Spectroscopic identification of individual fluorophores using photoluminescence excitation spectra

We report a high throughput method for measuring the photoluminescence excitation spectra of individual fluorophores using a tunable supercontinuum laser and prism‐type total internal reflection fluorescence microscope. We used the system to measure and sort the photoluminescence excitation spectra of individual Alexa dyes, fluorescent nanodiamonds (FNDs), and fluorescent polystyrene beads. We used a Gaussian mixture model with maximum likelihood estimation to objectively separate the spectra. Finally, we spectroscopically identified different species of fluorescent nanodiamonds with overlapping spectra and characterized the heterogeneity of fluorescent nanodiamonds of varying size. Lay description Single molecule experiments reveal details that are obscured in ensemble experiments. However, identification of single molecules can be ambiguous due to overlapping emission from other fluorophores of interest and non‐specific impurities. In this case, discrete detection windows created by optical filters are not sufficient to differentiate various fluorescent species. Photoluminescence excitation (PLE) techniques reduce this ambiguity by accurately identifying molecular species via their distinct spectroscopic features. We report a high throughput method for measuring the PLE spectra of individual emitters using a tunable supercontinuum laser and a single molecule total internal reflection fluorescence (TIRF) microscope. PLE spectra of individual Alexa555 dyes were measured....
Source: Journal of Microscopy - Category: Laboratory Medicine Authors: Tags: Original Article Source Type: research