Diffusion Analysis of NAnoscopic Ensembles: A Tracking‐Free Diffusivity Analysis for NAnoscopic Ensembles in Biological Samples and Nanotechnology

• Published in: Small (Weinheim an der Bergstrasse, Germany) Germany), 2023-04, Vol.19 (16), p.e2206722-n/a
• Main Authors: Wolf, Alexander, Volz‐Rakebrand, Pierre, Balke, Jens, Alexiev, Ulrike
• Format: Article
• Language: English
• Subjects: Biological properties; Blinking; Diffusion; Diffusion rate; Fluorescence; Lipids; Membranes; Microscopy, Fluorescence - methods; molecular interactions; nanocarriers; Nanoparticles; nanoscopic diffusion; Nanotechnology; single molecule fluorescence; Single Molecule Imaging - methods; Statistical analysis; Tracking; tracking‐free
• ISSN: 1613-6810; 1613-6829
• DOI: 10.1002/smll.202206722

The rapid development of microscopic techniques over the past decades enables the establishment of single molecule fluorescence imaging as a powerful tool in biological and biomedical sciences. Single molecule fluorescence imaging allows to study the chemical, physicochemical, and biological properties of target molecules or particles by tracking their molecular position in the biological environment and determining their dynamic behavior. However, the precise determination of particle distribution and diffusivities is often challenging due to high molecule/particle densities, fast diffusion, and photobleaching/blinking of the fluorophore. A novel, accurate, and fast statistical analysis tool, Diffusion Analysis of NAnoscopic Ensembles (DANAE), that solves all these obstacles is introduced. DANAE requires no approximations or any a priori input regarding unknown system‐inherent parameters, such as background distributions; a requirement that is vitally important when studying the behavior of molecules/particles in living cells. The superiority of DANAE with various data from simulations is demonstrated. As experimental applications of DANAE, membrane receptor diffusion in its natural membrane environment, and cargo mobility/distribution within nanostructured lipid nanoparticles are presented. Finally, the method is extended to two‐color channel fluorescence microscopy. A novel, accurate, and fast statistical analysis tool, Diffusion Analysis of Nanoscopic Ensembles (DANAE), for an assumption‐free determination of particle diffusivities without the need of “connecting the dots” is introduced. Precise determination of microscopic diffusion constants despite high particle densities, fast diffusion, or photobleaching/blinking of the molecule is feasible via a novel treatment of the background function.