Application of Nonlinear Microscopy in Life Sciences
Lingyan Shi, Robert R. Alfano in Deep Imaging in Tissue and Biomedical Materials, 2017
Forster resonance energy transfer is a process of nonradiative energy transfer between an excited donor molecule and an acceptor molecule in ground state within close proximity (several nanometers). Figure 6.8 depicts one of requirements of FRET—appreciable spectral overlap between donor emission and acceptor absorption. FRET is often used to study protein-protein interactions on the nanometer scale. The donor quenching and the increased emission of the acceptor due to FRET [75] can be measured in cultured cells, but is unreliable in tissues due to high levels of autofluorescence and spectrally dependent and variable light absorption in tissue. The shortening of the fluorescence lifetime of the donor fluorophores, on the other hand, can be measured precisely even deep in scattering and absorbing tissue. Point scanning multiphoton microscope with TCSPC detection is the prevalent instrumentation for FLIM-FRET measurements [76].
Toxicology Studies of Semiconductor Nanomaterials: Environmental Applications
Suresh C. Pillai, Yvonne Lang in Toxicity of Nanomaterials, 2019
Semiconductor QD-based biosensors target the specific biomolecules with the help of sensing ligands and the number of biomolecules is recognized from the change of luminescence. Besides the QD-based conventional biosensors, QD-based energy transfer sensors like FRET have also been developed. In QD-based FRET biosensors, QDs act as the donor and the fluorescence dye acts as acceptor and has been widely used for the detection of amino acids, insulin, intracellular pH, proteolytic activity assay, and monitoring DNA cleavage (Algar et al., 2014, Geissler et al., 2014). Similar to QD – FRET system, QD – BRET (bioluminescence resonance energy transfer) (Alam et al., 2014) and QD – CRET (chemiluminescence energy transfer) (Chen et al., 2014a) systems are also available (Figure 4.10).
Molecular Imaging of Viable Cancer Cells
Shoogo Ueno in Bioimaging, 2020
Förster resonance energy transfer (FRET) is energy transfer from the excited state of the fluorophore (donor) to an adjacent molecule (acceptor). In order to design FRET-based probes, it is important to select a fluorophore or chromophore pair so that the donor emission spectrum overlaps well with the acceptor absorbance spectrum. Since the energy transfer takes place only when donor and acceptor are in close vicinity to each other, most FRET-based probes are designed so that the distance between the two fluorophores changes before and after reaction with the target molecule. Combinations of fluorophore (donor) and quencher (acceptor) are used in the design of activatable probes targeted to cancer-related enzymes.
Discovery of RNA-targeted small molecules through the merging of experimental and computational technologies
Published in Expert Opinion on Drug Discovery, 2023
The three fluorescence-based assays that are often employed for screening of RNA binders are: (1) fluorescence resonance energy transfer (FRET)-based assay (Figure 3(a)), (2) time-resolved FRET (TR-FRET) assay, and fluorescent indicator displacement (FID). FRET refers to the transfer of energy from a donor fluorophore to an acceptor fluorophore conjugated to the target biomolecule. FRET-based assays are convenient and extremely sensitive and have become popular for screening small-molecule libraries against RNA targets. Simone et al. [92] used a FRET-based assay to screen for small-molecule stabilizers of the C9orf72 (G4C2)4 G-quadruplex RNA, which is a known cause of frontotemporal dementia and amyotrophic lateral sclerosis [116]. The authors monitored the changes in melting temperature of the 5’-FAM and 3’-TAMRA labeled (G4C2)4 RNA upon heating in the presence of small molecules and identified three structurally similar small molecules that stabilize the RNA. The small molecules were subsequently shown to reduce the frequency of RNA foci and the levels of dipeptide repeat protein in C9orf72 patient neurons. Furthermore, the most effective small molecule, DB1273, was found to improve survival and reduce levels of toxic poly-(glycine-arginine) in C9orf72 flies.
A combined “eat me/don’t eat me” strategy based on extracellular vesicles for anticancer nanomedicine
Published in Journal of Extracellular Vesicles, 2020
Zakia Belhadj, Bing He, Hailiang Deng, Siyang Song, Hua Zhang, Xueqing Wang, Wenbing Dai, Qiang Zhang
Lipid vesicles-exosomes fusion was monitored by FRET-based lipid exchange assay as described previously [44]. The FRET acceptor dye (DiI) and donor dye (DiO) were physically encapsulated into the lipid vesicles. A549 cells were then incubated with c(RGDm7)-LS and hybrid c(RGDm7)-LS for 4 h at 37°C. FRET images were visualized with a confocal laser scanning microscope (CLSM, TCS SP5, Leica, Germany). The FRET efficiency is defined as a difference of fluorescence intensity of the donor before and after photobleaching. The FRET efficiency is measured using this equation: FRETeff = (Dpost-Dpre)/Dpost, where Dpost and Dpre correspond to the fluorescence intensity of DiO after and before photobleaching, respectively. FRET occurs at DiO excitation wavelength (484 nm) and DiI emission wavelength (555–655 nm) [45].
Discovery of new non-pyrimidine scaffolds as Plasmodium falciparum DHFR inhibitors by fragment-based screening
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2021
Marie Hoarau, Jarunee Vanichtanankul, Nitipol Srimongkolpithak, Danoo Vitsupakorn, Yongyuth Yuthavong, Sumalee Kamchonwongpaisan
In a typical DSF experiment, a master mix containing buffer, PfDHFR and 8x SYPRO Orange dye were prepared in a microcentrifuge tube. Ligands of interest were dispensed in low-profile 96-well plates (Bio-Rad) to 1 mM final concentration, and the master mix was dispensed to a final volume of 50 μL per well. In these conditions, DMSO content was maintained constant at 2%. The microplate was sealed with adhesive film and mixed by shaking for 2 min at 800 rpm at RT. The microplate was then submitted to a DSF run on a CFX96 RT-PCR (Bio-Rad). DSF program was designed by starting with a 3 min equilibration phase at 30 °C, followed by a temperature gradient of 1 °C/min, recording fluorescence every 0.5 °C. Fluorescence was recorded using the FRET channel (λexc = 450–490 nm, λem = 560–580 nm). Curves were fitted using the Precision Melt Analysis software (Bio-Rad).
Related Knowledge Centers
- Absorption Spectroscopy
- Fluorescence
- Fluorophore
- Microscopy
- Quantum Yield
- Refractive Index
- Near & Far Field
- Radiative Transfer
- Emission Spectrum
- Molar Absorption Coefficient