Multi-scale tissue analysis: hybrid fluorescence-AFM

In partnership with Bruker JPK BioAFM, a research team at the University of Freiburg have demonstrated the practicality and value in analysis of native biological tissues across widely different length scales: macro- to nano-scale using a combination of fluorescence and atomic force microscopies.  

The model system for this was human articular cartilage (AC) with the aim of identifying relationships between early macroscopic markers of osteoarthritis (OA) and functional features at the nano-scale.

As has been shown with other correlative techniques which span length scales over many orders of magnitude such as fluorescence microscopy with cryo-EM the nucleus has been identified as a convenient and reliable fiduciary marker (Ellisman lab, UCSD and others).  The far-red fluorescing cell-permeant DNA dye DRAQ5™ has been chosen for these studies, as it has here in this work. Its properties enable the investigation of native, unfixed tissue (live cells) while excitation and emission light transmission is less perturbed by scatter permitting examination at greater sample depths.

Fluorescence microscopy at the millimetre sample scale showed the change in cellular organization associated with early OA and, thereby in the same sample loci, use of AFM to interrogate changes at the micro- and nano-scale such as collagen fibre modification, roughness of the AC surface and even functional properties such as elastic modulus.

This work represents another example of the breadth of application of DRAQ5™ in pursuit of correlative techniques that bridge the clinical observation with the structural and molecular background.

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Reference:

Tschaikowsky, Mathaeus, Tanja Neumann, Sofia Brander, Heiko Haschke, Bernd Rolauffs, Bizan N. Balzer, and Thorsten Hugel. "Hybrid fluorescence-AFM explores articular surface degeneration in early osteoarthritis across length scales." Acta Biomaterialia 126 (2021): 315-325.

Links to related blogposts on correlative microscopy techniques:

CryoChem - from fluorescence to µCT to SBEM

ChromEMT - labeling chromatin

Simplified CLEM method - from in vivo imaging to FIB/SEM

microCT-EM - non-destructive sample mounting

Non-professional phagocytosis and cell cycle

Scientists at Universitätsklinikum Erlangen led by Luitpold Distel have studied the possible importance of cell cycle position on the process of non-professional phagocytosis, specifically of necrotic cells.  

Several different cell types were studied for the capacity for phagocytosis, both epithelial and mesenchymal.  Epithelial cell lines showed greater propensity to phagocytose neighbouring necrotic cells. Similarly, active mitosis was positively implicated in a cell's ability to phagocytose.

The authors propose possible reasons for the increase in phagocytosis during the cell cycle e.g. re-modelling of the cytoskeleton and 'cannibalisation' to gain energy but further work will be required to uncover this. Nonetheless, this work adds to cell-in-cell knowledge, highly pertinent to cancers for example.

To track the engulfment of necrotic cells, homotypic cells were labelled with the fluorescent cell-permeant CyTRAK Orange™ (differentially labelling nucleus and cytoplasm) and heat-killed before being combined with counterpart living cells in 2D culture to permit real-time observation of the progress of phagocytosis.

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Reference:

Hofmann A, Putz F, Büttner-Herold M, Hecht M, Fietkau R, Distel LV (2021) Increase in non-professional phagocytosis during the progression of cell cycle. PLoS ONE 16(2): e0246402.

Cardiac Injury and Repair: transcriptomics for drug targets

A team led by Eva van Rooij at the prestigious Hubrecht Institute in Utrecht has undertaken a significant transcriptomics study in the search for drug targets for the encouragement of cardiac tissue repair following damage due to ischemia.

Using their tissue dissociation and FACS techniques they were able to analyse single adult cardiomyocytes of mice following cardiac ischemic injury.  To ensure that they sorted intact, live and nucleated cells the single cell suspension was stained with DAPI and DRAQ5 respectively.

The initial findings for cardiomyocytes, fibroblasts, neutrophils, endothelial cells and macrophages point to a significant role for beta-2 microglobulin, secreted from stressed cardiomyocytes and activating fibroblasts to stimulate repair for example.

The combination of DAPI and DRAQ5™ has become the benchmark for sorting of single cells for downstream molecular analysis, as exemplified in this work.  For a detailed analysis of the rationale see this earlier blog article and Ordoñez-rueda, et al.

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References:

Molenaar, Bas, et al. "Single-cell transcriptomics following ischemic injury identifies a role for B2M in cardiac repair." Communications Biology 4.1 (2021): 1-15.

Ordoñez‐Rueda, Diana, et al. "Apoptotic Cell Exclusion and Bias‐Free Single‐Cell Selection Are Important Quality Control Requirements for Successful Single‐Cell Sequencing Applications." Cytometry Part A 97.2 (2020): 156-167.

Cardiomyocyte Exophers - homeostasis for long-lived cells

Recent work of a multi-national effort led by Andrés Hidalgo at CNIO in Madrid has identified an important mechanism in the healthy maintenance of cardiomyocytes (CM), extremely long-lived cells, within the myocardium.

Importantly, they show that defective or worn-out mitochondria are expelled from these energy-intensive cells, by an autophagic process, inside vesicles called exophers. These exophers are tagged with phosphatidyl serine which labels them for engulfment and rapid destruction along with their contents by cardiac macrophages (cMacs).

Failure of CM to expel these failing organelles will obviously lead to poor cardiac function while a failure of macrophage engulfment of expelled exophers may lead to inflammatory processes amongst other effects and thereby indirectly to reduced cardiac function.

In addition, the group have most recently published a detailed protocol for the isolation of exophers from cardiac tissue.  In the final step of the isolation procedure exophers are purified by FACS, based on size and the exclusion of both endothelial cell-derived (via CD31-negativity) and DNA-containing (via DRAQ5-negativity).

DRAQ5™ was employed at a final concentration of 1 µM as the final step, without washing, prior to the sorting or analysis.

This novel application of the cell-permeant far-red DNA dye DRAQ5™ will enable the downstream investigation in cell-based and molecular analyses of exophers, to unpick the critical role of their expulsion and disposal by cMacs in the homeostasis of CM biology and perhaps in better treatments for heart disorders.

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References:

Nicolás-Ávila, José A., et al. "A network of macrophages supports mitochondrial homeostasis in the heart." Cell 183.1 (2020): 94-109.

Nicolás-Ávila, José Ángel, María Sánchez-Diaz, and Andrés Hidalgo. "Isolation of exophers from cardiomyocyte-reporter mouse strains by fluorescence-activated cell sorting." STAR protocols 2.1 (2021): 100286.