Optimization of a single molecule fluorescence microscopy setup

Abstract

Many single molecule fluorescence imaging modalities use a laser beam to excite fluorescent structures or fluorescent markers attached to molecules of interest. This excitation laser shows an inherent gaussian intensity distribution across its profile, which is disadvantageous due to the decreasing intensity at the periphery of the beam. A common way of tackling this issue is to crop the gaussian beam with an adjustable rectangular aperture in the excitation beam path with concomitant loss of overall intensity as the field of view decreases. In this thesis, a new beam path was assembled by utilizing a Top Hat beamshaper element which transforms the gaussian beam profile into a homogeneous intensity distribution with no significant loss of intensity. The standard deviation of this uniform intensity distribution decreased by 30% compared to the gaussian laser beam. Another issue that was addressed in this thesis is the stability of the microscope setup. Single molecule fluorescence microscopy (SMFM) experiments can be time consuming, as the particles of interest are often not just imaged momentarily but tracked over time to investigate the dynamic behavior. Also in single molecule localization microscopy (SMLM) thousands of images are recorded to finally achieve image resolution down to a few nanometers. During that time span of up to hours, drifts along the optical axis can occur resulting in de-focused images or even the loss of the fluorescence signal. Thus, the equipment with an automatic focus stabilizing system for long term measurements is substantial. The implementation and characterization of such a Focus Hold System (FHS) is presented in this thesis. A cost effective and hardware based approach, utilizing the back reflection of an external infrared laser diode to register movements between the microscope’s objective and the sample plane, was realized. The implemented FHS proved to maintain the z-position of the microscope within +/-100nm of the set value.