Fluorolog-3 v. 2.2 (10 Sep 2002)
Optimizing Data Acquisition
4-3
Data collection techniques
Selecting the collection method
The two basic collection methods are right-angle and front-face. In right-angle detection, the
fluorescence is collected at 90° to the incident exciting beam. Right-angle detection is used
primarily for clear solutions. Front-face detection is used for optically dense solutions such
as hemoglobin and for solid samples. In front-face detection, fluorescence is collected off
the front surface of the cuvette or the solid sample. Inner-filter and re-absorption characteris-
tics of opaque samples preclude right-angle detection. The optional front-face accessory for
cuvettes is required for solutions. For solid samples, use the Model 1933 Solid Sample
Holder.
Determining optimal wavelengths
The optimum excitation and emission wavelengths are known for many samples. If you are
running a sample whose wavelength positions are unknown, you must determine these
wavelengths to obtain the best possible results when you run the sample.
The method used to determine the excitation and emission wavelengths consists of first run-
ning an emission scan and observing the peak emission value. Once this value has been ob-
tained, it is necessary to conduct an excitation scan using the peak emission value deter-
mined by running an emission scan.
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