Microfluidic protein isolation and sample preparation for transmission electron microscopy

The knowledge of atomic structures is essential to understand the mechanics and chemistry of proteins in fundamental research and is often the base for drug development. During the last decades, X-ray crystallography has been the primary method for determining atomic models providing an impressive number of molecular structures. Nevertheless, the technique is limited by the fact that the complexes of interest have to be crystallized. Nuclear magnetic resonance (NMR), which is used as an alternative to solve biomolecules in solution, has the drawback of consuming large amounts of protein, being labour intensive and challenging for large molecules. In recent years, cryogenic electron microscopy (cryo-EM) has evolved as an important tool for protein structure determination. Technical advances in the instrumentation and increased computational power combined with better processing algorithms caused a massive improvement in the resolution of obtained structures. For these achievements Jacques Dubochet, Joachim Frank and Richard Henderson were awarded with a Nobel Prize in 2017. However, sample preparation methods lack behind and did not change a lot. A significant complication is the production of target proteins in sufficient amounts and quality. Although only some thousands to a few million protein particles must be imaged to solve a protein structure, much larger quantities are required to prepare specimens for cryo-EM. Conventional sample preparation methods are very wasteful with proteins and more than 99% of protein is lost during a paper blotting step. Thus, considerable amounts of purified proteins have to be produced using complex and costly procedures usually including several chromatography steps. In this thesis, a novel sample preparation and purification system consuming only minute amounts of biological material is presented. The system allows the purification of proteins and the subsequent preparation of isolated targets for negative stain and cryo-EM. We constructed corresponding hardware and software described in Chapters 1 & 2. The application of the system on biological samples is demonstrated in Chapters 3 & 4. As an example, we purified endogenous human 20S proteasome starting with <1 μL HeLa cytosol and determined it’s 3D structure at a resolution of 3.5Å. In Chapter 5, we show the purification of recombinantly expressed proteins by the use of a novel crosslinker that was developed during the course of this thesis

Validating device-based physical activity indicators with observation physical activity indicators in Swiss pre-school children

Purpose Accelometry is a very important tool to measure physical activity (PA) in preschoolers, as it can measure 24-hour PA and detect activity which cannot be captured by other methods. However, accelerometer validation for preschoolers is lacking (Altenburg et al. 2022). Specifically, for the ActiGraph wGT3X-BT and Move 4 (ActiGraph LLC; Move4 activity sensor, movisens GmbH) only a few, respectively no preschooler validation studies, were found. Thus, the purpose was to validate these two accelerometers in Swiss preschoolers with the System for Observing Children’s Activity and Relationships during Play (SOCARP; Ridgers et al., 2010). Methods and Design Preschoolers (2-5 years old) from two Swiss Sunday activity programs (MiniMove &amp; Ä Halle wo’s fägt) were randomly selected as part of a larger program evaluation. PA was assessed SOCARP for a duration of 12 minutes per child. During the observation, the children wore both an ActiGraph and a Move4 device taped to their right hip to record steps. Step-counts from the ActiGraph and Move4 were correlated with each other and with moderate-to-vigorous (MV)PA from SOCARP (as SOCARP does not count steps). Results Valid PA data was available for 45/58 (77.6%) children (49% girls) for SOCARP and for 47/58 (81%) children (51% girls) for accelometry. Step count correlations between the accelerometers (Actigraph and Move4) and %MVPA (SOCARP) was medium and positive (r(43) = .34, p = .03 and r(43) = .37, p = .02; respectively). There was a strong step count correlation between the two devices (r(45) = .90, p &lt; .001), although ActiGraph measured significantly more steps than Move4 (m = 557.74, SD = 255.77 versus m = 397.81, SD = 164.10); t(46)=8.47, p &lt; .001). Discussion PA measurement in preschool children can be challenging. However, the correlation between step counts and observed %MVPA indicates criterion validity for both devices. The step-counts of Actigraph and Move4 validate each other, but there is a difference in the absolute number of measured steps. Due to different outcome parameters, calculation algorithms, and inaccessibility to raw acceleration the comparison of the two devices on movement intensity was not possible. Although promising preliminary indications of validity of device-based measurement of PA in Swiss preschool children, further investigations into the methodological approaches of comparing measurements of movement intensity are warranted. References Altenburg, T. M., de Vries, L., op den Buijsch, R., Eyre, E., Dobell, A., Duncan, M., &amp; Chinapaw, M. J. M. (2022). Cross-validation of cut-points in preschool children using different accelerometer placements and data axes. Journal of Sports Sciences, 40(4), 379-385. https://doi.org/10.1080/02640414.2021.1994726 Ridgers, N. D., Stratton, G., &amp; McKenzie, T. L. (2010). Reliability and validity of the system for observing children’s activity and relationships during play (SOCARP). Journal of Physical Activity and Health, 7(1), 17–25. https://doi.org/10.1123/jpah.7.1.1

