Imaging across multiple scales can provide valuable insights into complex biological systems, thereby enhancing the understanding of physiology in healthy and diseased states. Electron microscopy (EM) is a technique that resolves the nanoscale structure of tissues and cells on millimeter length scales, thus making it an effective tool for studying intricate biological processes. Recently, several EM techniques have been established that reveal the three-dimensional structure, collectively referred to as volume electron microscopy (volume EM).

Traditionally, 3D reconstructions of tissue and cells are achieved by cutting serial thin sections of resin-embedded samples, mounting them on support grids, and imaging with transmission EM. Today, volume EM includes several complementary techniques, each with different resolutions and field-of-view. For example, in array tomography, serial sections are placed on a solid substrate and imaged with scanning EM. In serial block-face scanning EM, a thin tissue slice is removed by an in situ ultramicrotome, and the exposed tissue block face is imaged. With a focused ion beam in a scanning EM, an even thinner slice can be precisely removed. The expanded toolkit has extended volume EM beyond its original application in neuroscience to a wide range of fields.

Advances in volume EM have largely been made possible by improvements in instrumentation, such as more automated workflows and faster and sensitive detectors. Nevertheless, the limited throughput of EMs remains a major bottleneck, especially for large volume imaging. Recent methodological innovations are, however, making possible the imaging of millimeter-sized samples and small organisms. In transmission EM, the throughput is limited by time-consuming sample grid replacement, stage movements and limited fieldof- view at high magnification. Reel translation systems with transparent tape, faster sample stages, larger camera arrays and advanced beam deflection have solved these bottlenecks and increased throughput…

Cryogenic electron tomography (cryo-ET) is a powerful technique to investigate bio-logical structures at molecular resolution, which is essential to understand complex processes that occur within cells. Among imaging techniques, cryo-ET stands out as it can reveal intricate structural details without the need for external labels or markers. However, its utility is often limited by the difficulties in preparing high-quality bio-logical samples. A major challenge is the production of ultra-thin, frozen-hydrated sections, or lamellae, ideally between 100 and 200 nm thick, which must remain below the inelastic mean-free path of electrons in vitreous ice. Achieving such thin, artifact-free sections is crucial for high resolution imaging.

The primary method for producing lamellae is through cryogenic focused ion beam (cryo-FIB), where the ion beam is used to fabricate the lamella, carefully re-moving cellular material to expose a cross-section of the cell for imaging with transmission electron microscope (TEM). This process is delicate and requires numerous steps to be performed with precision. Despite several improvements in cryo-FIB workflows, the accurate targeting of specific regions of interest for milling, particularly in complex biological specimens, remains a major hurdle.

In recent years, various improvements and refinements have been made to the cryo-FIB milling workflow, enhancing throughput, reliability, sample yield, and quality. Different approaches to fluorescence imaging have been incorperated into the cryo-FIB workflow to aid in selecting target cells and identifying regions of interest for milling. The aim of this dissertation is to further develop in-situ fluorescence microscopy for the cryo-FIB milling workflow through integration, and coincidence imaging, thus gaining additional insights while milling, and exploring new prospects and applications in structural biology.

Chapter 2 describes the experimental setup that was designed and built to prepare frozen-hydrated lamellae using in-situ fluorescence microscopy to guide the milling. By integrating a small cryogenic cooler, a custom positioning stage, and an inverted widefield fluorescence microscope into an existing focused ion beam scanning electron microscope, a three-beam cryogenic correlative microscope is created. As a result, fluorescence microscopy can guide targeting at each milling step, which is confirmed by transmission electron microscope tomogram reconstructions. Being able to observe the sample during and after milling improves the success rate and efficiency of producing lamellae for high-resolution imaging.

While integrating fluorescence microscopy (FM) into the cryo-FIB setup helps guide the process by identifying specific cells or subcellular regions, the refractive index mismatches between different materials during fluorescence microscopy lead to registration errors and distortions, making it difficult to precisely localize the target which can result in sub optimal milling and poor sample quality. To address this we develop a depth-dependent, non-linear scaling theory in Chapter 3, generally applicable in the field of optical microscopy. This analytical theory allows the calculation of a depth-dependent re-scaling factor based on the numerical aperture, the refractive indices, and the wavelength. It is validated through wave-optics calculations and experimental data obtained using a measurement scheme for different numerical apertures and refractive index mismatch values. The depth-dependent axial scaling theory is used to correct high resolution 3D data, acquired under various refractive index mismatch conditions. This shows the importance of correcting axial distortions during fluorescence microscopy, which arise from refractive index mismatches when imaging into frozen-hydrated samples, and correcting these is crucial for accurate targeting, ensuring that regions of interest are precisely selected for milling.

Another critical challenge is obtaining reliable, real-time feedback on lamella thick-ness, uniformity, and quality during the milling process. Typically, scanning electron microscopes (SEMs) are used to assess lamella thickness, but this approach assumes the lamella consists of homogeneous material, which is often not the case for cellular samples. Moreover, many current methods require pre-calibration before each imaging session, adding to the complexity and limiting throughput. Chapter 4 presents a set of solutions to these challenges by introducing three complementary methods for determining lamella thickness during focused ion beam (FIB) milling: (i) the application of quantitative 4D-scanning transmission electron microscopy (q4STEM) to frozen-hydrated lamellae, benchmarked against energy filtered transmision electron microscopy (EFTEM); (ii) the estimation of lamella thickness using reflected light microcopy (RLM), which accounts for the milling geometry; and (iii) exploiting thin-film interference to create real-time, per-pixel thickness maps. Together, these techniques provide immediate feedback on the thickness, lateral uniformity, and condition of the protective Pt layer during the milling process. Integrating these innovations into the cryo-FIB workflow not only improves the precision and reliability of lamella preparation but also enhances the reproducibility and yield of high-quality lamellae. By providing real-time feedback on key parameters such as thickness, uniformity, and Pt layer integrity, our approach reduces the complexity of the process and makes it more accessible for routine use in high-resolution cryo-electron microscopy studies. The ability to target regions of interest based on fluorescence, combined with thickness and quality control, enables more efficient, automated workflows for cryo-ET sample preparation.

