Morphometric analysis of aerobic Eimeria bovis sporogony using live cell 3D holotomographic microscopy imaging

AbstractM onoxenous Eimeria species are widespread enteropathogenic apicomplexan protozoa with a high economic impact on livestock. In cattle, tenacious oocysts shed by E. bovis-infected animals are ubiquitously found and making infection of calves almost inevitable. To become infectious oocysts, exogenous oxygen-dependent E. bovis sporogony must occur leading to the formation of sporulated oocysts containing four sporocysts each harboring two sporozoites. Investigations on sporogony by live cell imaging techniques of ruminant Eimeria species are still absent in literature as commonly used fluorescent dyes do not penetrate resistant oocyst bi-layered wall. Sporogonial oocysts were daily analyzed by a 3D Cell Explorer Nanolive microscope to explore ongoing aerobic-dependent sporogony as close as possible to an in vivo situation. Subsequently, 3D holotomographic images of sporulating E. bovis oocysts were digitally stained based on refractive indices (RI) of oocyst bi-layered wall and sub-compartments of circumplasm using STEVE software (Nanolive), and the cellular morphometric parameters were obtained. Overall, three different E. bovis sporogony phases, each of them divided into two sub-phases, were documented: (i) sporoblast/sporont transformation into sporogonial stages, (ii) cytokinesis followed by nuclear division, and finally (iii) formation of four sporocysts with two fully developed sporozoites. Approximately 60% of sporulating E. bovis oocysts accomplished aerobic sporogony in a synchronized manner. E. bovis sporogony was delayed (i.e., 6 days) when compared to an in vivo situation where 2–3 days are required but under optimal environmental conditions. Live cell 3D holotomography analysis might facilitate the evaluation of either novel disinfectants- or anti-coccidial drug-derived effects on ruminant/avian Eimeria sporogony in vitro as discrimination of sporogony degrees based on compactness, and dry mass was here successfully achieved. Main changes were observed in the oocyst area, perimeter, compactness, extent, and granularity suggesting those parameters as an efficient tool for a fast evaluation of the sporulation degree.

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Imaging Microphysiological Systems: A Review

AJP Cell Physiology ◽

10.1152/ajpcell.00186.2020 ◽

2020 ◽

Author(s):

Samantha Peel ◽

Mark Jackman

Keyword(s):

Drug Discovery ◽

Animal Studies ◽

Imaging Techniques ◽

Quantitative Information ◽

In Vitro Models ◽

Microscopy Imaging ◽

In Vivo Models ◽

Microphysiological Systems

Microphysiological Systems (MPS), often referred to as 'organ-on-chips' are microfluidic-based in vitro models that aim to recapitulate the dynamic chemical and mechanical microenvironment of living organs. MPS promise to bridge the gap between in vitro and in vivo models, and ultimately improve the translation from pre-clinical animal studies to clinical trials. However, despite the explosion of interest in recent years, and the obvious rewards for such models which could improve R&D efficiency and reduce drug attrition in the clinic, the pharmaceutical industry has been slow to fully adopt this technology. The ability to extract robust, quantitative information from MPS at scale is a key requirement if these models are to impact drug discovery and the subsequent drug development process. Microscopy imaging remains a core technology that enables the capture of information at the single cell level and with subcellular resolution. Furthermore, such imaging techniques can be automated, increasing throughput, enabling compound screening. In this review we discuss a range of imaging techniques that have been applied to MPS of varying focus, such as organoids and organ-chip-type models. We outline the opportunities these technologies can bring in terms of understanding mechanistic biology, but also how they could be used in higher-throughput screens, widening the scope of their impact in drug discovery. We discuss the associated challenges of imaging these complex models and the steps required to enable full exploitation. Finally, we discuss the requirements for MPS, if they are to be applied at a scale necessary to support drug discovery projects.

