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Single-molecule fluorescence spectroscopy uses the fluorescence of a molecule for obtaining information on its environment, structure, and position. The technique affords the ability of obtaining information otherwise not available due to ensemble averaging (that is, a signal obtained when recording many molecules at the same time represents an ...
Single-molecule fluorescence (or Förster) resonance energy transfer (or smFRET) is a biophysical technique used to measure distances at the 1-10 nanometer scale in single molecules, typically biomolecules. It is an application of FRET wherein a pair of donor and acceptor fluorophores are excited and detected at a single molecule level. In ...
A complementary technique is absorption spectroscopy. In the special case of single molecule fluorescence spectroscopy, intensity fluctuations from the emitted light are measured from either single fluorophores, or pairs of fluorophores. Devices that measure fluorescence are called fluorometers.
Within a single reaction, the amplification of multiple nucleic acid sequences can be monitored simultaneously by using fluorophores (e.g. FAM, VIC, Cy5) with distinguishable excitation and emission spectra; this is known as multiplexed qPCR. Fluorescence is also used for analyses of nucleic acids in techniques such as microarray and in ...
TIRF can also be used to observe the fluorescence of a single molecule, [6] [7] making it an important tool of biophysics and quantitative biology. TIRF microscopy has also been applied in the single molecule detection of DNA biomarkers and SNP discrimination. [8]
Many analytical procedures involve the use of a fluorometer, usually with a single exciting wavelength and single detection wavelength. Because of the sensitivity that the method affords, fluorescent molecule concentrations as low as 1 part per trillion can be measured. [81]
Fluorescence correlation spectroscopy (FCS) is a statistical analysis, via time correlation, of stationary fluctuations of the fluorescence intensity. Its theoretical underpinning originated from L. Onsager's regression hypothesis. The analysis provides kinetic parameters of the physical processes underlying the fluctuations.
For quantum dots, prolonged single-molecule microscopy showed that 20-90% of all particles never emit fluorescence. [5] On the other hand, conjugated polymer nanoparticles (Pdots) show almost no dark fraction in their fluorescence. [6] Fluorescent proteins can have a dark fraction from protein misfolding or defective chromophore formation. [7]