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. 

Fluorescensmikroskopi är en kraftfull observationsteknik som gör det möjligt för forskare att visualisera biologiska strukturer och processer i celler och vävnader med hög kontrast. Utvecklingen av fluorescensmikroskopitekniker fortskrider snabbt, med ökande spatial och temporal upplösning. Möjligheten att observera levande prover i tre dimensioner är särskilt värdefull eftersom det gör det möjligt att studera komplexa dynamiska processer i hela organismer. 

Under de senaste åren har flera superupplösningsmikroskopitekniker utvecklats. Dessa tekniker kringgår den fysiska diffraktionsgränsen för upplösning och möjliggör observation av finare detaljer. Dessa tekniker är dock vanligtvis långsammare än diffraktionsbegränsade motsvarigheter. Multifokusmikroskopi är en ljusfältsmikroskopiteknik som möjliggör samtidig avbildning av flera fokalplan och kan kombineras med superupplösningsmikroskopi. För analys av större, upp till cm-stora prover, har light-sheetmikroskopi utvecklats till en kraftfull teknik som erbjuder förbättrad axiell kontrast genom optisk snittning och minimerad fototoxicitet tack vare dess ortogonala belysnings- och detektionsgeometri. För ljusfältsmikroskopi som saknar optisk snittningsförmåga kan avfaltning användas för att ändå ge denna funktionalitet. Detta förutsätter att det finns en noggrann karakterisering av mikroskopets punktspridningsfunktion (PSF), som beskriver hur ljus fortplantas genom avbildningssystemet. 

Denna avhandling beskriver bidrag till området genom utveckling av avancerade fluorescensmikroskopitekniker och databehandling. I artikel I konstruerade vi ett multifokus-strukturerad-belysningsmikroskop (MF-SIM) med en teoretisk volumetrisk avbildningshastighet på 7 Hz och lateral upplösning på 105 nm. Vi visar dess kapacitet genom att avbilda dynamiken i endoplasmatiskt retikulum och mikrotubuli. I artikel II konstruerade vi ett 25-plans multifokusmikroskop där de 25 fokusplanen registreras simultant på varsin kamera. Vi når en volumetrisk avbildningshastighet på 125 Hz och avbildar dynamiken hos levande Drosophila- larver och C. elegans samt anpassar djupinlärningsmetoder för analysen av bilddata. I artikel III avancerade vi designen av open-source light- sheetmikroskopet descSPIM för att nå högre spatial upplösning och bildkvalitet och visar flerfärgs 3D-avbildning av njur- och lungvävnad. I artikel IV gör vi analyser med ett nyligen utvecklat verktyg för punktspridningsfunktion och ger en experimentell validering med hjälp av data från ljusfältsmikroskopi av fluorescerande kulor och C. elegans. 

High-speed volumetric imaging of whole-organism dynamics is often constrained by trade-offs between speed, resolution, and imaging depth. We present the M25 microscope, a 25-camera-array, aberration-corrected refocusing multifocus imaging system that captures 3D volumes simultaneously across 25 focal planes using a synchronized array of machine-vision cameras. Each camera incorporates a custom-blazed grating to correct chromatic dispersion, enabling a simplified, sensitive, and scalable multifocus setup for large fields of view while maintaining high spatial resolution across the imaging volume. M25 achieves imaging speeds of >100 volumes per second over imaging volumes of 180 × 180 × 50 µm. This method enables both noninvasive, label-free brightfield and highly sensitive fluorescence imaging. We demonstrate its capabilities in 3D particle tracking, fluorescent and brightfield imaging of D. melanogaster larval dynamics, and C. elegans locomotion and neural activity. This method enables fast and sensitive 3D imaging for biological studies and has potential applications across a broad range of diffractive imaging modalities.

Image reconstruction in fluorescence microscopy is highly sensitive to the accuracy of the impulse response, defined as the point spread function (PSF), of the optical system under which the image to reconstruct was acquired. In our previous work, we developed a MATLAB toolbox for accurately calculating realistic vector Fourier-based PSF accounting for any type of aberrations [arXiv, arXiv:2301.13515 (2023)10.48550/arXiv.2301.13515]. In this work, we present a fundamental experimental validation of these numerical methods. The simulated results are found to fit experimental data under different image acquisition conditions at an accuracy higher than 0.97 in normalized cross-correlation. These methods enable a relative contrast of up to 95%.

Studying the nanoscale dynamics of subcellular structures is possible with 2D structured illumination microscopy (SIM). The method allows for acquisition with improved resolution over typical widefield. For 3D samples, the acquisition speed is inherently limited by the need to acquire sequential two-dimensional planes to create a volume. Here, we present a development of multifocus SIM designed to provide high volumetric frame rate by using fast synchronized electro-optical components. We demonstrate the high volumetric imaging capacity of the microscope by recording the dynamics of microtubule and endoplasmatic reticulum in living cells at up to 2.3 super resolution volumes per second for a total volume of 30 × 30 × 1.8 µm3,.

We have designed and constructed a 3D microscope that combines Multifocus Microscopy with Structured Illumination Microscopy for live 3D super-resolution biological imaging. Imaging multiple focal planes simultaneously requires a new approach to image reconstruction.