Although it's logical to assume equal extinction coefficients with use of the same fluorophore
in reference solution and analytes, accurate measurements are crucial for reliable quantitative flow cytometry.
The fluorescence polarization that we observe strongly indicates that the fluorophore
is arranged with its major absorbing and emission resonance planes (dipoles) oriented parallel to the long axis of the crystal.
When one end of the oligonucleotide is labeled with a donor fluorophore
and the other end is labeled with an acceptor dye, the folding of the molecule in the G-quartet structure brings the donor-acceptor pair into close proximity, allowing an interaction between the two labels which results in quenching of donor fluorescence or a change in other fluorescence properties which are the result of the interaction of two dyes in close proximity.
The reactions are then set up nearly identically, with the key difference being that different fluorophore
labels are used in the two reactions.
The former utilizes a FRET (fluorescent resonance energy transfer)-based peptide substrate and the latter, a fluorophore
Until recently, single-molecule fluorescence detection was not a trivial task, because the signals generated from an individual molecule (for example, a fluorophore
attached to a protein or DNA) were extremely weak.
The quantitation of fluorescence radiance may at first suggest the need to obtain the number of fluorophore
that are responsible for the measured fluorescence radiance.
The small molecule fluorophore
is selected from pyrene, 1-pyrenebutyric acid, pyrene-1-boronic acid and 1-pyrenebutyric acid N-hydroxysuccinimide ester.
While unexpected reasons for fluorescence increase during the assay can exist, such as nonspecific nuclease activity degrading probes and releasing active fluorophore
for detection, the kinetics of such processes generally looks quite different from an actual true positive with its exponential and plateau (sigmoidal) curve.
In the absence of its complementary "target", the single-stranded probe comes together in a hair-pin-like manner, with the complementary sequences at either end of the strand binding together, bringing the fluorophore
and quencher close to each other and turning down, or "quenching", the fluorophore
Slide images are acquired using a microarray scanner with appropriate settings for the fluorophore
KEY WORDS: polycyclic aromatic hydrocarbon, fluorophore
, Deepwater Horizon, Callinectes sapidus megalopae