Optical tweezers combined with various microscopy techniques are a versatile tool for single-molecule force spectroscopy. However, some combinations may compromise measurements. Here, we combined optical tweezers with total-internal-reflection-fluorescence (TIRF) and interference-reflection microscopy (IRM). Using a light-emitting diode (LED) for IRM illumination, we show that single microtubules can be imaged with high contrast. Furthermore, we converted the IRM interference pattern of an upward bent microtubule to its three-dimensional (3D) profile calibrated against the optical tweezers and evanescent TIRF field. In general, LED-based IRM is a powerful method for high-contrast 3D microscopy.

Biocompatible surfaces are important for basic and applied research in life science with experiments ranging from the organismal to the single-molecule level. For the latter, examples include the translocation of kinesin motor proteins along microtubule cytoskeletal filaments or the study of DNA−protein interactions. Such experiments often employ single-molecule fluorescence or force microscopy.In particular for force measurements, a key requirement is to prevent nonspecific interactions of biomolecules and force probes with the surface, while providing specific attachments that can sustain loads. Common approaches to reduce nonspecific interactions include supported lipid bilayers or PEGylated surfaces. However, fluid lipid bilayers do not support loads and PEGylation may require harsh chemical surface treatments and have limited reproducibility. Here, we developed and applied a supported solid lipid bilayer (SSLB) as a platform for specific, load bearing attachments with minimal nonspecific interactions. Apart from single-molecule fluorescence measurements, anchoring molecules to lipids in the solid phase enabled us to perform force measurements of molecular motors and overstretch DNA. Furthermore, using a heating laser, we could switch the SSLB to its fluid state allowing for manipulation of anchoring points. The assay had little nonspecific interactions, was robust, reproducible, and time-efficient, and required less hazardous and toxic chemicals for preparation. In the long term, we expect that SSLBs can be widely employed for single-molecule fluorescence microscopy, force spectroscopy, and cellular assays in mechanobiology.

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 1-10 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.

Gold nanoparticles are intriguing because of their unique size- and shape-dependent chemical, electronic and optical properties. Gold nanorods (AuNRs) are particularly promising for various sensor applications due to their tip-enhanced...

Optical tweezers combined with various microscopy techniques are a versatile tool for single-molecule force spectroscopy. However, some combinations may compromise measurements. Here, we combined optical tweezers with total-internal-reflection-fluorescence (TIRF) and interference-reflection microscopy (IRM). Using a light-emitting diode (LED) for IRM illumination, we show that single microtubules can be resolved with high contrast. Furthermore, we converted the IRM interference pattern of an upward bent microtubule to its three-dimensional (3D) profile calibrated against the optical tweezers and evanescent TIRF field. In general, LED-based IRM is a powerful method for high-resolution 3D microscopy.OCIS codes: (180.3170) Interference microscopy; (120.4570) Optical design of instruments (350.4855); Optical tweezers or optical manipulation.