The Benchtop mesoSPIM: a next-generation open-source light-sheet microscope for large cleared samples

In 2015, we launched the mesoSPIM initiative (www.mesospim.org), an open-source project for making light-sheet microscopy of large cleared tissues more accessible. Meanwhile, the demand for imaging larger samples at higher speed and resolution has increased, requiring major improvements in the capabilities of light-sheet microscopy. Here, we introduce the next-generation mesoSPIM ("Benchtop") with significantly increased field of view, improved resolution, higher throughput, more affordable cost and simpler assembly compared to the original version. We developed a new method for testing objectives, enabling us to select detection objectives optimal for light-sheet imaging with large-sensor sCMOS cameras. The new mesoSPIM achieves high spatial resolution (1.5 μm laterally, 3.3 μm axially) across the entire field of view, a magnification up to 20x, and supports sample sizes ranging from sub-mm up to several centimetres, while being compatible with multiple clearing techniques. The new microscope serves a broad range of applications in neuroscience, developmental biology, and even physics

Digital Pathology: The Time Is Now to Bridge the Gap between Medicine and Technological Singularity

Digitalization of the imaging in radiology is a reality in several healthcare institutions worldwide. The challenges of filing, confidentiality, and manipulation have been brilliantly solved in radiology. However, digitalization of hematoxylin- and eosin-stained routine histological slides has shown slow movement. Although the application for external quality assurance is a reality for a pathologist with most of the continuing medical education programs utilizing virtual microscopy, the abandonment of traditional glass slides for routine diagnostics is far from the perspectives of many departments of laboratory medicine and pathology. Digital pathology images are captured as images by scanning and whole slide imaging/virtual microscopy can be obtained by microscopy (robotic) on an entire histological (microscopic) glass slide. Since 1986, services using telepathology for the transfer of images of anatomic pathology between detached locations have benefited countless patients globally, including the University of Alberta. The purpose of specialist recertification or re-validation for the Royal College of Pathologists of Canada belonging to the Royal College of Physicians and Surgeons of Canada and College of American Pathologists is a milestone in virtual reality. Challenges, such as high bandwidth requirement, electronic platforms, the stability of the operating systems, have been targeted and are improving enormously. The encryption of digital images may be a requirement for the accreditation of laboratory services—quantum computing results in quantum-mechanical phenomena, such as superposition and entanglement. Different from binary digital electronic computers based on transistors where data are encoded into binary digits (bits) with two different states (0 and 1), quantum computing uses quantum bits (qubits), which can be in superpositions of states. The use of quantum computing protocols on encrypted data is crucial for the permanent implementation of virtual pathology in hospitals and universities. Quantum computing may well represent the technological singularity to create new classifications and taxonomic rules in medicine

A scanning device for wide band infrared reflectography

Diagnostics in the cultural heritage field is an important resource to investigate art history, issues, execution techniques, materials and state of conservation of an artwork. In this field the main concern is preservation and, for this reason, new non-invasive and non-destructive technologies have been developed. At the Department of Physics in Ferrara, imaging for cultural heritage, using electromagnetic radiation, from visible light to X-rays is applied and studied. A diagnostics protocol has been defined to standardize the study approach on paintings considering that each painting is a particular case, and the protocol must be adapted to the needs that the artwork itself requires. This work consists in the development of a scanning devices for wide band infrared Reflectography, to extend the applications of the reflectographic technique, and how it is inserted in the diagnostic protocol. Infrared reflectography use the electromagnetic radiation of Near-IR to investigate the underdrawing in paintings. The success of IR Reflectography to reveal the underdrawing in paintings since XIV up to XVI century depends on the peculiar technique of painters in that period. Thin and uniform pictorial layers, covering high contrast drawings on white priming allow a good detection of underdrawing details. Paintings of late sixteenth century have dark preparations and thick paint layers, so Reflectography doesn’t get the same good results. Extension of the spectral band to longer wavelengths, up to 2,5 μm, is a tool to improve reflectographic capability

