Session 2

Confocal Endoscopy in Medicine and Research

Lindsay Bussai, Optiscan

The talk will start with an introduction to endomicroscopy, also known as ‘virtual histology’, ‘optical biopsy’ or 'confocal laser endomicroscopy' (CLE). Endomicroscopy is a technique for obtaining cellular and subcellular images from inside the living body or tissue sample in real-time. Confocal endomicroscopy uses the tip of an optical fiber for both illumination and detection pinholes, simplifying the design and enabling miniaturisation of confocal microscopes. The technology has applications in both medical imaging and academic research. After the introduction, the talk will provide a short summary of the technological developments in the field and then focus on the latest applications of endomicroscopy.

AX/AX R Confocal laser scanning microscope system

Baharak Mahyad, Nikon

Solution for large sample image acquisition and visualization

Georgia Gofis, Andor

In this talk we will introduce Andor Dragonfly high speed confocal microscope system for large size sample imaging and Imaris software for visualization of the 3D image result.

Dr Emma Gordon, Institute for Molecular Bioscience

Blood vessels are a critical component of the body, supplying oxygen and nutrients to all tissues. During vessel sprouting, endothelial cells behave in a collective manner and display heterogeneous gene expression, morphology and movement. A balance between adhesion and migration allows cells to actively migrate past one another in a fluid but controlled fashion, yet how this is controlled remains unclear. Using novel imaging approaches in cell and mouse models, we have identified the tyrosine kinase c-Src as a key mediator of cell adhesion and vascular sprouting, highlighting the importance of cellular signalling during blood vessel growth and function.

Intravital imaging of mammary stem cells during organogenesis

Dr Caleb Dawson, Postdoc, WEHI

Organogenesis is a vitally important process but it is difficult to capture by live imaging in vivo. Mammary gland development provides a unique opportunity for studying organogenesis because it occurs in adolescence and is far more accessible than embryonic tissues. We developed a surgical technique to reveal the migrating tips of mammary ducts in puberty and filmed these using long-term multiphoton intravital imaging. We combined this with mosaic ‘confetti’ tracing of distinct mammary stem cell subsets to uncover single-cell behaviours that contribute to duct elongation. Custom-designed optical filters and alternative excitation allowed us to image the four confetti proteins and two additional markers in six separate channels. This revealed microenvironment interactions and tissue context as well as single-cell tracking. We then combined intravital imaging with cell behaviour modelling and subsequent immunostaining and 3D imaging. The resulting high-dimensional quantitative datasets have given us important new insights into mammary stem cell dynamics during organ development. This approach can also be applied to multiphoton imaging more broadly to help uncover complex tissue dynamics at the single-cell level.

Establishing a functional organoid model of placental development

Mrs Claire Richards, PhD Candidate, University of Technology Sydney

Co Authors
Dr Amy Bottomley, University of Technology Sydney
Dr Louise Cole, University of Technology Sydney
Dr Kristine McGrath, University of Technology Sydney
Dr Lana McClements, University of Technology Sydney

Introduction: Preeclampsia is a cardiovascular disorder of pregnancy with no effective prevention or treatment. Research into the pathogenesis of preeclampsia has been limited due to ethical limitations and a lack of reliable models of the disease. Inadequate trophoblast invasion and remodelling of maternal uterine arteries are major contributing factors in the development of preeclampsia, thus a 3D trophoblast model would aid future research.

Aim: To establish a simple, cost-effective, reproducible and low-risk 3D cell model of the early placenta using a custom-made first trimester trophoblast cell line.
Methods/Results: Trophoblasts were either manually seeded in Matrigel droplets or bioprinted in an equivalent matrix and allowed to form 3D organoids in their normal culture medium. Organoids were imaged using an Incucyte Live-Cell Analysis System for 14 days and demonstrated invasive capabilities. Some organoids were sectioned and stained by haematoxylin and eosin (H&E) or immunofluorescence (IF) for visualisation. Others were fixed in situ and probed for IF imaging using Nikon TiE2 wide-field and Nikon A1 inverted confocal microscopy. In situ images were denoised and clarified by NIS Elements Ver 5.3 software. Heterogeneous cell morphologies were seen within the organoids by H&E and probing for subtype-specific markers such as E-cadherin for villous trophoblasts and human leukocyte antigen G (HLA-G) for extravillous trophoblasts.

Conclusions: Collectively, these data support the establishment of a 3D organoid model that has been characterised to show morphology of early placental tissue development. This model presents an opportunity to investigate key factors involved in trophoblast proliferation, differentiation and cellular function important for placental development