It’s all about technique

HookeMicroscopeImageTechniques are at the heart of understanding in botany (and any science, whether it be an –ology or not *). For, as techniques advance so should our understanding – often to the point of over-turning the ‘truth’ that previously obtained (and which is lovingly cherished and jealously guarded by its devotees and advocates). That’s what we call progress in science; new wisdom replacing the old. And those who previously clung to the old, but who respect the scientific method, begin to embrace the new when its veracity – however ephemeral that may be – is demonstrated. So – and with no apologies for a cellular bias, and in the belief that structure is what gives function a reason to exist – here’s a collection of techniques for looking at plants and their parts in more detail **.

Although it is said that seeing is believing, one shouldn’t rely on one source of information; corroboration from multiple sources is an important aspect of evidence gathering. To that end, use of different microscopical techniques, each of which brings its own insights, can give a more informative view than reliance upon one technique alone. Whilst that may mean using different preparation techniques to allow a structure to be imaged by different technologies – e.g. light and transmission electron microscopy, more defensible is to use different techniques to examine the same material. And if that means just making better use of an existing technology, then so much the better; even more information for no more tissue preparation effort.

And that is what David Collings proposes with his “Optimisation approaches for concurrent transmitted light imaging during confocal microscopy” (Plant Methods (2015) 11:40; DOI 10.1186/s13007-015-0085-3). Recognising that transmitted light detectors present on most modern confocal microscopes (Adaobi Nwaneshiudu et al., Journal of Investigative Dermatology (2012) 132, e3. doi:10.1038/jid.2012.429), are an underutilised tool for the live imaging (which also has the advantage of avoiding tissue-preparation-induced artefacts that can confound interpretation of the living state) of plant cells, Collings advocates their use to provide “cellular and organismal context for fluorescence optical sections generated confocally”. Importantly, he also stresses the importance of ensuring the microscope is set up properly to get the best out of it(!). Another advantage of this approach is that, because essentially the same optics as the microscope’s confocal capability are used, the transmitted light images have spatial and temporal registration with the fluorescence images (unlike images taken with a separately-mounted camera).

A paper that demonstrates the enhanced value of combining comparatively new techniques with more established, ‘traditional’ even, technologies is Pavani Nadiminti et al’s study of the cuticle of Triticum aestivum (wheat), Zea mays (maize), and Lupinus angustifolius (lupin) (Protoplasma 252:1475–1486, 2015; DOI 10.1007/s00709-015-0777-6). Exploiting the image analysis and quantification power of confocal microscopy and the resolution of the scanning electron microscope (SEM), they developed and used novel image data analysis procedures for quantifying epicuticular wax in a multifaceted approach that has led to a better understanding of cuticular structure and provided new insights into leaf surface architecture. And as a – sorry, the – major barrier between the aerial parts of a plant and the external environment (both abiotic and biotic) (e.g. Trevor Yeats and Jocelyn Rose, Plant Physiol. 163(1): 5–20, 2013; doi:  10.1104/pp.113.222737), the cuticle is a structure of some considerable importance and research interest.

And, taking microscopy to the next level, George Komis et al. provide a timely review of the past, present and future of super-resolution microscopy ** of plants (e.g. structured-illumination microscopy (SIM), photoactivation localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), and stimulated emission depletion microscopy (STED) (Trends in Plant Science 20: 834-843, 2015; http://dx.doi.org/10.1016/j.tplants.2015.08.013).

Bridging the gap between molecule-based immunolocalisation work and the cellular context within which that occurs, Taras Pasternak et al. present “an improved and universal procedure for whole‑mount immunolocalization in plants” (Plant Methods (2015) 11:50; DOI 10.1186/s13007-015-0094-2). Whilst recognising the important contribution that immunolocalisation techniques make to plant biology, they also acknowledge that their value can be compromised by problems in adequate tissue preservation. In an attempt to improve such matters they present a detailed protocol that should permit robust immunolocalisation of proteins and allow better resolution and 3-D reconstruction for whole plants organs, and which is applicable to a wide range of plant species (e.g. Medicago sativa (lucerne, alfalfa), Triticum aestivum, Nicotaina [sic. ] tabacum (tobacco), Lycopersium [sic.] esculentum (tomato), Hedera helix (ivy), and even Arabidospis [sic.] (thale cress)…).

Finally, and to prove that it’s not just microstructural imaging techniques that catch my eye, Takashi Fujii et al. announce a technique that permits extraction of the contents of a single cell and its analysis by mass spectrometry (MS) (Nature Protocols 10: 1445-1456, 2015; doi:10.1038/nprot.2015.084). While that’s impressive enough, the major newsworthiness here is that they used plant cells from Raphanus sativus (radish, not Arabidopsis thaliana – which is refreshingly different in itself (though it is in the same family – the Brassicaceae…)). The present paper builds upon previous work of the team based at the Laboratory for Single Cell Mass Spectrometry at Japan’s RIKEN’s Quantitative Biology Center, under the Leadership of Tsutomu Masujima, which previously examined Pelargonium zonale (geranium – Mónica Tejedor et al., Anal. Chem. 84(12): 5221–5228, 2012; DOI: 10.1021/ac202447t). Sadly – and notwithstanding their importance as cellular organics that orchestrate metabolism via their role as enzymes, proteins are not detected with this technique because of insufficient sensitivity. Nevertheless, live single-cell mass spectrometry (live MS – Tsutomu Masujima, Analytical Sciences 25(8): 953-960, 2009) generates thousands of metabolite peaks from a single live plant cell within minutes. That, however, is only half the story. Although the inventory of metabolites arguably tells us what’s taking place inside the cell, the peaks still have to be identified – and the majority are as yet unknown as Sixue Chen of the University of Florida reminds us.

But, if the cell can be likened to the stage upon which the cytobiological drama is played out, the metabolites can be considered the ‘dramatis personae’ that act out that story.  And, as with a human drama, if you know the players you can work out the title of the play, so too with the cell – if the molecular assemblage is known the cell’s role can be inferred. Thus, as the authors optimistically conclude, this procedure “is expected to reveal undiscovered molecules and dynamic molecular mechanisms.” On the cusp of 2016, we wonder what new – botanical! – cellular dramas await to be premiered to a waiting world: Happy New Year!

 

* And here we are reminded of that wonderful advert from the 1980s dealing with this very notion. And for those who remark that the word botany is not an ‘ology’, we remind them that its alternative name of phytology is!

** But, if further justification for this microscopical medley is needed, let us never forget that it was Robert Hooke and his seminal microscopical researches on sections through cork that ultimately gave us the concept of the cell.

*** And, notably, development of super-resolution microscope techniques earned Stefan Hell, Eric Betzig, and William Moerner, the 2014 Nobel Prize for … Chemistry (Jennifer Lippincott-Schwartz, PNAS 112: 2630–2632, 2015; www.pnas.org/cgi/doi/10.1073/pnas.1500784112).

 

[Image from The National Library of Wales]