-4 & FluoroMax
-4P with USB v. B (23 Oct 2009)
measurements at discrete wavelength pairs. The data are acquired as
single points at a user-defined set of excitation-emission wavelength
pairs for a user-defined number of samples. These data are displayed in
either spreadsheet format, or in a plot. This application is for Micro-
Max or FluoroMax
-4 users who routinely perform assays on a large
number of samples.
The spin-paired ground or excited state. The process of absorption
generally produces the first excited singlet state, which takes time to
fluoresce, and may undergo intersystem crossing to form a triplet state.
The accuracy of a monochromator with respect to its wavelength
alignment. This is a measure of the monochromator being at the correct
wavelength when it is set there. Monochromators are traditionally cali-
brated using line-spectra sources, such as mercury lamps. Spectrofluo-
rometers may be calibrated by performing two scans, one of the source,
and one of a standard (such as water) to calibrate all of the monochro-
mators. For HORIBA Scientific spectrofluorometers, the xenon-lamp
scan is performed on the excitation with the 467.1-nm peak assigned as
such in the software. The water Raman band is scanned with 350-nm
excitation, and the 397-nm peak is assigned as such in the software for
the emission monochromators.
The removal of the wavelength sensitivity of detectors, optics, sources,
and backgrounds from the spectrum taken on a sample. When spectral
correction has been properly performed, the true theoretical spectra
from a sample should be all that remains. Spectral correction is accom-
plished with a variety of options on HORIBA Scientific spectrofluo-
rometers. Excitation and emission correction factor files are provided
to remove the wavelength sensitivity of detectors and their optics. The
reference detector is present to remove the lamp and excitation optics
response. Blank subtraction and dark offset are used to remove back-
ground levels and responses.
All detectors have a higher sensitivity to some wavelengths than to
others. The spectral response of a detector is often expressed graphical-
ly in a plot of responsivity versus wavelength.
An analytical instrument used to measure the fluorescence properties
of a molecule or substance. The device consists of at least two mo-
nochromators, a source, a sample compartment and detection electron-
ics. The instruments may be scanned on the excitation, emission or
both to provide insight on the characteristics of the sample being stu-
died. Newer spectrofluorometers provide many more automated op-
tions, including polarization, temperature, titer plates, pressure, and
many more. Today, these instruments are computer-controlled, allow-
ing easy control of assays and complex experiments.
Generally, the energy-difference between the absorption peak of lowest