The work presented shows a comprehensive set of tools and techniques for improving the workflow of cryo-FIB lamella fabrication. By addressing critical challenges in thickness measurement, fluorescence-based targeting, and axial distortion correction, this work paves the way for more automated, high-throughput, and reliable processes in cryo-electron microscopy (EM) sample preparation. In Chapter 5 we review the prospects and implementation in structural biology and showcase two examples of using direct targeting from fluorescence imaging, as part of ongoing investigations in collaboration with the groups of Arjen Jakobi and Dimphna Meijer at the Kavli Institute of Nanoscience in Delft, concluding with an overview of further developments and possible improvements.

This dissertation addresses one of the longstanding challenges in modern biomedical science: how to rapidly and precisely control the function of proteins in living cells. Traditionally, scientists have relied on genetic modifications to tweak proteins and improve their performance, but this approach has some inherent limitations in speed and adaptability. Instead, the work presented here explores an alternative strategy: manipulating the environment in which a protein operates to alter its behavior, rather than rewriting its genetic code. The study revolves around a genetically encoded voltage indicator known as QuasAr6a, a protein used to optically monitor electrical activity in cells, neurons in particular....

Optogenetics has revolutionized neuroscience in the last decade. In contrast to traditional electrode-based electrophysiology, optogenetics increases the throughput of targeted neurons by orders of magnitude. Genetically targeted populational neuron activities can thus be monitored and manipulated with high temporal and spatial resolution, thanks to joint efforts from both biological and optical sides. Optogenetics has become an attractive and reliable method for studying neuroscience problems.

In optogenetics, the most widely used protein to report action potentials (AP) is genetically encoded calcium indicators (GECI), which change the green fluorescence level when there is a calcium influx in the neuron. However, it is not a directmeasure ofmembrane potential, which makes them incapable of reporting sub-threshold events. Moreover, they have slow kinetics that can not distinguish a single AP.

To truly report membrane voltage dynamics, genetically encoded voltage indicators (GEVIs) were developed. GEVIs use either voltage-sensing domains (VSD) or microbial rhodopsins to detect the change in membrane potential. This change is reflected through the fluorescence emission difference from the linked fluorescent proteins or the microbial rhodopsins themselves. GEVIs based on different scaffolds have evolved through several iterations to make them brighter and faster, and voltage imaging using GEVIs has provided insights into neuroscience problems in vivo. However, the performance is still quite limited: although the VSD-based GEVIs are bright, they require blue laser excitation for the fluorescent proteins. Because of this, they suffer more from scattering in deep tissue, and their transduction time from VSD to fluorescence emission limits the speed; The microbial rhodopsin based GEVIs show a sub-millisecond response. On the other side, the biggest issue is their orders of magnitude lower fluorescence. These drawbacks would result in a poor signal-to-noise ratio (SNR) of measured signals, which is discussed in Chapter 1.

The goal of my PhD is to develop better tools to increase the SNR of voltage imaging. This dissertation achieves this goal from different disciplinary perspectives: optical engineering, software development, and protein engineering through rational design and directed evolution…

In this thesis, the author investigates the extended Schottky source for the application in a multi-beam source (MBS). The work has been done in both theoretical work and experimental work.

Interference of light can be used to determine the concentration of a gas, called gas sensing. The absorption of the light by the gas molecules is measured based on the phase change of the light. In this report, a hollow core photonic crystal fiber is treated. The gas sample is inserted into the hollow core. Photonic crystal fibers possess the characteristic to have electromagnetic modes confined to the core with low attenuation. This means that there can be a high interference rate between the gas and light, which is desirable for gas sensing.
First, less complicated optical fibers were studied. The analytical solution of the electric and magnetic field for the TE and TM modes of the simple fiber were derived and for the TE modes of the step-index fiber. These results were compared to simulation done with COMSOL Multiphysics. Photonic crystal fiber with circular core was then simulated with COMSOL Multiphysics, but the results did not match with the literature. Therefore another photonic crystal fiber was simulated with a star-shaped core. The simulation results found modes concentrated in the core, with low attenuation.
It was attempted to get similar results with a three layer step-index fiber, by varying the imaginary refractive index of the middle layer. The attenuation of the three layer fiber was much higher than that of the photonic crystal fiber for all simulations. This indicates that the three layer step-index fiber does not support propagation modes that are confined to the core. Further research could be done by studying the effect of the radius of the middle layer and the real part of the refractive index.

Optogenetics is a powerful addition to the spectrum of techniques available in neuroscience to investigate neurophysiology and unravel how neural circuit structure is related to circuit function. This technique relies on introducing lightsensitive proteins or molecules as actuators to transduce an optical signal into a physiological perturbation of a living cell in vitro or in a living animal. To date, optogenetics has allowed remote control of neural activity in living and awake animals at different scales from single cells to complex networks of neurons to the investigation of animal behaviours. This wide range of experimental scales has been accomplished through joint progress on engineering the biological sensors and the optical design of instruments capable of manipulating with cellular spatial precision and millisecond temporal resolution.