Microfluidic protein isolation and sample preparation for high-resolution cryo-EM

High-resolution structural information is essential to understand protein function. Protein-structure determination needs a considerable amount of protein, which can be challenging to produce, often involving harsh and lengthy procedures. In contrast, the several thousand to a few million protein particles required for structure determination by cryogenic electron microscopy (cryo-EM) can be provided by miniaturized systems. Here, we present a microfluidic method for the rapid isolation of a target protein and its direct preparation for cryo-EM. Less than 1 mu L of cell lysate is required as starting material to solve the atomic structure of the untagged, endogenous human 20S proteasome. Our work paves the way for high-throughput structure determination of proteins from minimal amounts of cell lysate and opens more opportunities for the isolation of sensitive, endogenous protein complexes

Differential Visual Proteomics: Enabling the Proteome-Wide Comparison of Protein Structures of Single-Cells

Proteins are involved in all tasks of life, and their characterization is essential to understand the underlying mechanisms of biological processes. We present a method called "differential visual proteomics" geared to study proteome-wide structural changes of proteins and protein-complexes between a disturbed and an undisturbed cell or between two cell populations. To implement this method, the cells are lysed and the lysate is prepared in a lossless manner for single-particle electron microscopy (EM). The samples are subsequently imaged in the EM. Individual particles are computationally extracted from the images and pooled together, while keeping track of which particle originated from which specimen. The extracted particles are then aligned and classified. A final quantitative analysis of the particle classes found identifies the particle structures that differ between positive and negative control samples. The algorithm and a graphical user interface developed to perform the analysis and to visualize the results were tested with simulated and experimental data. The results are presented, and the potential and limitations of the current implementation are discussed. We envisage the method as a tool for the untargeted profiling of the structural changes in the proteome of single-cells as a response to a disturbing force

Total Sample Conditioning and Preparation of Nanoliter Volumes for Electron Microscopy

Electron microscopy (EM) entered a new era with the emergence of direct electron detectors and new nanocrystal electron diffraction methods. However, sample preparation techniques have not progressed and still suffer from extensive blotting steps leading to a massive loss of sample. Here, we present a simple but versatile method for the almost lossless sample conditioning and preparation of nanoliter volumes of biological samples for EM, keeping the sample under close to physiological condition. A microcapillary is used to aspirate 3-5 nL of sample. The microcapillary tip is immersed into a reservoir of negative stain or trehalose, where the sample becomes conditioned by diffusive exchange of salt and heavy metal ions or sugar molecules, respectively, before it is deposited as a small spot onto an EM grid. We demonstrate the use of the method to prepare protein particles for imaging by transmission EM and nanocrystals for analysis by electron diffraction. Furthermore, the minute sample volume required for this method enables alternative strategies for biological experiments, such as the analysis of the content of a single cell by visual proteomics, fully exploiting the single molecule detection limit of EM

Miniaturizing EM Sample Preparation: Opportunities, Challenges, and "Visual Proteomics"

This review compares and discusses conventional versus miniaturized specimen preparation methods for transmission electron microscopy (TEM). The progress brought by direct electron detector cameras, software developments and automation have transformed transmission cryo-electron microscopy (cryo-EM) and made it an invaluable high-resolution structural analysis tool. In contrast, EM specimen preparation has seen very little progress in the last decades and is now one of the main bottlenecks in cryo-EM. Here, we discuss the challenges faced by specimen preparation for single particle EM, highlight current developments, and show the opportunities resulting from the advanced miniaturized and microfluidic sample grid preparation methods described, such as visual proteomics and time-resolved cryo-EM studies

Miniaturizing EM Sample Preparation: Opportunities, Challenges, and "Visual Proteomics"

This review compares and discusses conventional versus miniaturized specimen preparation methods for transmission electron microscopy (TEM). The progress brought by direct electron detector cameras, software developments and automation have transformed transmission cryo-electron microscopy (cryo-EM) and made it an invaluable high-resolution structural analysis tool. In contrast, EM specimen preparation has seen very little progress in the last decades and is now one of the main bottlenecks in cryo-EM. Here, we discuss the challenges faced by specimen preparation for single particle EM, highlight current developments, and show the opportunities resulting from the advanced miniaturized and microfluidic sample grid preparation methods described, such as visual proteomics and time-resolved cryo-EM studies

