Advancing fluorescence microscopy for improved spatio-temporal resolution

QC 2025-01-08

Abstract

Fluorescence microscopy is a powerful observation technique that allows researchers to visualize biological structures and processes within cells and tissues with high contrast. Development of fluorescence microscopy techniques is rapidly progressing, with increasing spatial and temporal resolution. The ability to observe live samples in three dimensions is particularly valuable as it enables studies of complex dynamic processes in whole organisms. 

In recent years several super-resolution microscopy techniques have been developed. Those techniques circumvent the physical diffraction limit for resolution and allow observation of finer details. However, these techniques are usually slower than their diffraction-limited counterparts. Multifocus microscopy is a widefield microscopy technique that enables simultaneous imaging of multiple focal planes and can be combined with super resolution microscopy. For the analysis of larger, up to cm-sized samples, light-sheet microscopy has arisen as a powerful technique offering improved axial contrast through optical sectioning and minimized phototoxicity due to its orthogonal illumination and detection geometry. For widefield techniques lacking optical sectioning capabilities, computational deconvolution can provide this functionality, provided there is an accurate characterization of the microscope’s point spread function (PSF), which describes how light propagates through the imaging system. 

This thesis describes contributions to the field by the development of advanced fluorescent microscopy techniques and computational post processing. In paper I, we constructed a multifocus structured illumination microscope (MF-SIM) with theoretical volumetric imaging speed of 7 Hz and lateral resolution of 105 nm. We demonstrate its capabilities by imaging endoplasmatic reticulum dynamics and microtubule dynamics. In paper II we constructed a 25-plane multifocus widefield microscope where each focus plane is captured by its own camera. We reach volumetric imaging speed of up to 125 Hz and show dynamics of living Drosophila larvae and C. elegans and apply deep learning methods to analyze the imaging data. In paper III we advanced the design of the open-source light-sheet microscope descSPIM for higher spatial resolution and image quality and show multicolor 3D imaging of kidney and lung tissue. In paper IV we apply a recently developed point spread function tool and provide experimental validation based on data from widefield imaging of fluorescent bead and C.elegans. 

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