Spectral imaging of single molecules by transmission grating-based epi-fluorescence microscopy

A spectral imaging method of single protein molecules labeled with a single fluorophore is presented. The method is based on a transmission grating and a routine fluorescence microscope. The bovine serum alubmin (BSA) and antiBSA molecules labeled with Alexa Fluor 488 and Alexa Fluor 594, respectively, are used as the model proteins. The fluorescence of single molecules is dispersed into zeroth-order spectrum and first-order spectrum by the transmission grating. Results show that the fluorescence emission spectrum of single molecule converted from the first-order spectral imaging is in good agreement with the bulk fluorescence spectrum. The spectral resolution of 2.4 nm/pixel is obtained, which is sufficient for identifying the molecular species in a multicomponent system.