Spectral precision improvement with demagnifying spectral images in spectroscopic nanoscopy

Abstract

Spectroscopic nanoscopy (SN) has been recognized as a key functional imaging tool in cell biology and chemistry because it offers the unique capability to simultaneously obtain the spatial and spectral information for single molecules. However, it has an intrinsic issue in using the limited photon budget from single emitters divided into two imaging channels to concurrently acquire spatial and spectral images. Accordingly, this issue lowers the spatial localization and spectral precision. Although several techniques have been introduced to improve the spatial precision in SN, improving the spectral precision has been overlooked so far. Here we propose a method to improve the spectral precision by optically manipulating the width of the spectroscopic signatures using a demagnifier. We evaluate its performance using numerical simulations with systematic investigations of several underlying optimal parameters such as the demagnification factor and the integration width in the proposed configuration. We also present achievable spectral precision values with different signal and background levels. Compared to the existing SN system, the 3× demagnifier-based configuration shows an approximate 35% improvement, from 2.9 nm to 1.9 nm, in the spectral precision at the 1000 photons signal level.