Characterization of Quantum Dots with Hyperspectral Fluorescence Microscopy for Multiplexed Optical Imaging of Biomolecules

Abstract

The optical properties of quantum dots were extensively characterized using a hyperspectral fluorescence microscopy system. The system provides a single excitation wavelength in the ultraviolet and 311 emission wavelength channels in the visible. This allows detection of multiple fluorophores (e.g. different quantum dots) with a high spectral resolution in one go which is not achievable with a conventional fluorescence microscope where different filter sets have to be used. A spectral library was established based on the spectral profiles of six types of quantum dots. Notably, a slight spectral shift was observed for all samples while the sample was drying. Subsequently, two quantum dot mixture samples were studied. Using the spectral unmixing approach, the relative proportions of each quantum dot within a homogeneous mixture and the spatial distribution of each quantum dot within a heterogeneous mixture were calculated. The calculated values match well with the theoretical predictions. Hence, the analysis method presented here can be used for simultaneous imaging of multiple fluorophores using hyperspectral imaging technology. The results provide valuable information for the realization of real-time multi-channel in vivo fluorescent imaging of biomolecules.