Emission spectra correction factors

The fluorescent components are denoted by I (intensity) followed by a capitalized subscript (D, A or s, for respectively Donors, Acceptors, or Donor/ Acceptor FRET pairs) to indicate the particular population of molecules responsible for emission of/and a lower-case superscript (d or, s) that indicates the detection channel (or filter cube). For example, / denotes the intensity of the donors as detected in the donor channel and reads as Intensity of donors in the donor channel, etc. Similarly, properties of molecules (number of molecules, N quantum yield, Q) are specified with capitalized subscript and properties of channels (laser intensity, gain, g) are specified with lowercase superscript. Factors that depend on both molecular species and on detection channel (excitation efficiency, s fraction of the emission spectrum detected in a channel, F) are indexed with both. Note that for all factorized symbols it is assumed that we work in the linear (excitation-fluorescence) regime with negligible donor or acceptor saturation or triplet states. In case such conditions are not met, the FRET estimation will not be correct. See Chap. 12 (FRET calculator) for more details. 

Alternatively, a standard fluorescent compound can be used whose corrected emission spectrum has been reported3 . Comparison of this spectrum with the technical spectrum recorded with the detection system provides the correction factors. The wavelength range must obviously cover the fluorescence spectrum to be corrected. Unfortunately, there is a limited number of reliable standards. 

In order to minimize the effects of possible inaccuracy of the correction factors for the emission spectrum, the standard is preferably chosen to be excitable at the same wavelength as the compound, and with a fluorescence spectrum covering a similar wavelength range. 

The lux meter is used in photometry and is simply a radiometer with a spectral responsiveness that closely matches the visual response of the human eye, thus measuring incident radiant power in the visible region of the electromagnetic spectrum. In this case, the unit of measurement is the illuminance in lux and is calibrated against a specific tungsten lamp. The lux meter should be provided with a set of correction factors to enable compensation for differences in spectral response for lamps with emission spectra different from the calibration lamp. 

Rate of Emission of Photons by the UV Lamp The rate of emission of photons by the lamp, so-called lamp characterization can be developed in the LTU, as described in Chapter III. In the LTU, a radiometer is placed at a fixed distance form the lamp s axis. A radiomenter correction factor of 1.41 (equation 3-1) is used which relates the tnie absolute reading to the lamp emission spectrum and the radiometer normalized spectral response. Thus the radiometric measurement allowed for the determination of the spatial distribution of the lamp radiative flux, qe y i. 

The fluorescence spectrum (emission spectrum) of a sample is obtained by scanning M2 while keeping the excitation wavelength constant. Since the efficiency of M2 is wavelength dependent, and the detector (see below) also has a sensitivity that varies with wavelength, such a fluorescence spectrum is normally uncorrected , i.e., it will depend upon the instrument as well as the sample. The correction of emission spectra is less easy to achieve than for excitation spectra, and is less often performed, so published emission spectra often vary from instrument to instrument. At least three methods are available for emission correction. A sound but tedious method is to calibrate the emission system (i.e., M2 plus the detector) with a standard light source of known emission profile. Such devices are available from NIST and other standards bodies. Comparison of the output of the fluorescence spectrometer with the certified output of the lamp then provides a correction factor at each wavelength, which can be applied to subsequent sample spectra. A related technique is to... 

The donor contribution in the acceptor channel (crosstalk) should be as low as possible the impact of this contribution on a bioassay is not obvious to anticipate starting from a lanthanide complex emission spectrum, since many instmmental factors, such as the filter settings (bandpass width), have to be considered. The intensity distribution between the emission lines is critical, particularly for europium complexes, with a strong impact of the ligand structure and symmetry (for terbium complexes, this impact is reduced). Care must be exercised in comparing published emission spectra, since many of the published spectra are not corrected for the photomultiplicator sensitivity (which falls off rapidly between 650 and 800 nm even using a red PMT ). The consequence is that the 690-nm ( Dq p4) band seems much smaller than its true value. Some articles do indeed show spectra corrected for the sensitivity of the detection system (which contains contributions from the PMT, but also from the monochromators and optics). Whenever such corrections have been applied, this is usually indicated in the experimental section of the article. 

If you do not have a calibrated lamp, one simple and fairly reliable method is to obtain the necessary correction factor by comparing the emission spectrum of a standard substance obtained with your own instmment with that of the spectmm of the same substance, but corrected, as shown in the literature. The main standards and their corrected spectra, which cover the region between 300 and 800 nm, are listed in [1]. 

There is, of course, also an uncertainty on the half-life and, if a decay correction is made, the uncertainty on that should also be included. The commercial spectrum analysis program libraries may only allow a single nuclide uncertainty factor to be accounted. If so, it will be necessary to devise a single factor, taking into account the likely magnitude of any decay correction and the various emission probability uncertainties for each nuclide. 

As was the case for XPS, to understand the correct use of sensitivity factors in AES it is necessary to consider briefly the origin of the various terms that give rise to the Auger intensity in the electron spectrum. The intensity of emission of Auger electrons originating from a particular electronic transition in a uniformly illuminated specimen of element a is... 

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