Single-molecule imaging with SPAD array detection

Projektbeschreibung

Fortschritte in der Einzelmolekül-Mikroskopie

Konventionelle optische Mikroskope erreichen eine maximale Auflösung von 250 nm, was der Forschung in der Strukturbiologie enge Grenzen setzt. Hauptziel des EU-finanzierten Projekts SM-SPAD ist es, eine 3D-Fluoreszenzlebensdauer-Mikroskopie für Einzelmoleküle zu entwickeln, mit der sich Makromoleküle abbilden lassen, die kleiner als 100 nm sind. Der Ansatz umgeht Größenbeschränkungen, indem einzelne fluoreszente Moleküle zufällig angeschaltet werden und ihre Position auf der Bildebene bestimmt wird. Mit einer Kombination verschiedener Technologien mit sehr hoher räumlicher und zeitlicher Auflösung, die für die Untersuchung komplexer biologischer Proben ideal geeignet sind, wollen die Forschenden in SM-SPAD diese Technik noch verbessern. In der Neurowissenschaft könnte sie dann eingesetzt werden, um große Proteinkomplexe zu finden, die für die Entstehung neurodegenerativer Erkrankungen wie der Alzheimer- oder Parkinson-Krankheit verantwortlich sind.

Ziel

The resolution limit of about 250 nm in conventional optical microscopes is problematic in the study of structural biology, since proteins, macromolecules and nuclear acids are typically much smaller than 100 nm. Single-molecule localization microscopy is able to circumvent this limit by sequentially and stochastically switching on/activating single fluorescent molecules and determining their position in the image plane. E.g. MINFLUX demonstrated a 3D resolution of 6 nm. However, the point-detection system in MINFLUX does not allow directly recording the image of the fluorescent molecules in the image plane, which is required for the localization, thus a rather complex and slow beam-scanning approach is required. Secondly, the technique does not leverage the fluorescence lifetime information, which can provide nanometer-scale information on the structure of interest, e.g. via Förster resonance energy transfer. In this project, both limitations will be tackled:

The main goal of the SM-SPAD project is to develop a 3D single-molecule fluorescence lifetime imaging technique for structural biology. The goal will be reached by combining the concept of MINFLUX with three new ideas: (i) 3D motionless structured illumination and structured detection for improved spatial resolution in all three dimensions; (ii) fluorescence antibunching analysis to speed up the data acquisition by enabling simultaneous localization of several active molecules; (iii) SM level fluorescence lifetime analysis to extract the maximum amount of information from the sample.

The proposed molecular-scale imaging technique, with very high spatial and temporal resolution, is perfectly suited for the study of complex biological samples. SM-SPAD is, in particular, promising in the field of neuroscience research, in which the organization of large protein complexes are of high interest, as they may play a crucial role in the development of neurodegenerative diseases, such as Alzheimer’s and Parkinson’s .