| Description |
In fluorescence microscopy a sample is tagged with light emitting molecules called fluorophores. In this study, we introduce a way of extracting the wavelength of fluorophores via point spread function manipulation using a custom phase plate. We simulated the effect of the phase plate with Monte Carlo methods and a photon propogation model. Then, we compared the simulated images with computationally defined reference functions to extract the wavelength or peak wavelength of both monochromatic and polychromatic sources respectively. We also attempted to use our set up as a spectrometer and find the spectra of fluorophores in the Alexa Fluor family. We found that there were two interesting wavelength ranges. In the first, named the asymmetry interval, wavelength could be accurately determined with a low number of photons (< 1000). The other, the symmetry interval, required a very large number of photons (> 3000). We also studied the effect of magnification on this method and concluded that it is more accurate at higher magnifications but the increase is minimal after around 100x magnification. At a photon count of 1000, a majority (> 95%) of wavelength extractions were within five nanometers of the true value. Our attempt to find the spectra of the Alexa Fluors was not successful. We could not achieve a fitted spectrum that reliably matched the known one. Further study may yield a satisfactory result. In future experiments we want to obtain the wavelength of fluorophores with this method while also determining their position to a high precision. |
