OpenFIBSEM: A universal API for FIBSEM control.

This paper introduces OpenFIBSEM, a universal API to control Focused Ion Beam Scanning Electron Microscopes (FIBSEM). OpenFIBSEM aims to improve the programmability and automation of electron microscopy workflows in structural biology research. The API is designed to be cross-platform, composable, and extendable: allowing users to use any portion of OpenFIBSEM to develop or integrate with other software tools. The package provides core functionality such as imaging, movement, milling, and manipulator control, as well as system calibration, alignment, and image analysis modules. Further, a library of reusable user interface components integrated with napari is provided, ensuring easy and efficient application development. OpenFIBSEM currently supports ThermoFisher and TESCAN hardware, with support for other manufacturers planned. To demonstrate the improved automation capabilities enabled by OpenFIBSEM, several example applications that are compatible with multiple hardware manufacturers are discussed. We argue that OpenFIBSEM provides the foundation for a cross-platform operating system and development ecosystem for FIBSEM systems. The API and applications are open-source and available on GitHub (https://github.com/DeMarcoLab/fibsem).

Automated cryo-lamella preparation for high-throughput in-situ structural biology.

Cryo-transmission electron tomography (cryo-ET) in association with cryo-focused ion beam (cryo-FIB) milling enables structural biology studies to be performed directly within the cellular environment. Cryo-preserved cells are milled and a lamella with a typical thickness of 200-300 nm provides an electron transparent window suitable for cryo-ET imaging. Cryo-FIB milling is an effective method, but it is a tedious and time-consuming process, which typically results in ~10 lamellae per day. Here, we introduce an automated method to reproducibly prepare cryo-lamellae on a grid and reduce the amount of human supervision. We tested the routine on cryo-preserved Saccharomyces cerevisiae, mammalian 293 T cells, and lysozyme protein crystals. Here we demonstrate that our method allows an increased throughput, achieving a rate of 5 lamellae/hour without the need to supervise the FIB milling. We demonstrate that the quality of the lamellae is consistent throughout the preparation and their compatibility with cryo-ET analyses.

Photon-counting, energy-resolving and super-resolution phase contrast X-ray imaging using an integrating detector.

This work demonstrates the use of a scientific-CMOS (sCMOS) energy-integrating detector as a photon-counting detector, thereby eliminating dark current and read-out noise issues, that simultaneously provides both energy resolution and sub-pixel spatial resolution for X-ray imaging. These capabilities are obtained by analyzing visible light photon clouds that result when X-ray photons produce fluorescence from a scintillator in front of the visible light sensor. Using low-fluence monochromatic X-ray projections to avoid overlapping photon clouds, the centroid of individual X-ray photon interactions was identified. This enabled a tripling of the spatial resolution of the detector to 6.71 ± 0.04 µm. By calculating the total charge deposited by this interaction, an energy resolution of 61.2 ± 0.1% at 17 keV was obtained. When combined with propagation-based phase contrast imaging and phase retrieval, a signal-to-noise ratio of up to 15 ± 3 was achieved for an X-ray fluence of less than 3 photons/mm2.

GoldDigger and Checkers, computational developments in cryo-scanning transmission electron tomography to improve the quality of reconstructed volumes.

In this work, we present a pair of tools to improve the fiducial tracking and reconstruction quality of cryo-scanning transmission electron tomography (STET) datasets. We then demonstrate the effectiveness of these two tools on experimental cryo-STET data. The first tool, GoldDigger, improves the tracking of fiducials in cryo-STET by accommodating the changed appearance of highly defocussed fiducial markers. Since defocus effects are much stronger in scanning transmission electron microscopy than in conventional transmission electron microscopy, existing alignment tools do not perform well without manual intervention. The second tool, Checkers, combines image inpainting and unsupervised deep learning for denoising tomograms. Existing tools for denoising cryo-tomography often rely on paired noisy image frames, which are unavailable in cryo-STET datasets, necessitating a new approach. Finally, we make the two software tools freely available for the cryo-STET community.

Assembly and Imaging set up of PIE-Scope.

Cryo-Electron Tomography (cryo-ET) is a method that enables resolving the structure of macromolecular complexes directly in the cellular environment. However, sample preparation for in situ Cryo-ET is labour-intensive and can require both cryo-lamella preparation through cryo-Focused Ion Beam (FIB) milling and correlative light microscopy to ensure that the event of interest is present in the lamella. Here, we present an integrated cryo-FIB and light microscope setup called the Photon Ion Electron microscope (PIE-scope) that enables direct and rapid isolation of cellular regions containing protein complexes of interest. The PIE-scope can be retrofitted on existing microscopes, although the drawings we provide are meant to work on ThermoFisher DualBeams with small mechanical modifications those can be adapted on other brands.

Emphysema quantified: mapping regional airway dimensions using 2D phase contrast X-ray imaging.

We have developed an analyser-based phase contrast X-ray imaging technique to measure the mean length scale of pores or particles that cannot be resolved directly by the system. By combining attenuation, phase and ultra-small angle X-ray scattering information, the technique was capable of measuring differences in airway dimension between lungs of healthy mice and those with mild and severe emphysema. Our measurements of airway dimensions from 2D images showed a 1:1 relationship to the actual airway dimensions measured using micro-CT. Using 80 images, the sensitivity and specificity were measured to be 0.80 and 0.89, respectively, with the area under the ROC curve close to ideal at 0.96. Reducing the number of images to 11 slightly decreased the sensitivity to 0.75 and the ROC curve area to 0.90, whilst the specificity remained high at 0.89.

