Spectrofluorometers - Signal to Noise Ratio and the HORIBA Scientific FluoroLog-3

The FluoroLog-3 is a unique, modular system which allows the researcher to interchange a versatile range of accessories to correspond perfectly with the characteristics of a given sample. From analysis of steady-state or molecular dynamics to IR probes, the FluoroLog-3 comes equipped with a wide range and limitless configuration of accessories to enhance the accuracy and speed of your application.

The FluoroLog-3 from HORIBA Scientific.

Figure 1. The FluoroLog-3 from HORIBA Scientific.

Features of the FluoroLog-3

The FluoroLog-3 from HORIBA Scientific features:

  • World's most sensitive spectrofluorometer
  • Can detect 50-femtomolar fluorescein
  • Unique, modular system
  • Interchange wide range of computer-controlled accessories: spectrometers, detectors, sources, and more!
  • Totally computer-controlled
  • All-reflective optics

Signal to Noise Ratio Measurement

Water Raman Test

The water Raman test is a good measure of relative sensitivity between different instruments, provided the experimental conditions used to compare the systems are the same.

Unfortunately, there are a number of different ways of handling the data, all of which are valid but which will give quite different numbers. Therefore, it is important not only to know how the water Raman S/N values is measured, but also how the data were treated.

In general, the water Raman S/N test method combines a value for system sensitivity (in the presence of a signal) with a value for system noise (in the absence of signal) to show the overall performance of the instrument.

Signal to Noise Ratio Definition

At HORIBA Scientific we define the S/N ratio as the difference of peak and background signal, divided by the square root of the Background signal. The peak signal is measured at the water Raman peak (397 nm for 350 nm excitation) and the noise in a region (450 nm) where no Raman signal is present, and an "ideal" system would give a signal value of zero.

Another commonly used method is to divide the difference (Peak signal - Background signal) by the rms value of the noise on the background signal. This second method is used by a few other manufacturers.

FluoroLog Data

Some actual data from our FluoroLog FL3-11 system (this was a typical system, a few years old) will serve to show the difference between the two methods.

The experimental conditions were as follows:

  • Excitation 350 nm with 5 nm bandpass
  • Emission 360 - 450 nm with 5nm bandpass
  • Interval 1nm
  • Integration 1s
  • No smoothing of data points

Standard room temperature, red sensitive, detector (Note: make sure the test is carried out with the actual detector you will be using. All HORIBA Scientific systems are specified with a R928P PMT at room temperature).

Signal to Noise Ratio Measurements

The measurements provided the following data:

  • Peak signal (at 397nm) = 501,500 cps
  • Background (at 450 nm) = 10,500 cps

Peak to peak noise of background (at 450 nm) = 223c (measured with a separate kinetic scan), which gives an rms noise of the background signal of 223/5 = 44.6

Therefore, the HORIBA Scientific method gives a water Raman S/N of (501500-10500)/ (10500)½ = 4791

The second method similarly gives a water Raman S/N of (501500-10500)/ 44.6 = 11008

HORIBA Scientific feels that the first method is correct although it gives a lower number. The second method only takes into account the detector noise and the shot noise of the electronics.

On the other hand, by using the background total intensity as a measure of noise, the HORIBA Scientific method is more representative of a real "live" experiment where noise is also influenced by factors like the quality of the optics and scattered light in the system. These additional factors will influence the ability to measure a very low signal from a sample and should not be left out.

This information has been sourced, reviewed and adapted from materials provided by HORIBA Scientific.

For more information on this source, please visit HORIBA Scientific.

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