Waveguide platform and methods for super-resolution fluorescence microscopy of sub-cellular structures

Super-resolution fluorescence microscopy is widespread, owing to its demonstrated ability to resolve dynamical processes within cells and to identify the structure and position of specific proteins in the interior of protein complexes. Nowadays, subcellular features can be routinely resolved at the nanoscopic scale thanks to the accessibility of straightforward sample-preparation protocols, simple hardware tools, and open source software. Building on its ability to investigate large-scale macromolecules networks in their natural environment with high resolution, fluorescence microscopy is further evolving by the development of quantitative and high-throughput methods to characterize such networks. Previous implementations of high-throughput microscopy made use of imaging sequentially smaller fields of view (FOV), which makes axial alignment a challenge and extends the imaging time. In our work, we circumvent these problems with our large FOV systems, which are based on flat-field sample illumination over large areas, combined with a CMOS-camera. In this thesis, I present a waveguide platform designed to image a wide area with low background by mean of total internal reflection fluorescence (TIRF) excitation. The waveguide chips for this platform were fabricated at the center of micro-nano technology (CMi) at EPFL, in collaboration with the group of Aleksandra Radenovic (specifically with Evgenii Glushkov). The resulting waveguide-TIRF system is specifically optimized for applications where easy and repetitive buffer exchange is needed. To achieve large and uniform TIRF excitation, I studied some fundamental parameters of the waveguide, developing specific code to simulate, at the first order, its behavior. I then extended light propagation solutions adopted in the field of integrated photonics to our waveguide chip fabrication process. To easily integrate the chip within the commercial stage of an upright microscope, I designed a novel chip holder that ensures aqueous solution sealing, mitigates the presence of scatter light in the imaging area, and facilitates the waveguide alignment during the input beam-coupling phase. On the analysis side, the need for computational tools that are specific to fluorescence microscopy is continuously growing, due to the fact that this technique heavily relies on the treatment of large quantities of data. The automated analysis of images is a fundamental step of the measurement process, necessary for unbiased quantification and statistical validation, especially where repetitive visual inspection would be impractically long. This is particularly critical for single molecule localization microscopy (SMLM), where the quality of the reconstructed super-resolved image actually is a trade-off between the algorithm localization precision and its speed, a key element considering the need of processing tens of thousands of large images to generate the final, super-resolved one. In this work, I present a series of computational tools for CMOS camera characterization developed for large flat-field STORM microscopy, a 3D SMLM reconstruction software specific for Double-Helix (DH) point spread function (PSF) and a set of cell shape analysis tools to study C.Crescentus shape dynamics

High performance, LED powered, waveguide based total internal reflection microscopy.

Total internal reflection fluorescence (TIRF) microscopy is a rapidly expanding optical technique with excellent surface sensitivity and limited background fluorescence. Commercially available TIRF systems are either objective based that employ expensive special high numerical aperture (NA) objectives or prism based that restrict integrating other modalities of investigation for structure-function analysis. Both techniques result in uneven illumination of the field of view and require training and experience in optics. Here we describe a novel, inexpensive, LED powered, waveguide based TIRF system that could be used as an add-on module to any standard fluorescence microscope even with low NA objectives. This system requires no alignment, illuminates the entire field evenly, and allows switching between epifluorescence/TIRF/bright field modes without adjustments or objective replacements. The simple design allows integration with other imaging systems, including atomic force microscopy (AFM), for probing complex biological systems at their native nanoscale regimes

Super-condenser enables labelfree nanoscopy.

Labelfree nanoscopy encompasses optical imaging with resolution in the 100 nm range using visible wavelengths. Here, we present a labelfree nanoscopy method that combines Fourier ptychography with waveguide microscopy to realize a 'super-condenser' featuring maximally inclined coherent darkfield illumination with artificially stretched wave vectors due to large refractive indices of the employed Si$_3$N$_4$ waveguide material. We produce the required coherent plane wave illumination for Fourier ptychography over imaging areas 400 $\mathrm{\mu}$m$^2$ in size via adiabatically tapered single-mode waveguides and tackle the overlap constraints of the Fourier ptychography phase retrieval algorithm two-fold: firstly, the directionality of the illumination wave vector is changed sequentially via a multiplexed input structure of the waveguide chip layout and secondly, the wave vector modulus is shortend via step-wise increases of the illumination light wavelength over the visible spectrum. We validate the method via in silico and in vitro experiments and provide details on the underlying image formation theory as well as the reconstruction algorithm

Super-condenser enables labelfree nanoscopy.

Labelfree nanoscopy encompasses optical imaging with resolution in the 100 nm range using visible wavelengths. Here, we present a labelfree nanoscopy method that combines coherent imaging techniques with waveguide microscopy to realize a super-condenser featuring maximally inclined coherent darkfield illumination with artificially stretched wave vectors due to large refractive indices of the employed Si3N4 waveguide material. We produce the required coherent plane wave illumination for Fourier ptychography over imaging areas 400 μm2 in size via adiabatically tapered single-mode waveguides and tackle the overlap constraints of the Fourier ptychography phase retrieval algorithm two-fold: firstly, the directionality of the illumination wave vector is changed sequentially via a multiplexed input structure of the waveguide chip layout and secondly, the wave vector modulus is shortend via step-wise increases of the illumination light wavelength over the visible spectrum. We test the method in simulations and in experiments and provide details on the underlying image formation theory as well as the reconstruction algorithm. While the generated Fourier ptychography reconstructions are found to be prone to image artefacts, an alternative coherent imaging method, rotating coherent scattering microscopy (ROCS), is found to be more robust against artefacts but with less achievable resolution

Optically-controlled platforms for transfection and single- and sub-cellular surgery

