Confocal reconstructions of the head, thorax and tail regions of the Zebrafish (Danio rerio)

The Zebrafish (Danio rerio) is, in many ways, the perfect model for microscopists. Not only does it share 70% genetic homology with man, but its larvae are born in large, transparent broods all year round and develop extremely quickly (a single cell develops into something resembling a fish within 24 hours!)  This means that developmental events can be visualised in vivo in real-time down the microscope. On top of this, their genome has been sequenced and it is easily amenable to molecular manipulation- again, these manipulations can be followed closely under the microscope lens.

Over the last few years we have been collaborating with Dr Chrissy Hammond at Bristol University, a fish biologist who shares an interest in skeletal development and disease. In our joint studies, we have used a variety of imaging techniques (brightfield, DIC, polarising, epifluorescence, confocal, TEM, radiography and microCT) to investigate skeletal development, growth and ageing in  this animal model.

One of the many interesting findings from our studies is that ageing fish undergo degenerative changes to their spine that resemble osteoarthritis (for example, spinal curvature, osteophyte formation, and connective tissue degeneration). This opens up the possibility  that they could be used to experimentally model aspects of the human disease. So it’s not just fishy tails!

AJH

Find out more:

• Guardian article: How the diminutive zebrafish is having a big impact on medical research
• Arthritis Research UK: Fishy tales

Further Reading:

• Hayes, et al. (2013) Expression of glycosaminoglycan epitopes during zebrafish skeletogenesis. Developmental Dynamics 242: 778-789
• Hayes et al. (2013) Spinal deformity in aged zebrafish is accompanied by degenerative changes to their vertebrae that resemble osteoarthritis. PLoS ONE 8 : e75787

Dr Simon Pope and his research group in CHEMY have been collaborating with the BIOSI Bioimaging Unit for over 8 years. Their research is focussed upon the development of new, metal-based, fluorescent probes for cell imaging applications, and forms part of a larger study on the use of metal complexes, which include rhenium and gold, as multi-modal imaging agents with therapeutic potential. In these studies, we have performed confocal imaging to (1) assess the cytotoxicity of the new probes, (2) evaluate their cellular uptake and determine their cytoplasmic localisation, and (3) characterise their fluorescent emissions via spectral (wavelength or lambda) scanning. The collaboration has yielded a number of high impact publications (see below) as well as a journal cover! With the improved potential of the new super-resolution confocal system we anticipate a lot more to come.

AJH

Find out more:

• Langdon-Jones et al. (2014) Fluorescent Rhenium-Naphthalimide conjugates as cellular imaging agents. Inorganic Chemistry 53: 3788-3797.
• Lloyd, D., Coogan, M. P. and Pope, S. J. 2012. Novel metal-based luminophores for biological imaging. In: Geddes, C. D. ed. Reviews in Fluorescence 2010. Reviews in Fluorescence, Vol. 7. New York:  Springer, pp. 15-44.
• Thorp-Greenwood et al. (2011) A ‘Sleeping Trojan Horse’ which transports metal ions into cells, localises in nucleoli, and has potential for bimodal fluorescence/PET imaging. Chem Commun 47: 3096-3098.
• Fernandez-Moreira et al. (2010) Uptake and localisation of rhenium fac-tricarbonyl polypyridyls in fluorescent cell imaging experiments. Org Biomol Chem 8:3888-901.
• Coogan et al. (2010) Probing intracellular oxygen by quenched phosphorescence lifetimes of nanoparticles containing polyacrylamide-embedded [Ru(dpp(SO3Na)2)3]Cl2. Photochem Photobiol Sci 9: 103-109.
• Amoroso et al. (2008) 3-Chloromethylpyridyl bipyridine fac-tricarbonyl rhenium: a thiol-reactive luminophore for fluorescence microscopy accumulates in mitochondria. New J Chem 32: 1097-1102.
• Amoroso et al (2007) Rhenium fac tricarbonyl bisimine complexes: biologically useful fluorochromes for cell imaging applications. Chem Commun (Camb) 2007: 3066-3068.

