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

The lab of Prof. Jochen Herms (LMU-Munich) has developed a simplified CLEM sample-processing workflow to allow study of a single region of interest (ROI) - the tripartite synapse - from in vivo 2-P microscopy through to FIB/SEM.  The alignment and registration of the tissue section through the various stages was achieved by slide mounting and natural landmarks without the need for complex EM labelling techniques known to be associated with deterioration or obscuration of target and proximal ultrastructure.

Instead, natural landmarks - blood vessels, nuclei and myelinated axons - were utilised, and identified by a variety of light microscopy techniques, combined with a simplified "flat embedding" which preserved the various landmarks for alignment between the LM and EM images.

For high resolution nuclear imaging by confocal laser scanning microscopy DRAQ5™ was chosen as nuclear counterstain, as a proven vital (cell permeant) DNA dye that therefore eliminates the need for tissue permeabilization.

In some cases the three landmarks could be identified in a label-free manner utilising DIC and SCoRe (spectral confocal reflectance) microscopy, albeit at lower resolution. In all cases registration of the various LM images could be achieved with SEM of the carbon-coated sample.

In the example on mouse brain tissue, 50 µm vibratome sections were slide mounted under coverslip and spacer for LM and then re-exposed on-slide for FIB/SEM sample processing.  Thereby, sample orientation remained the same throughout and the various landmarks used as fiducial points for overlaying the LM and EM images to enable localisation of the region of interest (ROI), in this case the tripartite synapse.

Here, far-red DRAQ5™ again demonstrates its utility to reliably mark nuclei without resort to unwanted sample permeabilization and processing in already challenging and complex workflows.  Based on the evidence of a number of papers from leading laboratories DRAQ5™ appears to be the nuclear counterstain of choice for CLEM!

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Reference: Luckner, M., Burgold, S., Filser, S., Scheungrab, M., Niyaz, Y., Hummel, E., ... & Herms, J. (2018). Label-free 3D-CLEM using endogenous tissue landmarks. iScience 6:92-101.

Read more articles on DRAQ5™ in exciting new EM techniques: 

• CryoChem for CLEM - preservation of high definition in fluorescence & EM dimensions
• ChromEMT for EM-Tomography of chromatin - direct labelling of chromatin

Pneumococcal infections - dependence upon pneumolysin expression

Scientists led by Assoc. Prof. Anirban Banerjee, IIT, Mumbai have made a significant and deep investigation into infection by Streptococcus pneumoniae where tissue-destination and persistence are highly dependent upon the expression level of the major virulence factor pneumolysin (Ply), a pore-forming toxin.  

Even in an isogenic culture Ply was shown to be expressed over a wide range.  Bacterial constructs recapitulating the Ply-low and Ply-high expression gave markedly different capacities for evasion of the bactericidal response mechanisms in human host brain microvascular endothelial cells and for resulting transcytosis across the blood brain barrier, as detected in an animal model.

In one part of the study to explore the response of infected host cells, human host brain microvascular endothelial cells were stably transduced with GFP and mStrawberry to permit real-time tracking of LC3 and Ubiquitin (Ubq), respectively.  To track intracellular wildtype S pneumoniae and their complex interactions with LC3 and Ubq, the bacteria were pre-stained with far-red cell permeant DNA counterstain DRAQ5™.  DRAQ5™ has spectral compatibility with GFP and mStrawberry as shown in the figure below.  We believe this might represent the first example of three-colour, real-time imaging of intracellular bacterial infections.

This publication begins a more complete elucidation of the role Ply has in the varied nature of pneumococcal infections and the complexity of interactions with host cell defences.

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Reference:
Surve MV, Bhutda S, Datey A, Anil A, Rawat S, Pushpakaran A, et al. (2018) Heterogeneity in pneumolysin expression governs the fate of Streptococcus pneumoniae during blood-brain barrier trafficking. PLoS Pathog 14(7): e1007168.

Figure: emission spectrum for eGFP (LC3), mStrawberry (Ubq) and DRAQ5™

Searchlight™is provided courtesy of Semrock/IDEX Health & Science, LLC

Light Sheet Microscopy & DRAQ probes

Since the development of Light Sheet Microscopy by the laboratory of Prof. Ernst Stelzer more than 10 years ago there have been many publications utilising DRAQ5™ and DRAQ7™.  A selection of these are presented below with brief notes on the samples that were interrogated.