PIE-scope, integrated cryo-correlative light and FIB/SEM microscopy.

Cryo-electron tomography (cryo-ET) is emerging as a revolutionary method for resolving the structure of macromolecular complexes in situ. However, sample preparation for in situ Cryo-ET is labour-intensive and can require both cryo-lamella preparation through cryo-focused ion beam (FIB) milling and correlative light microscopy to ensure that the event of interest is present in the lamella. Here, we present an integrated cryo-FIB and light microscope setup called the Photon Ion Electron microscope (PIE-scope) that enables direct and rapid isolation of cellular regions containing protein complexes of interest. Specifically, we demonstrate the versatility of PIE-scope by preparing targeted cryo-lamellae from subcellular compartments of neurons from transgenic Caenorhabditis elegans and Drosophila melanogaster expressing fluorescent proteins. We designed PIE-scope to enable retrofitting of existing microscopes, which will increase the throughput and accuracy on projects requiring correlative microscopy to target protein complexes. This new approach will make cryo-correlative workflow safer and more accessible.

Laryngeal closure impedes non-invasive ventilation at birth.

BACKGROUND: Non-invasive ventilation is sometimes unable to provide the respiratory needs of very premature infants in the delivery room. While airway obstruction is thought to be the main problem, the site of obstruction is unknown. We investigated whether closure of the larynx and epiglottis is a major site of airway obstruction. METHODS: We used phase contrast X-ray imaging to visualise laryngeal function in spontaneously breathing premature rabbits immediately after birth and at approximately 1 hour after birth. Non-invasive respiratory support was applied via a facemask and images were analysed to determine the percentage of the time the glottis and the epiglottis were open. HYPOTHESIS: Immediately after birth, the larynx is predominantly closed, only opening briefly during a breath, making non-invasive intermittent positive pressure ventilation (iPPV) ineffective, whereas after lung aeration, the larynx is predominantly open allowing non-invasive iPPV to ventilate the lung. RESULTS: The larynx and epiglottis were predominantly closed (open 25.5%±1.1% and 17.1%±1.6% of the time, respectively) in pups with unaerated lungs and unstable breathing patterns immediately after birth. In contrast, the larynx and the epiglottis were mostly open (90.5%±1.9% and 72.3%±2.3% of the time, respectively) in pups with aerated lungs and stable breathing patterns irrespective of time after birth. CONCLUSION: Laryngeal closure impedes non-invasive iPPV at birth and may reduce the effectiveness of non-invasive respiratory support in premature infants immediately after birth.

CT dose reduction factors in the thousands using X-ray phase contrast.

Phase-contrast X-ray imaging can improve the visibility of weakly absorbing objects (e.g. soft tissues) by an order of magnitude or more compared to conventional radiographs. Combining phase retrieval with computed tomography (CT) can increase the signal-to-noise ratio (SNR) by up to two orders of magnitude over conventional CT at the same radiation dose, without loss of image quality. Our experiments reveal that as the radiation dose decreases, the relative improvement in SNR increases. We show that this enhancement can be traded for a reduction in dose greater than the square of the gain in SNR. Upon reducing the dose 300 fold, the phase-retrieved SNR was still up to 9.6 ± 0.2 times larger than the absorption contrast data with spatial resolution in the tens of microns. We show that this theoretically reveals the potential for dose reduction factors in the tens of thousands without loss in image quality, which would have a profound impact on medical and industrial imaging applications.

X-ray specks: low dose in vivo imaging of lung structure and function.

Respiratory health is directly linked to the structural and mechanical properties of the airways of the lungs. For studying respiratory development and pathology, the ability to quantitatively measure airway dimensions and changes in their size during respiration is highly desirable. Real-time imaging of the terminal airways with sufficient contrast and resolution during respiration is currently not possible. Herein we reveal a simple method for measuring lung airway dimensions in small animals during respiration from a single propagation-based phase contrast x-ray image, thereby requiring minimal radiation. This modality renders the lungs visible as a speckled intensity pattern. In the near-field regime, the size of the speckles is directly correlated with that of the dominant length scale of the airways. We demonstrate that Fourier space quantification of the speckle texture can be used to statistically measure regional airway dimensions at the alveolar scale, with measurement precision finer than the spatial resolution of the imaging system. Using this technique we discovered striking differences in developmental maturity in the lungs of rabbit kittens at birth.

Real-time measurement of alveolar size and population using phase contrast x-ray imaging.

Herein a propagation-based phase contrast x-ray imaging technique for measuring particle size and number is presented. This is achieved with an algorithm that utilizes the Fourier space signature of the speckle pattern associated with the images of particles. We validate this algorithm using soda-lime glass particles, demonstrating its effectiveness on random and non-randomly packed particles. This technique is then applied to characterise lung alveoli, which are difficult to measure dynamically in vivo with current imaging modalities due to inadequate temporal resolution and/or depth of penetration and field-of-view. We obtain an important result in that our algorithm is able to measure changes in alveolar size on the micron scale during ventilation and shows the presence of alveolar recruitment/de-recruitment in newborn rabbit kittens. This technique will be useful for ventilation management and lung diagnostic procedures.