Dielectric-sphere-based microsystem for optical super-resolution imaging

Well-established imaging techniques proved that features below the diffraction limit can be observed optically using so-called super-resolution microscopies, which overcome Abbe's resolution limit. In traditional far-field microscopy, the introduction of fluorescent samples and engineered light paths was key for this breakthrough. In parallel, near-field techniques with similar performance were developed, but they suffered from a limited field-of-view. The merge of the two approaches was already demonstrated ~15 years ago, when micrometer-sized dielectric objects positioned on a sample were found to be able to image the sample with super-resolution. By observing the sample through the micro-object with a classical optical microscope, the latter could capture a virtual image showing sub-diffraction details. Although this way the near-field information transfer into the far-field by the micro-object was proven and found to be key for enabling super-resolution imaging, the limited field-of-view, as determined by the size of the micro-object, remained an issue. In this dissertation, a novel method is presented that provides a microscopy technique capable of achieving super-resolution without field-of-view restrictions. Based on previous studies, dielectric microspheres were chosen for this imaging technique. First, the working principle of these microspheres was explored by investigating both the illumination and the reflected light path. These findings provided a better understanding on the phenomena working behind microsphere-assisted imaging and allowed to create an engineering toolbox that can be used to design microsphere-based optical systems. This was followed by an investigation on microfabrication techniques, in order to create a microchip that can serve as a bridge between a single microsphere and the macro-sized-components of a classical optical microscope. The resulting chip was later embedded in a custom fixation system that allowed scanning of this microsphere over the sample, while keeping its position fixed compared to the microscope objective. The microscope mounted camera recorded pictures during the scan, which were used to generate a large field-of-view super-resolution image by stitching. After initial successes, the setup was improved in terms of robustness and application range. The new version allowed field-of-view in the millimeter range, while it could be operated in both oil- and water-immersion. Parallel imaging with an array of microspheres was also implemented, which further enhanced the imaging speed. The algorithmic background (including an automated scanning and image reconstruction protocol) of this microscopy method was developed in-house. Its validation showed superior performance compared to existing software. Future developments (e.g. employment of 3D-printing for mass-production, imaging in vivo biological samples, metrology applications) are envisioned. The findings presented here may pave the road for an easy-to-use, generalized super-resolution imaging system

Local features for visual object matching and video scene detection

Local features are important building blocks for many computer vision algorithms such as visual object alignment, object recognition, and content-based image retrieval. Local features are extracted from an image by a local feature detector and then the detected features are encoded using a local feature descriptor. The resulting features based on the descriptors, such as histograms or binary strings, are used in matching to find similar features between objects in images. In this thesis, we deal with two research problem in the context of local features for object detection: we extend the original local feature detector and descriptor performance benchmarks from the wide baseline setting to the intra-class matching; and propose local features for consumer video scene boundary detection. In the intra-class matching, the visual appearance of objects semantic class can be very different (e.g., Harley Davidson and Scooter in the same motorbike class) and making the task more difficult than wide baseline matching. The performance of different local feature detectors and descriptors are evaluated over three different image databases and results for more advance analysis are reported. In the second part of the thesis, we study the use of Bag-of-Words (BoW) in the video scene boundary detection. In literature there have been several approaches to the task exploiting the local features, but based on the author’s knowledge, none of them are practical in an online processing of user videos. We introduce an online BoW based scene boundary detector using a dynamic codebook, study the optimal parameters for the detector and compare our method to the existing methods. Precision and recall curves are used as a performance metric. The goal of this thesis is to find the best local feature detector and descriptor for intra-class matching and develop a novel scene boundary detection method for online applications