Miniaturized Sample Preparation for Transmission Electron Microscopy

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein complexes to atomic resolution. However, protein isolation techniques and sample preparation methods for EM remain a bottleneck. A relatively small number (100,000 to a few million) of individual protein particles need to be imaged for the high-resolution analysis of proteins by the single particle EM approach, making miniaturized sample handling techniques and microfluidic principles feasible. A miniaturized, paper-blotting-free EM grid preparation method for sample pre-conditioning, EM grid priming and post processing that only consumes nanoliter-volumes of sample is presented. The method uses a dispensing system with sub-nanoliter precision to control liquid uptake and EM grid priming, a platform to control the grid temperature thereby determining the relative humidity above the EM grid, and a pick-and-plunge-mechanism for sample vitrification. For cryo-EM, an EM grid is placed on the temperature-controlled stage and the sample is aspirated into a capillary. The capillary tip is positioned in proximity to the grid surface, the grid is loaded with the sample and excess is re-aspirated into the microcapillary. Subsequently, the sample film is stabilized and slightly thinned by controlled water evaporation regulated by the offset of the platform temperature relative to the dew-point. At a given point the pick-and-plunge mechanism is triggered, rapidly transferring the primed EM grid into liquid ethane for sample vitrification. Alternatively, sample-conditioning methods are available to prepare nanoliter-sized sample volumes for negative stain (NS) EM. The methodologies greatly reduce sample consumption and avoid approaches potentially harmful to proteins, such as the filter paper blotting used in conventional methods. Furthermore, the minuscule amount of sample required allows novel experimental strategies, such as fast sample conditioning, combination with single-cell lysis for "visual proteomics," or "lossless" total sample preparation for quantitative analysis of complex samples

Miniaturized Sample Preparation for Transmission Electron Microscopy

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein complexes to atomic resolution. However, protein isolation techniques and sample preparation methods for EM remain a bottleneck. A relatively small number (100,000 to a few million) of individual protein particles need to be imaged for the high-resolution analysis of proteins by the single particle EM approach, making miniaturized sample handling techniques and microfluidic principles feasible.A miniaturized, paper-blotting-free EM grid preparation method for sample pre-conditioning, EM grid priming and post processing that only consumes nanoliter-volumes of sample is presented. The method uses a dispensing system with sub-nanoliter precision to control liquid uptake and EM grid priming, a platform to control the grid temperature thereby determining the relative humidity above the EM grid, and a pick-andplunge-mechanism for sample vitrification. For cryo-EM, an EM grid is placed on the temperature-controlled stage and the sample is aspirated into a capillary. The capillary tip is positioned in proximity to the grid surface, the grid is loaded with the sample and excess is re-aspirated into the microcapillary. Subsequently, the sample film is stabilized and slightly thinned by controlled water evaporation regulated by the offset of the platform temperature relative to the dew-point. At a given point the pick-and-plunge mechanism is triggered, rapidly transferring the primed EM grid into liquid ethane for sample vitrification. Alternatively, sample-conditioning methods are available to prepare nanoliter-sized sample volumes for negative stain (NS) EM.The methodologies greatly reduce sample consumption and avoid approaches potentially harmful to proteins, such as the filter paper blotting used in conventional methods. Furthermore, the minuscule amount of sample required allows novel experimental strategies, such as fast sample conditioning, combination with single-cell lysis for "visual proteomics," or "lossless" total sample preparation for quantitative analysis of complex samples

Total Sample Conditioning and Preparation of Nanoliter Volumes for Electron Microscopy

Electron microscopy (EM) entered a new era with the emergence of direct electron detectors and new nanocrystal electron diffraction methods. However, sample preparation techniques have not progressed and still suffer from extensive blotting steps leading to a massive loss of sample. Here, we present a simple but versatile method for the almost lossless sample conditioning and preparation of nanoliter volumes of biological samples for EM, keeping the sample under close to physiological condition. A microcapillary is used to aspirate 3-5 nL of sample. The microcapillary tip is immersed into a reservoir of negative stain or trehalose, where the sample becomes conditioned by diffusive exchange of salt and heavy metal ions or sugar molecules, respectively, before it is deposited as a small spot onto an EM grid. We demonstrate the use of the method to prepare protein particles for imaging by transmission EM and nanocrystals for analysis by electron diffraction. Furthermore, the minute sample volume required for this method enables alternative strategies for biological experiments, such as the analysis of the content of a single cell by visual proteomics, fully exploiting the single molecule detection limit of EM