Hello!
The aim of my experiment is to optimize and test an optical module for fluorescence polarization measurements. I am testing this setup using various fluorescein dilutions as samples. My measurements for parallel signal recordings are as follows:
[Concentration (nmol/l), Signal Amplitude (V)] : (0 nmol/l, 0.66 V); (0.1 nmol/l, 0.68 V); (0.5 nmol/l, 0.7 V); (1 nmol/l, 0.85 V); (2 nmol/l, 0.9 V); (5 nmol/l, 1.3 V); (10 nmol/l; 2.1 V)
The perpendicular intensities are close to the parallel intensities, so I haven't mentioned them. The problem is that while calculating the signal-to-background ratio, I found that the ratio is quite low (0.3 for conc of 1 nmol/l and 2.45 for conc of 10 nmol/l). My question: Does the cuvette geometry influence the intensity of bacground light that reaches the detector? (I use a cylindrical cuvette. So, I just thought that the curvature of the cuvette might just be a matter of concern)
It is more usual to use a square cuvette for fluorescence measurements to reduce reflected excitation light from entering the emission detector.
To improve the sensitivity of detecting low concentrations of the fluorophore, you can increase the bandwidths of the monochromators and use a cuvette with a longer pathlength. Increasing the detector gain can be useful if it is set too low. Using a brighter source of illumination and a more sensitive detector would also be useful if the equipment is available. Make sure your lamp is properly focused.
A good way to evaluate the sensitivity of the polarization measurement is to calculate the polarization as a function of the fluorophore concentration. As the fluorophore concentration increases, the polarization will decrease, until it levels off. This is because the fluorescence intensities are dominated by highly polarized stray light when the fluorophore concentration is very low. Once the fluorophore concentration gets high enough that the stray light becomes insignificant, the polarization levels off at the value expected for the free fluorophore.
Geometry of cuvette is propably not the reason.
But
1st idea: What is about the photon energy distribution/bandwidth of your excitation source and that of the detector section. There may be some spectral overlap such that spectral separation is not sufficient.
2nd idea: dark signal arising from detector part: which signal amplitude do you have when excitation source is switched off? or any other light sources present, e.g. ambient light not sufficiently suppressed (switch off the lamp(s) in your lab while performing the measurement).
It is more usual to use a square cuvette for fluorescence measurements to reduce reflected excitation light from entering the emission detector.
To improve the sensitivity of detecting low concentrations of the fluorophore, you can increase the bandwidths of the monochromators and use a cuvette with a longer pathlength. Increasing the detector gain can be useful if it is set too low. Using a brighter source of illumination and a more sensitive detector would also be useful if the equipment is available. Make sure your lamp is properly focused.
A good way to evaluate the sensitivity of the polarization measurement is to calculate the polarization as a function of the fluorophore concentration. As the fluorophore concentration increases, the polarization will decrease, until it levels off. This is because the fluorescence intensities are dominated by highly polarized stray light when the fluorophore concentration is very low. Once the fluorophore concentration gets high enough that the stray light becomes insignificant, the polarization levels off at the value expected for the free fluorophore.
Yes, I agree you should try to get rid of the background signal. We had good success using bandpass filters. So we used a short pass filter for the excitation and a long pass filter for the fluorescence detection which where absolutely exclusive in the spectra. There are such filters at Thorlabs for about 50 Euros/each. After applying we did not have any more background at all. Of course you need an optimal pair of filters for each sample then.
An example where we did so is found here:
Article Characterization of Frex as an NADH sensor for in vivo appli...
The geometry of the measurement must be kept very strictly, because it strongly affects the luminescent measurements. Particularly great is the role of scattering. For example, the limits of phenol hardening in water are limited by strong Raman scattering of water.
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