Improving the resolution of biological research to the single- or sub-cellular level is of critical importance in a wide variety of processes and disease conditions. Most obvious are those linked to aging and cancer, many of which are dependent upon stochastic processes where individual, unpredictable failures or mutations in individual cells can lead to serious downstream conditions across the whole organism. The traditional tools of biochemistry struggle to observe such processes: the vast majority are based upon ensemble approaches analysing the properties of bulk populations, which means that the detail about individual constituents is lost. What are required, then, are tools with the precision and resolution to probe and dissect cells at the single-micron scale: the scale of the individual organelles and structures that control their function. In this review, we highlight the use of highly-focused laser beams to create systems providing precise control and specificity at the single cell or even single micron level. The intense focal points generated can directly interact with cells and cell membranes, which in conjunction with related modalities such as optical trapping provide a broad platform for the development of single and sub-cellular surgery approaches. These highly tuneable tools have demonstrated delivery or removal of material from cells of interest, but can simultaneously excite fluorescent probes for imaging purposes or plasmonic structures for very local heating. We discuss both the history and recent applications of the field, highlighting the key findings and developments over the last 40 years of biophotonics researc

Selected Advances of Quantum Biophotonics – a Short Review

This article discusses four fields of study with the potential to revolutionize our understanding and interaction with biological systems: quantum biophotonics, molecular and supramolecular bioelectronics, quantum-based approaches in gaming, and nano-biophotonics. Quantum biophotonics uses photonics, biochemistry, biophysics, and quantum information technologies to study biological systems at the sub-nanoscale level. Molecular and supramolecular bioelectronics aim to develop biosensors for medical diagnosis, environmental monitoring, and food safety by designing materials and devices that interface with biological systems at the molecular level. Quantum-based approaches in gaming improve modeling of complex systems, while nanomedicine enhances disease diagnosis, treatment, and prevention using nanoscale devices and sensors developed with quantum biophotonics. Lastly, nano-biophotonics studies cellular structures and functions with unprecedented resolution

Selected Advances of Quantum Biophotonics – a Short Review

This article discusses four fields of study with the potential to revolutionize our understanding and interaction with biological systems: quantum biophotonics, molecular and supramolecular bioelectronics, quantum-based approaches in gaming, and nano-biophotonics. Quantum biophotonics uses photonics, biochemistry, biophysics, and quantum information technologies to study biological systems at the sub-nanoscale level. Molecular and supramolecular bioelectronics aim to develop biosensors for medical diagnosis, environmental monitoring, and food safety by designing materials and devices that interface with biological systems at the molecular level. Quantum-based approaches in gaming improve modeling of complex systems, while nanomedicine enhances disease diagnosis, treatment, and prevention using nanoscale devices and sensors developed with quantum biophotonics. Lastly, nano-biophotonics studies cellular structures and functions with unprecedented resolution

Multiparametric Optical Characterization of Biological Nanoparticles using Evanescent Field Sensing

In light of the increasingly realized dependence of many biological functions on nanoscopic supramolecular assemblies, also including novel biotechnological applications, there is a need for advanced analysis methods capable of accurately quantifying different characteristics of these elusive entities. The prime aim of this thesis is the development and utilization of surface-based bioanalytical sensing methods for quantitative characterization of biological nanoparticles. The possibility to construct and use a waveguide-based evanescent light scattering microscopy instrument for investigation of various nanoparticle properties is explored through the study of liposomes and mRNA-containing lipid nanoparticles as well as polystyrene and silica nanoparticles. It is shown that through analysis of scattered light from such particles, single-particle-resolved information on their size, refractive index and interactions with surrounding protein solutions is obtainable, thus providing multiparametric characterization beyond the ensemble average. Additionally, this is combined with information gained from fluorescent labeling of certain biomolecular components, allowing nanoparticle content to be correlated with the other particle properties. The aforementioned systems were additionally investigated using a range of complementary methods, including nanoparticle tracking analysis, surface plasmon resonance sensing, and quartz crystal microbalance with dissipation monitoring. It was concluded that the waveguide microscopy method provides quantitative information in good agreement with established methods, but offers certain key advantages, such as the possibility to provide single-particle resolved label-free information on protein binding kinetics combined with simultaneous evanescent light fluorescence microscopy measurements, thus providing new insights regarding nanoparticle heterogeneity

Technical implementations of light sheet microscopy

Fluorescence-based microscopy is among the most successful methods in biological studies. It played a critical role in the visualization of subcellular structures and in the analysis of complex cellular processes, and it is nowadays commonly employed in genetic and drug screenings. Among the fluorescence-based microscopy techniques, light sheet fluorescence microscopy (LSFM) has shown a quite interesting set of benefits. The technique combines the speed of epi-fluorescence acquisition with the optical sectioning capability typical of confocal microscopes. Its unique configuration allows the excitation of only a thin plane of the sample, thus fast, high resolution imaging deep inside tissues is nowadays achievable. The low peak intensity with which the sample is illuminated diminishes phototoxic effects and decreases photobleaching of fluorophores, ensuring data collection for days with minimal adverse consequences on the sample. It is no surprise that LSFM applications have raised in just few years and the technique has been applied to study a wide variety of samples, from whole organism, to tissues, to cell clusters, and single cells. As a consequence, in recent years numerous set-ups have been developed, each one optimized for the type of sample in use and the requirements of the question at hand. Hereby, we aim to review the most advanced LSFM implementations to assist new LSFM users in the choice of the LSFM set-up that suits their needs best. We also focus on new commercial microscopes and do-it-yourself strategies; likewise we review recent designs that allow a swift integration of LSFM on existing microscopes