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Live-cell imaging with Aspergillus fumigatus-specific fluorescent siderophore conjugates

Scientific Reports ◽

10.1038/s41598-020-72452-2 ◽

2020 ◽

Vol 10 (1) ◽

Cited By ~ 1

Author(s):

Joachim Pfister ◽

Alexander Lichius ◽

Dominik Summer ◽

Hubertus Haas ◽

Thines Kanagasundaram ◽

...

Keyword(s):

Aspergillus Fumigatus ◽

Live Cell Imaging ◽

Cell Imaging ◽

Fluorescent Dyes ◽

Iron Acquisition ◽

Live Cell ◽

Subcellular Localisation ◽

Gallium 68

Abstract Live-cell imaging allows the in vivo analysis of subcellular localisation dynamics of physiological processes with high spatial–temporal resolution. However, only few fluorescent dyes have been custom-designed to facilitate species-specific live-cell imaging approaches in filamentous fungi to date. Therefore, we developed fluorescent dye conjugates based on the sophisticated iron acquisition system of Aspergillus fumigatus by chemical modification of the siderophore triacetylfusarinine C (TAFC). Various fluorophores (FITC, NBD, Ocean Blue, BODIPY 630/650, SiR, TAMRA and Cy5) were conjugated to diacetylfusarinine C (DAFC). Gallium-68 labelling enabled in vitro and in vivo characterisations. LogD, uptake assays and growth assays were performed and complemented by live-cell imaging in different Aspergillus species. Siderophore conjugates were specifically recognised by the TAFC transporter MirB and utilized as an iron source in growth assays. Fluorescence microscopy revealed uptake dynamics and differential subcellular accumulation patterns of all compounds inside fungal hyphae.[Fe]DAFC-NBD and -Ocean Blue accumulated in vacuoles, whereas [Fe]DAFC-BODIPY, -SiR and -Cy5 localised to mitochondria. [Fe]DAFC -FITC showed a uniform cytoplasmic distribution, whereas [Fe]DAFC-TAMRA was not internalised at all. Co-staining experiments with commercially available fluorescent dyes confirmed these findings. Overall, we developed a new class of fluorescent dyes that vary in intracellular fungal targeting , thereby providing novel tools for live-cell imaging applications for Aspergillus fumigatus.

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Detecting apoptosis as a clinical endpoint for proof of a clinical principle

Ophthalmologica ◽

10.1159/000513584 ◽

2020 ◽

Author(s):

Piero Zollet ◽

Timothy E.Yap ◽

M Francesca Cordeiro

Keyword(s):

Drug Development ◽

Therapeutic Response ◽

Imaging Techniques ◽

Brain Diseases ◽

Promising Strategy ◽

Sight Loss ◽

Apoptosis Detection ◽

Novel Therapeutic Strategies

The transparent eye media represent a window through which to observe changes occurring in the retina during pathological processes. In contrast to visualising the extent of neurodegenerative damage that has already occurred, imaging an active process such as apoptosis has the potential to report on disease progression and therefore the threat of irreversible functional loss in various eye and brain diseases. Early diagnosis in these conditions is an important unmet clinical need to avoid or delay irreversible sight loss. In this setting, apoptosis detection is a promising strategy with which to diagnose, provide prognosis, and monitor therapeutic response. Additionally, monitoring apoptosis in vitro and in vivo has been shown to be valuable for drug development in order to assess the efficacy of novel therapeutic strategies both in the pre-clinical and clinical setting. Detection of Apoptosing Retinal Cells (DARC) technology is to date the only tool of its kind to have been tested in clinical trials, with other new imaging techniques under investigation in the fields of neuroscience, ophthalmology and drug development. We summarize the transitioning of techniques detecting apoptosis from bench to bedside, along with the future possibilities they encase.

