Advances in enhanced multi-plane 3D imaging and image scanning microscopy

Abstract

High-speed 3D optical microscopy is an indispensable requirement in studying rapid processes like signaling in neuronal networks, flagellar motion, or complex motion of living and highly dynamic subcellular components. However, the 3D imaging capability often compromises either the acquisition speed or the complexity of the imaging system. Multi-plane imaging offers a parallelized acquisition of several focal planes at the same time, which tremendously enhances the temporal resolution. Nonetheless, most of the current multi-plane approaches are complex and require several optical elements to create multiple beams with different optical path lengths, limiting their broad application. To address these problems, in this thesis, employing a multi-plane prism, we introduce three novel techniques that each improve the concept of rapid 3D optical microscopy in a different direction: First, we accomplish a multi-plane detection for a phase-contrast microscope resulting in a 3D, label-free, and non-complex imaging system with a ~ 4ms of temporal resolution. We applied this system in real-time studying isolated bare axonemes of Chlamydomonas beating in the vicinity of a surface. This enabled us to observe their non-zero torsional motion where the torsion sign slowly changes from negative at the basal end toward positive at the distal end of flagella. Second, straightforwardly, we combine an experimental spectral unmixing setup with the multi-plane detection and obtain an instant 3- color 3D microscope with a minimal axial color aberration of 140 nm in a ~ 2.5 μm axial range. The performance of such a microscope is verified in volumetric imaging of three different subcellular components in fixed COS-7 cells. Third, a single-color multi-plane fluorescence microscope has been employed as a rapid 3D particle tracking velocimetry method in microscale to study single particles tracing the so-called Marangoni flow. Using that, besides the velocity fields of single particles, we achieve a localization precision of ~ 60 nm in the lateral and ~ 300 nm in the axial direction over a 120 120 13 m3 field of view. Altogether, using these three techniques, we extend the temporal resolution, throughput, and applicability of 3D optical microscopy. In another project, we implemented image scanning microscopy simultaneously in two colors on a conventional wide-field microscope by applying an LED light source and a digital micro-mirror device. This permits us to realize an inexpensive, speckle noise-free, and easy-to-implement super-resolution add-on for a conventional epi-fluorescence microscope.