Spectrometer setup to minimize absorption of reflected light

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Discussion Overview

The discussion revolves around optimizing a spectrometer setup to minimize the interference of reflected blue light with the red fluorescence emitted by an oxygen-sensitive dye. Participants explore various methods to enhance the measurement of low dye concentrations, focusing on the challenges posed by overlapping light spectra.

Discussion Character

  • Exploratory
  • Technical explanation
  • Debate/contested

Main Points Raised

  • One participant describes their experimental setup and the issue of blue light overlapping with red fluorescence, complicating the measurement of quenching in low dye concentrations.
  • Another participant suggests using filters to reject unwanted light, drawing from their experience in astrophotography.
  • A different participant argues that filters may not effectively distinguish between reflected and fluoresced light due to their overlapping spectra.
  • One participant proposes checking the polarization of the blue and red peaks, suggesting that polarizers might help filter the desired spectrum.
  • Another participant suggests recording the spectrum of the reflected blue light and digitally subtracting it from the overall spectrum to isolate the red emission.
  • Participants confirm the use of a specific spectrometer model (oceanoptics jaz) for the measurements.
  • Concerns are raised about the effectiveness of polarizers in filtering out reflected light without also reducing the emitted light.
  • One participant expresses the difficulty of moving the spectrometer to isolate blue light due to changes in intensity, emphasizing the need for a solution within the current setup.

Areas of Agreement / Disagreement

Participants express differing views on the effectiveness of filters and polarizers in addressing the issue of overlapping light spectra. There is no consensus on the best approach to minimize the interference of blue light with red fluorescence.

Contextual Notes

Participants note the limitations of their current setup and the challenges of isolating specific wavelengths due to the overlapping nature of the light spectra involved.

caa4444
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I'm working on an experiment to measure the quenching of an oxygen-sensitive dye. The excitation light is blue and the light fluoresced is red. When I collect data, there is always a peak for blue and red, which makes it impossible to measure really the quenching in low dye concentrations because the blue overlaps the small red emission. I'm thinking of using a polarizing film, but don't know the best setup.

Below is the current setup:

[film w/ dye] — |glass wall| — spectrometer & blue light source

Can you guys tell me how to minimize the pickup of blue light without affecting red light pickup?
 
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I use filters to reject unwanted parts of the spectrum when I do astrophotography. Do you not have any filters?
 
a filter wouldn't help because I am concerned with the overlap of reflected and fluoresced light, and a filter wouldn't be able to distinguish between them
 
If the spectrum of the blue peak is overlapping the spectrum of the red peak, then I don't think you can do much with filters. There are edge pass filters that can block a desired part of the spectrum. Are you using a commercial spectrometer?

It might be worth checking if the blue peak and red peak have different polarizations; then you could use polarizers to filter the desired spectrum out.
 
caa4444 said:
a filter wouldn't help because I am concerned with the overlap of reflected and fluoresced light, and a filter wouldn't be able to distinguish between them

Ah ok I see the issue now. Well, I can't help with the setup, but have you considered recording the spectrum of the reflected blue light and digitally subtracting it from the spectrum of the whole experiment? I do something similar in astrophotography to get dark current and electronic bias removed from my images. I'm assuming that you are using a CCD or something similar to record the spectrum, is that correct?
 
yes. I am using the oceanoptics jaz
 
caa4444 said:
yes. I am using the oceanoptics jaz

Well, you could try the method I suggested. I'm not sure if a polarizer would filter the reflected light out or just reduce both the reflected and emitted light.
 
^i'd do that if I could, but I can't reliably get just the blue light because i'd have to move the spectrometer and that changes the intensity.

I'm just trying to get data for a stern-volmer plot, so if there's a way to do that with just this setup, that'd be great
 

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