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Optimization of X-ray Investigations in Dentistry Using Optical Coherence Tomography

Sensors ◽

10.3390/s21134554 ◽

2021 ◽

Vol 21 (13) ◽

pp. 4554

Author(s):

Ralph-Alexandru Erdelyi ◽

Virgil-Florin Duma ◽

Cosmin Sinescu ◽

George Mihai Dobre ◽

Adrian Bradu ◽

...

Keyword(s):

Optical Coherence Tomography ◽

Radiation Dose ◽

Imaging Techniques ◽

Image Resolution ◽

Optical Coherence ◽

X Rays ◽

High Quality ◽

X Ray

The most common imaging technique for dental diagnoses and treatment monitoring is X-ray imaging, which evolved from the first intraoral radiographs to high-quality three-dimensional (3D) Cone Beam Computed Tomography (CBCT). Other imaging techniques have shown potential, such as Optical Coherence Tomography (OCT). We have recently reported on the boundaries of these two types of techniques, regarding. the dental fields where each one is more appropriate or where they should be both used. The aim of the present study is to explore the unique capabilities of the OCT technique to optimize X-ray units imaging (i.e., in terms of image resolution, radiation dose, or contrast). Two types of commercially available and widely used X-ray units are considered. To adjust their parameters, a protocol is developed to employ OCT images of dental conditions that are documented on high (i.e., less than 10 μm) resolution OCT images (both B-scans/cross sections and 3D reconstructions) but are hardly identified on the 200 to 75 μm resolution panoramic or CBCT radiographs. The optimized calibration of the X-ray unit includes choosing appropriate values for the anode voltage and current intensity of the X-ray tube, as well as the patient’s positioning, in order to reach the highest possible X-rays resolution at a radiation dose that is safe for the patient. The optimization protocol is developed in vitro on OCT images of extracted teeth and is further applied in vivo for each type of dental investigation. Optimized radiographic results are compared with un-optimized previously performed radiographs. Also, we show that OCT can permit a rigorous comparison between two (types of) X-ray units. In conclusion, high-quality dental images are possible using low radiation doses if an optimized protocol, developed using OCT, is applied for each type of dental investigation. Also, there are situations when the X-ray technology has drawbacks for dental diagnosis or treatment assessment. In such situations, OCT proves capable to provide qualitative images.

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Thrombus Imaging Using 3D Printed Middle Cerebral Artery Model and Preclinical Imaging Techniques: Application to Thrombus Targeting and Thrombolytic Studies

Pharmaceutics ◽

10.3390/pharmaceutics12121207 ◽

2020 ◽

Vol 12 (12) ◽

pp. 1207

Author(s):

Andrea Vítečková Wünschová ◽

Adam Novobilský ◽

Jana Hložková ◽

Peter Scheer ◽

Hana Petroková ◽

...

Keyword(s):

Middle Cerebral Artery ◽

Cerebral Artery ◽

Imaging Techniques ◽

Barium Sulphate ◽

Flow Conditions ◽

Preclinical Imaging ◽

Blood Clots ◽

3D Printed

Diseases with the highest burden for society such as stroke, myocardial infarction, pulmonary embolism, and others are due to blood clots. Preclinical and clinical techniques to study blood clots are important tools for translational research of new diagnostic and therapeutic modalities that target blood clots. In this study, we employed a three-dimensional (3D) printed middle cerebral artery model to image clots under flow conditions using preclinical imaging techniques including fluorescent whole-body imaging, magnetic resonance imaging (MRI), and computed X-ray microtomography (microCT). Both liposome-based, fibrin-targeted, and non-targeted contrast agents were proven to provide a sufficient signal for clot imaging within the model under flow conditions. The application of the model for clot targeting studies and thrombolytic studies using preclinical imaging techniques is shown here. For the first time, a novel method of thrombus labeling utilizing barium sulphate (Micropaque®) is presented here as an example of successfully employed contrast agents for in vitro experiments evaluating the time-course of thrombolysis and thus the efficacy of a thrombolytic drug, recombinant tissue plasminogen activator (rtPA). Finally, the proof-of-concept of in vivo clot imaging in a middle cerebral artery occlusion (MCAO) rat model using barium sulphate-labelled clots is presented, confirming the great potential of such an approach to make experiments comparable between in vitro and in vivo models, finally leading to a reduction in animals needed.

