RESUMEN
Observation of highly dynamic processes inside living cells at the single molecule level is key for a better understanding of biological systems. However, imaging of single molecules in living cells is usually limited by the spatial and temporal resolution, photobleaching and the signal-to-background ratio. To overcome these limitations, light-sheet microscopes with thin selective plane illumination, for example, in a reflected geometry with a high numerical aperture imaging objective, have been developed. Here, we developed a reflected light-sheet microscope with active optics for fast, high contrast, two-colour acquisition of z -stacks. We demonstrate fast volume scanning by imaging a two-colour giant unilamellar vesicle (GUV) hemisphere. In addition, the high contrast enabled the imaging and tracking of single lipids in the GUV cap. The enhanced reflected scanning light-sheet microscope enables fast 3D scanning of artificial membrane systems and potentially live cells with single-molecule sensitivity and thereby could provide quantitative and molecular insight into the operation of cells.
Asunto(s)
Microscopía , Liposomas Unilamelares , Imagenología Tridimensional/métodos , Microscopía/métodos , FotoblanqueoRESUMEN
Mechanical vibrations in buildings are ubiquitous. Such vibrations limit the performance of sensitive instruments used, for example, for high-precision manufacturing, nanofabrication, metrology, medical systems, or microscopy. For improved precision, instruments and optical tables need to be isolated from mechanical vibrations. However, common active or passive vibration isolation systems often perform poorly when low-frequency vibration isolation is required or are expensive. Furthermore, a simple solution such as suspension from common bungee cords may require high ceilings. Here we developed a vibration isolation system that uses steel springs to suspend an optical table from a common-height ceiling. The system was designed for a fundamental resonance frequency of 0.5 Hz. Resonances and vibrations were efficiently damped in all translational and rotational degrees of freedom of the optical table by spheres, which were mounted underneath the table and immersed in a highly viscous silicone oil. Our low-cost, passive system outperformed several state-of-the-art passive and active systems in particular in the frequency range between 1 and 10 Hz. We attribute this performance to a minimal coupling between the degrees of freedom and the truly three dimensional viscous damping combined with a nonlinear hydrodynamic finite-size effect. Furthermore, the system can be adapted to different loads, resonance frequencies, and dimensions. In the long term, the excellent performance of the system will allow high-precision measurements for many different instruments.