Video sculpture:spatio-temporal warping

In this thesis the concept behind our notion of video sculpture is to imagine an image sequence or movie as a three dimensional volume. We then also imagine that there is a frameset that traverses the image sequence to give us what we commonly think of as a video or movie. In the ordinary sense this frameset moves through an image sequence in a completely timeparallel linear fashion. In video sculpture, we free the frameset from these bounds so that we can sample space and time in completely unorthodox ways. We can view the whenwhere in previously unforeseen perspectives. Slices of the video environment can simultaneously reveal both past and future actions within a single frame. Building on this free representation of video spacetime, we then wrest the frame once more from the present constraints of topography and/or topology. The frame can bend and twist and jump and dive. The view of a fading quadratic surface cutting through two scenes makes for a beautiful curtain transition. We present a framework and an implementation for modeling the frame as it passes through the image sequence volume object

Contactin 2 homophilic adhesion structure and conformational plasticity

The cell-surface attached glycoprotein contactin 2 is ubiquitously expressed in the nervous system and mediates homotypic cell-cell interactions to organize cell guidance, differentiation, and adhesion. Contactin 2 consists of six Ig and four fibronectin type III domains (FnIII) of which the first four Ig domains form a horseshoe structure important for homodimerization and oligomerization. Here we report the crystal structure of the six-domain contactin 2 Ig1-6 and show that the Ig5-Ig6 combination is oriented away from the horseshoe with flexion in interdomain connections. Two distinct dimer states, through Ig1-Ig2 and Ig3-Ig6 interactions, together allow formation of larger oligomers. Combined size exclusion chromatography with multiangle light scattering (SEC-MALS), small-angle X-ray scattering (SAXS) and native MS analysis indicates contactin 2 Ig1-6 oligomerizes in a glycan dependent manner. SAXS and negative-stain electron microscopy reveals inherent plasticity of the contactin 2 full-ectodomain. The combination of intermolecular binding sites and ectodomain plasticity explains how contactin 2 can function as a homotypic adhesion molecule in diverse intercellular environments

Characterisation and computational modelling of retinal stem cells in medaka (Oryzias latipes)

The central functional unit of the vertebrate eye is the retina, composed of neural retina (NR), retinal pigmented epithelium (RPE), and non-visual retina (NVR). In amphibians and fish, the retina grows throughout life via different pools of stem cells (SCs). In this work, I combined experimental and computational approaches to elucidate SC dynamics in the three retinal tissues of the teleost fish medaka (Oryzias latipes). I developed a cell centred agent based model to recapitulate post-embryonic growth of the NR and RPE. By accounting for 3D tissue geometry and continuous growth, the model reconciled conflicting hypotheses, demonstrating that competition between SCs is not mutually exclusive with lifelong coexistence of multiple SC lineages. To understand how NR and RPE regulate their proliferative output to coordinate growth rates, I developed quantitative methods to compare experiment and simulation. I tested the experimental data against simulations implementing two modes of feedback between cell proliferation and organ growth. Thus, I identified that the NR acts upstream to set the growth pace by sending an inductive growth signal, while the RPE responds downstream to this signal. Leveraging the model, I showed that NR SCs compete for niche space, but tissue geometry biases cells at certain positions to win this competition. Further, NR SCs modulate division axes and proliferation rate to change organ shape and retinal topology. Motivated by model predictions, I experimentally characterised the large SC population of the RPE, which consisted of both cycling and non-cycling quiescent cells. Putative sister cells exhibited similar temporal dynamics in local clusters, indicating that quiescence was the major mechanism for regulating proliferative output in the RPE. Finally, I experimentally showed that the NVR grows post-embryonically from a primordium, and shared all known markers for NR SCs in the same spatial distribution. Unlike NR and RPE, the NVR lacked a dedicated niche, instead proliferative cells were distributed throughout the tissue. Lineage tracing revealed a continuous relationship between RPE, NVR, and NR. Thus, the SCs of NR and RPE, and all cells of the NVR displayed plastic multipotency capable of generating all retinal tissues. By taking advantage of the positive feedback loop between experiment and simulation, this work shines a new light into a fundamental problem – growth coordination of different SC populations in a complex vertebrate organ