Alignment and Characterisation of Remote-Refocusing Systems

The technique of remote refocusing is used in optical microscopy to provide rapid axial scanning without mechanically perturbing the sample and in techniques such as oblique plane microscopy that build on remote refocusing to image a tilted plane within the sample. The magnification between the pupils of the primary (O1) and secondary (O2) microscope objectives of the remote-refocusing system has been shown previously by Mohanan and Corbett [J Microsc, 288(2):95-105 (2022)] to be crucial in obtaining the broadest possible remote-refocusing range. In this work, we performed an initial alignment of a remote-refocusing system and then studied the effect of axial misalignments of O1 and O2, axial misalignment of the primary tube lens (TL1) relative to the secondary tube lens (TL2), lateral misalignments of TL2 and changes in the focal length of TL2. For each instance of the setup, we measured the mean point spread function FWHMxy of 100 nm fluorescent beads, the normalised bead integrated fluorescence signal, and calculated the axial and lateral distortion of the system: all of these quantities were mapped over the remote-refocusing range and as a function of lateral image position. This allowed us to estimate the volume over which diffraction-limited performance is achieved and how this changes with the alignment of the system.Comment: 28 pages, 12 figure

Multiphoton Laser Microscopy and Fluorescence Lifetime Imaging for the Evaluation of the Skin

Multiphoton laser microscopy is a new, non-invasive technique providing access to the skin at a cellular and subcellular level, which is based both on autofluorescence and fluorescence lifetime imaging. Whereas the former considers fluorescence intensity emitted by epidermal and dermal fluorophores and by the extra-cellular matrix, fluorescence lifetime imaging (FLIM), is generated by the fluorescence decay rate. This innovative technique can be applied to the study of living skin, cell cultures and ex vivo samples. Although still limited to the clinical research field, the development of multiphoton laser microscopy is thought to become suitable for a practical application in the next few years: in this paper, we performed an accurate review of the studies published so far, considering the possible fields of application of this imaging method and providing high quality images acquired in the Department of Dermatology of the University of Modena

Galaxy correlations and the BAO in a void universe: structure formation as a test of the Copernican Principle

A suggested solution to the dark energy problem is the void model, where accelerated expansion is replaced by Hubble-scale inhomogeneity. In these models, density perturbations grow on a radially inhomogeneous background. This large scale inhomogeneity distorts the spherical Baryon Acoustic Oscillation feature into an ellipsoid which implies that the bump in the galaxy correlation function occurs at different scales in the radial and transverse correlation functions. We compute these for the first time, under the approximation that curvature gradients do not couple the scalar modes to vector and tensor modes. The radial and transverse correlation functions are very different from those of the concordance model, even when the models have the same average BAO scale. This implies that if void models are fine-tuned to satisfy average BAO data, there is enough extra information in the correlation functions to distinguish a void model from the concordance model. We expect these new features to remain when the full perturbation equations are solved, which means that the radial and transverse galaxy correlation functions can be used as a powerful test of the Copernican Principle.Comment: 12 pages, 8 figures, matches published versio

Active mesh and neural network pipeline for cell aggregate segmentation

Segmenting cells within cellular aggregates in 3D is a growing challenge in cell biology due to improvements in capacity and accuracy of microscopy techniques. Here, we describe a pipeline to segment images of cell aggregates in 3D. The pipeline combines neural network segmentations with active meshes. We apply our segmentation method to cultured mouse mammary gland organoids imaged over 24 h with oblique plane microscopy, a high-throughput light-sheet fluorescence microscopy technique. We show that our method can also be applied to images of mouse embryonic stem cells imaged with a spinning disc microscope. We segment individual cells based on nuclei and cell membrane fluorescent markers, and track cells over time. We describe metrics to quantify the quality of the automated segmentation. Our segmentation pipeline involves a Fiji plugin that implements active mesh deformation and allows a user to create training data, automatically obtain segmentation meshes from original image data or neural network prediction, and manually curate segmentation data to identify and correct mistakes. Our active meshes-based approach facilitates segmentation postprocessing, correction, and integration with neural network prediction

Ultrafast 3-D Super Resolution Ultrasound using Row-Column Array specific Coherence-based Beamforming and Rolling Acoustic Sub-aperture Processing: In Vitro, In Vivo and Clinical Study

The row-column addressed array is an emerging probe for ultrafast 3-D ultrasound imaging. It achieves this with far fewer independent electronic channels and a wider field of view than traditional 2-D matrix arrays, of the same channel count, making it a good candidate for clinical translation. However, the image quality of row-column arrays is generally poor, particularly when investigating tissue. Ultrasound localisation microscopy allows for the production of super-resolution images even when the initial image resolution is not high. Unfortunately, the row-column probe can suffer from imaging artefacts that can degrade the quality of super-resolution images as `secondary' lobes from bright microbubbles can be mistaken as microbubble events, particularly when operated using plane wave imaging. These false events move through the image in a physiologically realistic way so can be challenging to remove via tracking, leading to the production of 'false vessels'. Here, a new type of rolling window image reconstruction procedure was developed, which integrated a row-column array-specific coherence-based beamforming technique with acoustic sub-aperture processing for the purposes of reducing `secondary' lobe artefacts, noise and increasing the effective frame rate. Using an {\it{in vitro}} cross tube, it was found that the procedure reduced the percentage of `false' locations from $\sim$26\% to $\sim$15\% compared to traditional orthogonal plane wave compounding. Additionally, it was found that the noise could be reduced by $\sim$7 dB and that the effective frame rate could be increased to over 4000 fps. Subsequently, {\it{in vivo}} ultrasound localisation microscopy was used to produce images non-invasively of a rabbit kidney and a human thyroid

Microclusters of inhibitory killer immunoglobulin–like receptor signaling at natural killer cell immunological synapses

We report the supramolecular organization of killer Ig–like receptor (KIR) phosphorylation using a technique applicable to imaging phosphorylation of any green fluorescent protein–tagged receptor at an intercellular contact or immune synapse. Specifically, we use fluorescence lifetime imaging (FLIM) to report Förster resonance energy transfer (FRET) between GFP-tagged KIR2DL1 and a Cy3-tagged generic anti-phosphotyrosine monoclonal antibody. Visualization of KIR phosphorylation in natural killer (NK) cells contacting target cells expressing cognate major histocompatibility complex class I proteins revealed that inhibitory signaling is spatially restricted to the immune synapse. This explains how NK cells respond appropriately when simultaneously surveying susceptible and resistant target cells. More surprising, phosphorylated KIR was confined to microclusters within the aggregate of KIR, contrary to an expected homogeneous distribution of KIR signaling across the immune synapse. Also, yellow fluorescent protein–tagged Lck, a kinase important for KIR phosphorylation, accumulated in a multifocal distribution at inhibitory synapses. Spatial confinement of receptor phosphorylation within the immune synapse may be critical to how activating and inhibitory signals are integrated in NK cells

Single-shot phase contrast microscopy using polarisation-resolved differential phase contrast

We present a robust, low-cost single-shot implementation of differential phase microscopy utilising a polarisation-sensitive camera to simultaneously acquire four images from which phase contrast images can be calculated. This polarisation-resolved differential phase contrast (pDPC) microscopy technique can be easily integrated with fluorescence microscopy