Advanced imaging methods drive research breakthroughs in a wide range of disciplines at the UTS Microbial Imaging Facility

Session

Poster Session

Authors

Louise Cole (2), Yan Liao (2), Clara Liu Chung Ming (1), Laura Rangel-Sanchez (1), Bill Soderstrom (2)

Affiliations

1. Faculty of Engineering and Information Technology, University of Technology Sydney
2. Faculty of Science, University of Technology Sydney

Keywords

Live-cell imaging. Structured Illumination Microscopy. Archaeal cell division. Single Molecule Localisation Microscopy. Urinary tract infections. Uropathogenic E. Coli (UPEC). Infection related filamentation. Cardiac spheroids. Confocal microscopy. Myocardial ischaemic-reperfusion injury. Breast cancer. Tumoroids. Alginate-based Tissue Encapsulation (ALTEN).

Abstract text

The Microbial Imaging Facility (MIF) in the Faculty of Science at UTS facilitates world-class research in a wide range of microbiological and biomedical research applications by providing state-the-art advanced imaging microscopes, key advice on specimen preparation, expert training on MIF microscopes and downstream image analysis and visualisation methods. Here, we focus on several case studies that demonstrate advances using light microscopy methods, and how these techniques drive breakthroughs in both fundamental biology and translational research. Examples of microscopy-led research advances include: 

 

(i) How protein subcellular localization and dynamic protein tracking in live Archaeal cells can provide insights into how these cells control cell shape, drive cell division, and help unravel some of the mysteries of early cellular evolution and division [Y. Liao] (1,2). Live-cell, fluorescence, and 3D Structured Illumination Microscopy (SIM) methods have shed new light on the roles of the superfamily of tubulin-like proteins (CetZ and FtsZ) in Archaea using Haloferax volcanii as a model archaeon for studying membrane dynamics. 

 

3D-SIM of H. volcanii labelled CetZ1-mturquoise2 showed that CetZ1 is a key part of a dynamic cytoskeleton that reshapes cells during rod development (Roshali de Silva et al., unpublished data).

(ii) Fluorescence and single-molecule localisation microscopy (SMLM) approaches to the study of bacterial invasion behaviour, including infection-related filamentation (IRF) and the molecular mechanisms that cause these extreme morphology changes during urinary tract infections (UTIs) [B. Söderström] (3). While there are many different species of bacteria that can cause UTIs, the major causative infectious agent is uropathogenic Escherichia coli (UPEC). During the dispersal phase of the UPEC infection cycle, a subset of bacteria stop dividing and grow into long filaments, some hundreds of microns long. These filaments can later divide back into rods and reinitiate the infection cycle. Very little is known about how UPEC filaments reinitiate division to revert to rods. This IRF response in UPEC during UTIs can be readily observed in the lab using phase-contrast and fluorescence microscopy. Further to this, SMLM of proteins involved in the bacterial cell division machinery, using the well-characterised uropathogenic strain of E. coli UTI189 expressing the protein DamX tagged with photoconvertible green fluorescent protein mEos3.2, showed that DamX remains at the division site after membrane separation, suggesting its role in septal peptidoglycan regulation until building of the new cell wall is completed. DamX is then thought to switch function for reversal to assist in cell division in a manner that structurally resembles normal cell division. Recent studies of this IRF response using the UPEC infection model and microscopy methods, has revealed that this approach is becoming increasingly important for the study of morphology, division, and survival of pathogenic bacteria.

 

 SMLM (by Photo-Activated Localisation Microscopy) of mEos3.2-DamX ring at the division site in UT189 filaments collected from infection (3). 

 

Graph shows axial breadth of mEos3.2-DamX rings along length of filaments at various cell diameters. Average width 116.5±13.4 nm (n=122); values represent mean±SD. Red line represents linear fit to data: y=−0.009*x+116 (3).

(iii) Use of cardiac spheroids (CS), confocal microscopy and 3D-rendering methods to investigate the potential therapeutics against heart attack and drug-induced myocardial damage [C. Liu Chung Ming](4). Current preclinical in vitro and in vivo models of cardiac injury typical of myocardial infarction and drug-induced cardio-toxicity fail to fully replicate the complex scenario of the human cardiac pathophysiology. It has already been established that CS can be considered advanced in vitro 3D models of the human heart and therefore can be used to better mimic the molecular, cellular, and extracellular features of the cardiac microenvironment. Here, we present new findings relating to the roles of the different cell types in CS and their response to drug-mediated cardiotoxicity.

