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The Dye Tracing Pages | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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In the mid 1980's a new instrument became available that would eventually revolutionize
fluorescence analysis along with dye tracing methodologies. The synchronously scanning
spectrofluorophotometer (SSS), while still exploiting the same principles of fluorescence
as the filter fluorometer, did not need special filters to remove unwanted wavelengths
of light from the excitation beam. Instead it used a series of motorized
mirrors,
lenses, and thin slits to produce the precise wavelength range required for a particular
molecule to fluoresce. It also used the same technique to measure the wavelength of
the beam of light emitted from the sample. Another unique feature of
this instrument
was its ability measure the wavelength of both the excitation and emission spectra
thanks to its dual-monocromator design. With this design, the following types of
analyses could be performed: 1. Fixed excitation/fixed emission analysis -- For this type of analysis, the excitation beam can be set to a particular wavelength and the monocromator can be set to read the intensity emitted light from the sample that has a particular wavelength. This type of analysis is the same type performed on a fluorometer, just without the filters. 2. Fixed excitation/variable emission analysis -- For this type of analysis, the excitation beam is set to a particular wavelength and the emission monocromator is set to scan through a defined wavelength range measuring the intensity of any light emitted from the sample. This type of analysis produces a plot of fluorescence intensity vs. emission wavelength for that particular excitation wavelength. This plot allowed the user to determine what the maximum (peak) fluorescence intensity was for a particular excitation wavelength. Fluorometers cannot perform this type of analysis. 3. Variable excitation/fixed emission analysis -- For this type of analysis, the excitation beam (being controlled by the second monocromator) is set to scan through a defined wavelength range and the emission monocromator is set to measure the intensity of any light emitted from the sample that is of a particular wavelength. This type of analysis produces a plot of fluorescence intensity vs. excitation wavelength for that particular emission wavelength. This plot allowed the user to determine what the maximum (peak) fluorescence intensity was for a particular emission wavelength. Fluorometers cannot perform this type of analysis. 4. Variable excitation/variable emission analysis (called synchronous scanning) -- For this type of analysis, both the excitation and emission monocromators are programmed to scan through a defined wavelength range. In order to keep the emission monocromator from detecting scattered light from the excitation beam, the monocromators are typically programmed to scan slightly different wavelength ranges. Since fluorescent molecules typically absorb light at a lower wavelength range than they emit, the excitation monocromator is programmed to start its scan at a lower wavelength than the emission monocromator. This type of analysis produces a plot of fluorescence intensity vs. emission wavelength which is very different from the plot produced by a fixed excitation/variable emission analysis. This is the best type of analysis for identifying fluorescent molecules in an unknown sample. Fluorometers cannot perform this type of analysis. Today, synchronously scanning spectrofluorophotometers are in use at every major fluorescence analysis laboratory in the United States, including the US EPA's premier analysis laboratory in Las Vegas, Nevada. While filter fluorometers are still manufactured and still have their appropriate uses, the SSS has necessarily become the standard for fluorescence analysis. |
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