Characterization of Nanometric Qdots by AFM and Optical Microscopy

Quantum dots (Qdots) are fluorescent nanocrystals with exceptional brightness, tunability, and photostability, and huge potential in biomedical imaging from single-particle tracking, to multicolor imaging of cell structures, to real-time in vivo imaging of whole organisms. We have undertaken a combined optical and atomic force microscopy (AFM) investigation to better understand Qdots. We have developed multimodal instrumentations around AFM platforms and total internal reflection fluorescent microscopy (TIRFM) and Raman microscopy with environmental control for simultaneous characterization of the nanoscopic structural features and optical properties of individual particles and biomedical samples. Our new instruments allow fuller observation of Qdots nanocrystal core, biocompatible coating, oligomerization states, and their optical behavior, as they interact with the environment. For example, fluorescent time traces show that the fraction of “on”-time varied from a few percent to over 80% among isolated Qdots. Our high resolution AFM topological maps and super-resolution (below the diffraction limit) TIRFM localization detection reveal a varying degree of Qdot dimerization and oligomerization on glass and mica substrates. We also demonstrate direct fluorescence spectral mapping using a newly customized and integrated Raman-AFM instrument. By understanding the relationships between their topological, mechanical, electrostatic profiles and their optical properties, Qdots can be ultimately improved toward more quantitative biomedical imaging applications.