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Fluorescence Sensing

The Phos-4 has 2 key features that are useful for fluorescence sensing.:-

  • Selectable Filtering
  • Multi-Wavelength Excitation

Selectable Filtering

LED’s have two disadvantages when used as light sources for measuring fluorescent:-

  1. “Substrate fluorescence” – small amounts of light generated at wavelengths much greater than the peak;
  2. Spectrum width, limiting measurements to fluorophores with large Stoke’s shifts.

Simply adding a filter into the light path substantially reduces the optical power.

However the Phos-4 P provides an adjustable lens to allow the light to be focused through the filter and maximise the optical output.

Multi-Wavelength Excitation

Some fluorophores respond differently to different excitation wavelengths.  For example, see Wencel et al, 2009; (Sensors and Actuators B 139, 2009; 208–213).  By alternately exciting these fluorophores at two wavelengths, the ratio of the fluorescence can be used to measure pH.  The P4-VCGB-U00 LED module has 4 LED dies, two of which (405nm & 450nm) can be used to make ratiometric fluorescence-based pH measurements.

Case Study : Selectable Filtering with a 365nm LED for Fluorescence

The spectral output of the P4-365-510 LED is shown below in the figure below:

  • Figure (a) shows the spectral output without filtering on a linear axis;
  • Figure (b) shows the spectral output without filtering on a logarithmic axis;
  • Figure (c) shows the spectral output with a UG11 filter on a logarithmic axis.

Notice in (b) that there is some optical output in the range 475nm to 500nm at about 1/1000 of the peak, whereas in (c) that output has been removed.

In addition the filter has reduced the width of the spectrum allowing measurements from 400nm upward, so much smaller Stoke’s shifts can be detected.

Installing a Filter

The diagram shows how to install a new filter in the light path.

  • Slacken the Fibre Holder’s Dovetail clamping screw using a 2mm Hex Key and slide the fibre holder off the dovetail;
  • Prise the old filter from the filter pad from the top of the filter;
  • Install the new filter pushing firmly on to the filter pad;
  • Slide the Fibre Holder back on to the Dovetail and retighten its dovetail clamping screw;
  • Re-align the LED Module with the fibre connector by adjusting the position of the LED and Lens holders;