Universal Laser Engine: a uniform picosecond-to-continuous laser illumination to enable novel fast multimodal fluorescence microscopy techniques

Optical microscopy is one of the most important tools in many scientific fields, such as biomedical research. Here, the importance of illumination for image quality has been known since the early days of microscopy. For classical light microscopy, this was optimized with the famous Köhler illumination. Fluorescence microscopy offers new insights into cellular and subcellular mechanisms as individual proteins can be selectively labeled with fluorescent dyes. Currently, this technique uses laser-based illumination methods to achieve the required light yield. However, the high spatial and temporal coherence of laser sources leads to interference patterns, so-called speckles, which prevent uniform illumination. To provide a solution, the universal laser engine (ULE) is presented in this work. It is based on a novel method for generating a uniform illumination with sharp beam profile edges. In contrast to existing temporal-averaging solutions, the homogenization approach is also effective for fast temporal acquisitions within the microsecond range. The ULE has been developed into a turnkey device that replaces conventional laser-based illumination systems for most microscopy applications. The ULE is very versatile and compatible with multiple illumination modes, such as highly inclined and laminated optical sheet (HILO), total internal reflection fluorescence microscopy (TIRFM), widefield and confocal illumination. In addition to powerful continuous wave to microsecond-pulsable lasers, the ULE also contains picosecond-pulsed lasers. As demonstrated in this work, the ULE can be used for widefield microscopes, single-molecule localization microscopy (SMLM), fluorescence lifetime imaging microscopy (FLIM), but also for widefield FLIM, where the ULE can combine FLIM with homogeneous illumination. The ULE is the starting point for the further development of microscopy methods. The throughput of single-molecule fluorescence measurements using the ULE and zero-mode waveguides (ZMW)s could be increased by a factor of 80. In addition, 10 fluorescence correlation spectroscopy (FCS) measurements could be recorded simultaneously. Together with the further development of detectors, ULE has the potential to automate sensitive but labor-intensive techniques and to enable new biophysical experimental protocols.