The composition, conformation, and function of most macromolecular complexes depend on their cellular context and must be studied inside cells. Few cells are sufficiently thin to permit direct imaging with cryoelectron microscopy (cryo-EM). Focused ion beam milling enables cryo-EM to visualize macromolecules in cells at high resolution by generating thin sections of frozen-hydrated cells. We show how thin cellular sections can be prepared in a controlled fashion using an integrated light microscope coincident with electron and ion beams. The procedure provides live feedback on the thickness and uniformity of the prepared lamella, reducing complexity and increasing the success rate. Combined with its ability for fluorescence-based targeting, our procedure paves the way toward an automated workflow that allows for control over lamella quality, thickness, and target inclusion, facilitating the routine fabrication of frozen-hydrated cellular sections.

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.

In volume fluorescence microscopy, refractive index matching is essential to minimize aberrations. There are, however, common imaging scenarios where a refractive index mismatch (RIM) between immersion and a sample medium cannot be avoided. This RIM leads to an axial deformation in the acquired image data. Over the years, different axial scaling factors have been proposed to correct for this deformation. While some reports have suggested a depth-dependent axial deformation, so far none of the scaling theories has accounted for a depth-dependent, non-linear scaling. Here, we derive an analytical theory based on determining the leading constructive interference band in the objective lens pupil under RIM. We then use this to calculate a depth-dependent re-scaling factor as a function of the numerical aperture (NA), the refractive indices n₁ and n₂, and the wavelength λ. We compare our theoretical results with wave-optics calculations and experimental results obtained using a measurement scheme for different values of NA and RIM. As a benchmark, we recorded multiple datasets in different RIM conditions, and corrected these using our depth-dependent axial scaling theory. Finally, we present an online web applet that visualizes the depth-dependent axial re-scaling for specific optical setups. In addition, we provide software that will help microscopists to correctly re-scale the axial dimension in their imaging data when working under RIM.

A quantitative four-dimensional scanning transmission electron microscopy (4D-STEM) imaging technique (q4STEM) for local thickness estimation across amorphous specimen such as obtained by focused ion beam (FIB)-milling of lamellae for (cryo-)TEM analysis is presented. This study is based on measuring spatially resolved diffraction patterns to obtain the angular distribution of electron scattering, or the ratio of integrated virtual dark and bright field STEM signals, and their quantitative evaluation using Monte Carlo simulations. The method is independent of signal intensity calibrations and only requires knowledge of the detector geometry, which is invariant for a given instrument. This study demonstrates that the method yields robust thickness estimates for sub-micrometer amorphous specimen using both direct detection and light conversion 2D-STEM detectors in a coincident FIB-SEM and a conventional SEM. Due to its facile implementation and minimal dose reauirements, it is anticipated that this method will find applications for in situ thickness monitoring during lamella fabrication of beam-sensitive materials.

Cryogenic electron tomography (cryo-ET) combined with sub-tomogram averaging, allows in-situ visualization and structure determination of macromolecular complexes at sub-nanometre resolution. Cryogenic focused ion beam (cryo-FIB) micromachining is used to prepare a thin lamella-shaped sample out of a frozen-hydrated cell for cryo-ET imaging, but standard cryo-FIB fabrication is blind to the precise location of the structure or proteins of interest. Fluorescence-guided focused ion beam (FIB) milling at target locations requires multiple sample transfers prone to contamination, and relocation and registration accuracy is often insufficient for 3D targeting. Here, we present in-situ fluorescence microscopy-guided FIB fabrication of a frozen-hydrated lamella to address this problem: we built a coincident 3-beam cryogenic correlative microscope by retrofitting a compact cryogenic microcooler, custom positioning stage, and an inverted widefield fluorescence microscope (FM) on an existing focused ion-beam scanning electron microscope (FIB-SEM). We show FM controlled targeting at every milling step in the lamella fabrication process, validated with transmission electron microscope (TEM) tomogram reconstructions of the target regions. The ability to check the lamella during and after the milling process results in a higher success rate in the fabrication process and will increase the throughput of fabrication for lamellae suitable for high-resolution imaging.