of macromolecular complexes (Zhang et al, 2019).…”

Section: Introductionmentioning

confidence: 99%

“…Accurately predetermining the acceptor-donor quantum yield ratio (Q A /Q D ) and the acceptor-donor extinction coefficient ratio (K A /K D ) is the prerequisite for quantitative ExEm-spFRET imaging (Mustafa et al, 2013;Du et al, 2016;Zhang et al, 2018;Su et al, 2019). Most FRET-related researches directly cited the quantum yield values of fluorophores reported in the literature to obtain Q A /Q D values (Wlodarczyk et al, 2008;Elder et al, 2009;Mustafa et al, 2013).…”

Section: Introductionmentioning

confidence: 99%

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Measuring System Calibration Factors by Unmixing the Excitation–Emission Spectra of One Dish of Cells

et al. 2021

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Accurate predetermination of the quantum yield ratio (QA/QD) and the extinction coefficient ratio (KA/KD) between acceptor and donor is a prerequisite for quantitative fluorescence resonance energy transfer (FRET) imaging. We here propose a method to measure KA/KD and QA/QD by measuring the excitation–emission spectra (ExEm-spectra) of one dish of cells expressing m (≥3) kinds of FRET constructs. The ExEm-spectra images are unmixed to obtain the weight maps of donor (WD), acceptor (WA), and acceptor sensitization (WS). For each cell, the frequency distribution plots of the WS/WD and WS/WA images are fitted by using a single-Gaussian function to obtain the peak values of WS/WD (SD) and WS/WA (SA). The statistical frequency-SD/SA plots from all cells are fitted by using a multi-Gaussian function to obtain the peak values of both SD and SA, and then the ranges of WS/WD (RSD) and WS/WA (RSA) for each FRET construct are predetermined. Based on the predetermined RSD and RSA values of FRET constructs, our method is capable of automatically classifying cells expressing different FRET constructs. Finally, the WS/WD–WA/WD plot from different kinds of cells is linearly fitted to obtain KA/KD and QA/QD values.

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“…To accurately analyze the signal amplification, FRET is also coupled with spectral unmixing technique to separate the signal contributions from the donor and the acceptor [74][75] . Although spectral unmixing has been rarely explored for CRET, we speculated that spectral unmixing could be feasible for our DAS-CRET.…”

Section: Das-cret In Brain Homogenatesmentioning

confidence: 99%

Smart Chemiluminescence Probes and Dual-Amplification of Signal for Detection of Amyloid Beta Species in Alzheimer’s Disease Model

Yang¹,

Yin²,

Van³

et al. 2020

Preprint

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<div>Fluorescence and chemiluminescence imaging are the most widely applied optical emissive imaging</div><div>methods in biomedical research. “Smart” (turn-on) fluorescence imaging has been routinely used for in</div><div>vitro, cellular, and in vivo imaging; however, smart chemiluminescence imaging has been rarely explored.</div><div>In this report, we designed chemiluminescence probe ADLumin-1 and validated that ADLumin-1 was a</div><div>smart chemiluminescence probe for amyloid beta (Ab) species, evidenced by a 216-fold amplification of</div><div>chemiluminescence intensity upon mixing with Abs in vitro. In vivo two photon imaging indicated that</div><div>ADLumin-1 could efficiently cross blood-brain- barrier (BBB) and provided excellent contrast both for Ab</div><div>plaques and cerebral amyloid angiopathy (CAA). In vivo whole brain imaging showed that the</div><div>chemiluminescence signal of ADLumin-1 from 5-month-old transgenic AD (5xFAD) mice was 1.80-fold</div><div>higher than that from the age-matched wild-type mice. Moreover, we demonstrated that it was feasible to</div><div>further dually-amplify signal via chemiluminescence resonance energy transfer (DAS-CRET) using two</div><div>non-conjugated smart probes (ADLumin-1 and CRANAD-3) in solutions, brain homogenates, and in vivo</div><div>whole brain imaging. Our results showed that DAS-CRET could provide a 2.25-fold margin between 5-</div><div>month-old 5xFAD mice and wild type mice. To our knowledge, this is the first report that a</div><div>chemiluminescence probe could be used for detecting Ab species both in vitro and in vivo. Although</div><div>ADLumin-1 was designed for Abs, we believe that our strategy could be potentially extended to a wide</div><div>range of targets, including other aggregating-prone proteins. Notably, our results suggested that the</div><div>strategies for turning-on fluorescence could be used for amplifying chemiluminescence, and we believe that</div><div>our studies could inspire considerably more research on chemiluminescence imaging</div>

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Quantitative FRET measurement based on spectral unmixing of donor, acceptor and spontaneous excitation‐emission spectra

Cited by 7 publications

References 32 publications

Turn-on chemiluminescence probes and dual-amplification of signal for detection of amyloid beta species in vivo

Turn-on chemiluminescence probes and dual-amplification of signal for detection of amyloid beta species in vivo

Measuring System Calibration Factors by Unmixing the Excitation–Emission Spectra of One Dish of Cells

Smart Chemiluminescence Probes and Dual-Amplification of Signal for Detection of Amyloid Beta Species in Alzheimer’s Disease Model

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