Spectrometer setup to minimize absorption of reflected light

[caa4444]

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?

 

[Drakkith]

I use filters to reject unwanted parts of the spectrum when I do astrophotography. Do you not have any filters?

 

[caa4444]

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

 

[glapglipper]

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.

 

[Drakkith]

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?

 

[caa4444]

yes. I am using the oceanoptics jaz

 

[Drakkith]

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.

 

[caa4444]

^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