Part of the power of Light Sheet Microscopy is the ability to optically section 3-dimensional structures while limiting out of focus light and its bleaching effect on fluorophores.  Aiding this, red-excited, far-red-emitting DRAQ™ probes benefit from less light scatter and have remarkable resistance to photo-bleaching (see: Martin et al., 2005) and chemical degradation (e.g. DRAQ7™ in multi-day cell health assays).

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Verveer, P. J., Swoger, J., Pampaloni, F., Greger, K., Marcello, M., & Stelzer, E. H. (2007). High-resolution three-dimensional imaging of large specimens with light sheet–based microscopy. Nature methods, 4(4), 311.

• GFP-MDCK cysts and BxPC-3 spheroids counterstained with DRAQ5™

Keller, P. J., & Stelzer, E. H. (2010). Digital scanned laser light sheet fluorescence microscopy. Cold Spring Harbor Protocols, 2010(5), pdb-top78.

• BxPC-3 spheroid: counterstained with DRAQ5™

Ejsmont, R. K., Sarov, M., Winkler, S., Lipinski, K. A., & Tomancak, P. (2009). A toolkit for high-throughput, cross-species gene engineering in Drosophila. Nature methods, 6(6), 435.

• Drosophila embryo: anti-GFP stained and counterstained with DRAQ5™

Lorenzo, C., Frongia, C., Jorand, R., Fehrenbach, J., Weiss, P., Maandhui, A., ... & Lobjois, V. (2011). Live cell division dynamics monitoring in 3D large spheroid tumor models using light sheet microscopy. Cell division, 6(1), 1.

• Capan-2 cell spheroid: counterstained with DRAQ5™

Pampaloni, F., Ansari, N., & Stelzer, E. H. (2013). High-resolution deep imaging of live cellular spheroids with light-sheet-based fluorescence microscopy. Cell and tissue research, 352(1), 161-177.

• T-47D tumour spheroid: counterstained with DRAQ5™ & MitoView™ Green (mitochondria)
• BxPC-3 spheroid: counterstained with DRAQ5™

Schmitz, A., Fischer, S. C., Mattheyer, C., Pampaloni, F., & Stelzer, E. H. (2017). Multiscale image analysis reveals structural heterogeneity of the cell microenvironment in homotypic spheroids. Scientific Reports, 7, 43693.

• T-47D tumour spheroid: counterstained with DRAQ5™ and cleared with BABB

Glaser, A. K., Reder, N. P., Chen, Y., McCarty, E. F., Yin, C., Wei, L., ... & Liu, J. T. (2017). Light-sheet microscopy for slide-free non-destructive pathology of large clinical specimens. Nature biomedical engineering, 1(7), 0084.

• Prostate core biopsy: cleared with X-CLARITY and stained with DRAQ5™ and eosin Y ("D&E"; see Elfer, et al. PloS one, 11(10), e0165530.)

Lawson, P. J., Hu, B., Fasy, B. T., Wenk, C., & Brown, J. Q. (2018, March). Quantifying prostate cancer morphology in 3D using light sheet microscopy and persistent homology (Conference Presentation). In Diagnosis and Treatment of Diseases in the Breast and Reproductive System IV (Vol. 10472, p. 1047209). International Society for Optics and Photonics.

• Biobanked Prostate-biopsies: stained with DRAQ5™ and eosin Y ("D&E"; see Elfer, et al. PloS one, 11(10), e0165530.)

Rieckher, M., Psycharakis, S. E., Ancora, D., Liapis, E., Zacharopoulos, A., Ripoll, J., ... & Zacharakis, G. (2018). Demonstrating Improved Multiple Transport‐Mean‐Free‐Path Imaging Capabilities of Light Sheet Microscopy in the Quantification of Fluorescence Dynamics. Biotechnology journal, 13(1), 1700419.

• T-47D tumour spheroid: imaged following 24h. incubation with 1.5 µM DRAQ7™ to label dead cells in the intact 3D structure

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