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The Highly Conserved tRNAHis Guanylyltransferase Thg1p Interacts with the Origin Recognition Complex and Is Required for the G2/M Phase Transition in the Yeast Saccharomyces cerevisiae

Eukaryotic Cell ◽

10.1128/ec.4.4.832-835.2005 ◽

2005 ◽

Vol 4 (4) ◽

pp. 832-835 ◽

Cited By ~ 13

Author(s):

Terri S. Rice ◽

Min Ding ◽

David S. Pederson ◽

Nicholas H. Heintz

Keyword(s):

Saccharomyces Cerevisiae ◽

Origin Recognition Complex ◽

Nuclear Division ◽

Permissive Temperature ◽

Yeast Saccharomyces Cerevisiae ◽

M Phase ◽

And Migration ◽

Origin Recognition

ABSTRACT Here we show that the Saccharomyces cerevisiae tRNAHis guanylyltransferase Thg1p interacts with the origin recognition complex in vivo and in vitro and that overexpression of hemagglutinin-Thg1p selectively impedes growth of orc2-1(Ts) cells at the permissive temperature. Studies with conditional mutants indicate that Thg1p couples nuclear division and migration to cell budding and cytokinesis in yeast.

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A new visible-light-excitable ICT-CHEF-mediated fluorescence ‘turn-on’ probe for the selective detection of Cd2+ in a mixed aqueous system with live-cell imaging

Dalton Transactions ◽

10.1039/c4dt02463j ◽

2015 ◽

Vol 44 (12) ◽

pp. 5763-5770 ◽

Cited By ~ 58

Author(s):

Shyamaprosad Goswami ◽

Krishnendu Aich ◽

Sangita Das ◽

Chitrangada Das Mukhopadhyay ◽

Deblina Sarkar ◽

...

Keyword(s):

Visible Light ◽

Live Cell Imaging ◽

Cell Imaging ◽

Aqueous System ◽

Live Cell ◽

Selective Detection ◽

Turn On ◽

Fluorescence Turn On

A new quinoline based sensor was developed and applied for the selective detection of Cd2+ both in vitro and in vivo.

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Current and Future Advancements of Raman Spectroscopy Techniques in Cancer Nanomedicine

International Journal of Molecular Sciences ◽

10.3390/ijms222313141 ◽

2021 ◽

Vol 22 (23) ◽

pp. 13141

Author(s):

Elisabetta Canetta

Keyword(s):

Raman Spectroscopy ◽

Imaging Techniques ◽

Rapid Development ◽

Surface Enhanced Raman Spectroscopy ◽

Cancer Diagnostics ◽

Raman Signal ◽

Therapeutic Effects ◽

Cancer Nanomedicine

Raman scattering is one of the most used spectroscopy and imaging techniques in cancer nanomedicine due to its high spatial resolution, high chemical specificity, and multiplexity modalities. The flexibility of Raman techniques has led, in the past few years, to the rapid development of Raman spectroscopy and imaging for nanodiagnostics, nanotherapy, and nanotheranostics. This review focuses on the applications of spontaneous Raman spectroscopy and bioimaging to cancer nanotheranostics and their coupling to a variety of diagnostic/therapy methods to create nanoparticle-free theranostic systems for cancer diagnostics and therapy. Recent implementations of confocal Raman spectroscopy that led to the development of platforms for monitoring the therapeutic effects of anticancer drugs in vitro and in vivo are also reviewed. Another Raman technique that is largely employed in cancer nanomedicine, due to its ability to enhance the Raman signal, is surface-enhanced Raman spectroscopy (SERS). This review also explores the applications of the different types of SERS, such as SERRS and SORS, to cancer diagnosis through SERS nanoprobes and the detection of small-size biomarkers, such as exosomes. SERS cancer immunotherapy and immuno-SERS (iSERS) microscopy are reviewed.