 

 

Confocal, immunofluorescence and 3D-rendering methods revealed that cardiac cell types: cTNT-labelled cardiomyocytes (red); CD31-labelled endothelial cells (blue) and vimentin-labelled fibroblasts (green) were affected differently following myocardial ischaemic-reperfusion (I/R) injury in CS stained with ethidium homo-dimer (yellow) to identify dead cells. Following I/R injury (top image), most cardiomyocytes died and could not form spheroids, compared to control conditions (bottom image). 

 

Graph shows establishment of CS I/R injury using hypoxic conditions. Image-iT labelled CS were exposed to hypoxic conditions (0% O2) for 20 h to mimic the hypoxia typical of the ischemic event and imaged using EVOS FL system. CS were exposed to 5% O2 to mimic normoxic conditions. Image-iT is a reversible hypoxic fluorescent dye sensitive to changes in intracellular O2 levels (4).

(iv) Establishment of an ex vivo tissue culture device, ALTEN (ALginate-based Tissue ENcapsulation), for the culture of high-fidelity breast cancer tumoroids and development of a drug testing platform to infer patient therapeutic response. Current 3D ex vivo culture methods have a limited capacity to maintain the tissue ecosystem, they fail to recapitulate the native extracellular matrix components and original cellular configuration and exhibit extensive cell death. To address these limitations, the researchers have developed the ALTEN platform, a versatile and cost-effective ex vivo tissue system that will enable rapid screening and analysis of exogenous molecule perturbation and drug sensitivity testing on native tissue specimens in situ (5). 

 

Maximum Intensity Projection confocal image of intact ALTEN breast cancer tumoroid, cleared and immunolabelled with F-Actin (red), CD31 (yellow) and stained with DAPI (cyan) to show nuclei (L. Rangel-Sanchez, unpublished data).

Using a combination of tissue clearing techniques and 3D confocal imaging of labelled ALTEN tumoroids, the 3D-architecture of these tumoroids can be clearly seen in toto [L. Rangel-Sanchez]. It is hoped that this approach will allow the spatial positioning of molecular biomarkers and the functional interactions between cancer cells and the surrounding environment to be elucidated.

 

Together these case studies provide a unique snapshot of the impact of new and emerging microscopy-led research at the MIF facilitated by close partnership between MIF facility staff and researchers at UTS.

References

1. Duggin, I., Aylett, C., Walsh, J. et al. CetZ tubulin-like proteins control archaeal cell shape. Nature 519, 362–365 (2015). https://doi.org/10.1038/nature13983

 

2. Liao, Y., Ithurbide, S., Evenhuis, C. et al. Cell division in the archaeon Haloferax volcanii relies on two FtsZ proteins with distinct functions in division ring assembly and constriction. Nat Microbiol 6, 594–605 (2021). https://doi.org/10.1038/s41564-021-00894-z.

 

3. Söderström, B., Pittorino, M.J., Daley, D.O. et al. Assembly dynamics of FtsZ and DamX during infection-related filamentation and division in uropathogenic E. coli. Nat Commun 13, 3648 (2022). https://doi.org/10.1038/s41467-022-31378-1.

 

4. Sharma, P., Liu Chung Ming, C., Wang, X., Bienvenu, L. A., Beck, D., Figtree, G., Boyle, A., & Gentile, C. (2022). Biofabrication of advanced in vitro 3D models to study ischaemic and doxorubicin-induced myocardial damage. Biofabrication, 14(2), 025003. https://doi.org/10.1088/1758-5090/ac47d8.

 

5. Law, A. M. K., Chen, J., Colino-Sanguino, Y., Fuente, L. R., Fang, G., Grimes, S. M., Lu, H., Huang, R. J., Boyle, S. T., Venhuizen, J., Castillo, L., Tavakoli, J., Skhinas, J. N., Millar, E. K. A., Beretov, J., Rossello, F. J., Tipper, J. L., Ormandy, C. J., Samuel, M. S., Cox, T. R., … Gallego-Ortega, D. (2022). ALTEN: A High-Fidelity Primary Tissue-Engineering Platform to Assess Cellular Responses Ex Vivo. Advanced science (Weinheim, Baden-Wurttemberg, Germany), 9(21), e2103332. https://doi.org/10.1002/advs.202103332.