One-Dimensional STED Microscopy in Optical Tweezers.

Optical tweezers and fluorescence microscopy are powerful methods for investigating the mechanical and structural properties of biomolecules and for studying the dynamics of the biomolecular processes that these molecules are involved in. Here we provide an outline of the concurrent use of optical tweezers and fluorescence microscopy for analyzing biomolecular processes. In particular, we focus on the use of super-resolution microscopy in optical tweezers, which allows visualization of molecules at the higher molecular densities that are typically encountered in living systems. We provide specific details on the alignment procedures of the optical pathways for confocal fluorescence microscopy and 1D-STED microscopy and elaborate on how to diagnose and correct optical aberrations and STED phase plate misalignments.

Temperature Quantification and Temperature Control in Optical Tweezers.

Optical tweezers are widely used to investigate biomolecules and biomolecular interactions. In these investigations, the biomolecules of interest are typically coupled to microscopic beads that can be optically trapped. Since high-intensity laser beams are required to trap such microscopic beads, laser-induced heating due to optical absorption is typically unavoidable. This chapter discusses how to identify, quantify, and control thermal effects in optical tweezers. We provide a brief overview of the reported causes and effects of unwanted heating in optical tweezers systems. Specific details are provided on methods to perform a temperature-independent trap calibration procedure. Finally, an effective temperature-control system is presented, and we discuss the operation of this system as well as the methods to measure the temperature at the optically trapped particle.