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Efficacy of Potential Control Agents Against Rosellinia necatrix and Their Physiological Impact on Avocado

Plant Disease ◽

10.1094/pdis-08-20-1751-re ◽

2021 ◽

Author(s):

Phinda Magagula ◽

Nicky Taylor ◽

Velushka Swart ◽

Noëlani van den Berg

Keyword(s):

Integrated Management ◽

Integrated Approach ◽

Dry Mass ◽

Persea Americana ◽

Effective Control ◽

Rosellinia Necatrix ◽

Potential Control ◽

Greenhouse Trial

Rosellinia necatrix is the causal agent of white root rot (WRR), a fatal disease affecting many woody plants, including avocado (Persea americana). As with other root diseases, an integrated approach is required to control WRR. No fully effective control methods are available, and no chemical or biological agents against R. necatrix have been registered for use on avocado in South Africa. Fluazinam has shown promising results in the greenhouse and field in other countries, including Spain. The current study aimed to investigate the potential of a fumigant, chloropicrin, and biological control agents (B-Rus, Beta-Bak, Mity-Gro, and Trichoderma) against R. necatrix both in vitro and in vivo as compared with fluazinam. In a greenhouse trial, results showed that Trichoderma and B-Rus were as effective as fluazinam at inhibiting R. necatrix in vitro and suppressed WRR symptoms when applied before inoculation with R. necatrix. In contrast, Mity-Gro and Beta-Bak failed to inhibit the pathogen in vitro and in the greenhouse trial, despite application of the products to plants before R. necatrix infection. Fluazinam suppressed WRR symptoms in plants when applied at the early stages of infection, whereas chloropicrin rendered the pathogen nonviable when used as a preplant treatment. Plants treated with Trichoderma, B-Rus, and fluazinam sustained dry mass production and net CO2 assimilation by maintaining the green leaf tissues despite being infected with the pathogen. This study has important implications for the integrated management of WRR.

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Role of Aqueous-Phase Calcination in Synthesis of Ultra-Stable Dye-Embedded Fluorescent Nanoparticles for Cellular Probing

Applied Spectroscopy ◽

10.1177/00037028211027597 ◽

2021 ◽

pp. 000370282110275

Author(s):

Rachel Rex ◽

Soumik Siddhanta ◽

Ishan Barman

Keyword(s):

Silica Nanoparticles ◽

Cell Imaging ◽

Fluorescent Dyes ◽

Aqueous Media ◽

Clinical Diagnostics ◽

Live Cell ◽

Metal Nanoparticle ◽

Superior Performance ◽

Fluorescent Nanoparticles

Fluorescence imaging is a major driver of discovery in biology, and an invaluable asset in clinical diagnostics. To overcome quenching limitations of conventional fluorescent dyes and further improve intensity, nanoparticle-based constructs have been the subject of intense investigation, and within this realm, dye-doped silica-coated nanoparticles have garnered significant attention. Despite their growing popularity in research, fluorescent silica nanoparticles suffer from a significant flaw. The degradation of these nanoparticles in biological media by hydrolytic dissolution is underreported, leading to serious misinterpretations, and limiting their applicability for live cell and in vivo imaging. Here, the development of an ultra-stable, dye-embedded, silica-coated metal nanoparticle is reported, and its superior performance in long-term live cell imaging is demonstrated. While conventional dye-doped silica nanoparticles begin to degrade within an hour in aqueous media, by leveraging a modified liquid calcination process, this new construct is shown to be stable for at least 24 h. The stability of this metal-enhanced fluorescent probe in biologically relevant temperatures and media, and its demonstrated utility for cell imaging, paves the way for its future adoption in biomedical research.

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