Not just pretty pictures: an earthworm coelomocyte imaged by conventional confocal microscopy (left; DIC/fluorescence image) and via spectral scanning (centre) to analyse changes in riboflavin fluorescence caused by soil pollution (right).

The Research Techniques module run by Professor Pete Kille and Dr Carsten Muller (Introduction to Environmental Toxicology) makes for a busy week within the Bioimaging Research Hub. In the practical, students learn a range of advanced analytical research techniques as they aim to identify and characterize earthworm populations that have been sampled from land polluted by heavy metal – and I’m not talking about Axel’s Rose garden here : )

Pete is an expert in ecotoxicology and much of his research centers on how invertebrate species, such as the earthworm, deal with heavy metal pollutants, e.g., lead, in their environment. As it turns out, they seem to be pretty good at tolerating a lot of the nasty stuff that passes through them, but it does leave an indelible metabolic mark – making the organisms ideal for environmental toxicological testing. And here’s where it gets interesting: previous studies of the earthworm, Eisenia fetida, have shown that heavy metals affect riboflavin (vitamin B2) biosynthesis. Now, riboflavin happens to be (1) highly autofluorescent, and (2) neatly packaged within spheroidal organelles, or chloragosomes, within a sub-population of  immune cells, called coelomocytes, that are resident within the body cavity of the worm. Fortunately, earthworms can be gently persuaded to give up some of these cells for confocal microscopic analysis.

In the practical, we use confocal microscopy to image earthworm coelomocytes and, via spectral scanning, generate emission spectra of the riboflavin autofluorescence from within the chloragosomes. By comparing the autofluorescent signatures of coelomocytes from worms obtained from different sampling sites, we have asked the question: can riboflavin autofluorescence in this organism be used to assess soil pollution?

And the answer? Well, I’m not at liberty to say – the students reports aren’t in yet! (Answers on the back of a postcard to…)

AJH

Find out more:

Zimmerman et al. (2003) Spectral Imaging and its applications in live cell microscopy. FEBS Letters 546: 87-92.

Further Reading

• Koziol et al. (2006) Riboflavin as a source of autofluorescence in Eisenia fetida coelomocytes. Photochem Photobiol 82: 570-573
• Plytycz et al. (2009) Riboflavin content of coelomocytes in earthworms (Dendrodrilus rubidus) field populations as a molecular biomarker of soil metal pollution. Environ Pollut 157: 3042-3050

3D surface-rendered model of appendage (endopod of maxilliped) of Chinese mitten crab larva. Image produced on Zeiss LSM880 confocal and reconstructed using Bitplane Imaris.

Last week we undertook some confocal microscopy for the National History Museum to help characterise the arrangement of setae on the larval appendages of the Chinese mitten crab, Eriocheir sinesis * (now published, see Kamanli et al, 2017 below). The Mitten crab, so-named because of the tufts of ‘fur’ on the adult’s claws, is officially listed as one of the World’s most invasive species. The crabs out-compete and prey on native crab species, damage fishing nets and cause significant erosion of riverbanks, thus are of considerable economic importance.  They arrived in this country from China in the 1930’s via discharge of ballast water from trading ships and are now firmly established in many of Britain’s waterways. The National History Museum is investigating ways of reducing the population of Mitten crabs and this species is currently under evaluation as a potential food source in the UK (so if you can’t beat them, eat them!)

AJH

Find out more:

• National History Museum: Chinese Mitten crab research
• The Guardian: Gloves off as scientists go to war on Mitten crab
• The Independent: Will we soon be tucking into Mitten crabs fresh from the Thames

Further reading:

• Kamanli et al. (2017) A 3D imaging and visualization workflow, using confocal microscopy and advanced image processing for brachyuran rab larvae. J Micros 266: 307-323
• Klaus & Schawaroch (2006) Novel methodology utilizing confocal laser scanning microscopy for systematic analysis in arthropods (Insecta) Integ Comp Biol 46: 207-214