So image you have two groups one group consist out of 1 fluorescent particle and the other group consist out of multiple particles. However, the volume of the particles combined is the same for both groups. How is the overall fluorescence of each group affected?
I think it would depend on how the fluorescent moieties within the particles are arranged. That is because there are various effects of fluorescent molecules on each other that influence the quantum yield of the fluorophores. These include the inner filter effect, self-quenching, resonance energy transfer and excimer formation.
Thank you so much for the response!
So basically I am doing an experiment with fluorescent internally dyed polystyrene particles.
I want to know how the size affects the uptake of polystyrene particles by human cells. Therefore I added 1 micrometer fluorescent polystyrene particles to human cells. I also added 0.2 micrometer fluorescent polystyrene particles to a different group of cells.
Both particles were added at the same concentration, thus the same volume. However, there are thus more particle per cell available for the cells that were exposed to the 0.2 micrometer particles.
After exposure to these particles I measured the fluorescence of each cell. The results show that the fluorescence of the cells that were exposed to 1 micrometer particles is higher than the cells that were exposed to the 0.2 micrometer particles.
Does this mean that the 1 micrometer particles are better absorbed by the cells? Or does this mean that the 1 micrometer particle causes a higher fluorescence?
I think that the fluorescence of a cell exposed to the same volume of 0.2 micrometer particles causes a lower fluorescence intensity because, they are spread throughout the cell. The 1 micrometer particle will give a higher fluorescent intensity because it more concentrated at a single point, but is this true?
It is also possible that since these particles are internally dyed, the smaller particles will simply have less dye because, relatively there volume surface ratio is bigger than for the bigger particles.
To reduce the complications, I suggest that you use a high-resolution microscopy technique to count the number of fluorescent particles in the cells directly, rather than measuring the bulk fluorescence of the cells. This should be feasible as long as the number of particles taken up is not too large.
If that is impractical, perhaps you can find a way to calibrate the average fluorescence intensity per particle for each particle size. That would allow you to convert the fluorescence of the cells to the number of particles. This would require knowing the number of particles per unit volume of suspension.
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