Functional imaging of a model unicell: Spironucleus vortens as an anaerobic but aerotolerant flagellated protist

Lloyd, David ORCID: https://orcid.org/0000-0002-5656-0571, Millet, Coralie O., Williams, Catrin F. ORCID: https://orcid.org/0000-0001-8619-2581, Hayes, Anthony, Pope, Simon J. A. ORCID: https://orcid.org/0000-0001-9110-9711, Pope, Iestyn ORCID: https://orcid.org/0000-0002-4104-0389, Borri, Paola ORCID: https://orcid.org/0000-0002-7873-3314, Langbein, Wolfgang ORCID: https://orcid.org/0000-0001-9786-1023, Olsen, Lars Folke, Isaacs, Marc and Lunding, Anita 2020. Functional imaging of a model unicell: Spironucleus vortens as an anaerobic but aerotolerant flagellated protist. Advances in Microbial Physiology 76 , pp. 41-79. 10.1016/bs.ampbs.2020.01.002

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Abstract

Advances in optical microscopy are continually narrowing the chasm in our appreciation of biological organization between the molecular and cellular levels, but many practical problems are still limiting. Observation is always limited by the rapid dynamics of ultrastructural modifications of intracellular components, and often by cell motility: imaging of the unicellular protist parasite of ornamental fish, Spironucleus vortens, has proved challenging. Autofluorescence of nicotinamide nucleotides and flavins in the 400–580 nm region of the visible spectrum, is the most useful indicator of cellular redox state and hence vitality. Fluorophores emitting in the red or near-infrared (i.e., phosphors) are less damaging and more penetrative than many routinely employed fluors. Mountants containing free radical scavengers minimize fluorophore photobleaching. Two-photon excitation provides a small focal spot, increased penetration, minimizes photon scattering and enables extended observations. Use of quantum dots clarifies the competition between endosomal uptake and exosomal extrusion. Rapid motility (161 μm/s) of the organism makes high resolution of ultrastructure difficult even at high scan speeds. Use of voltage-sensitive dyes determining transmembrane potentials of plasma membrane and hydrogenosomes (modified mitochondria) is also hindered by intracellular motion and controlled anesthesia perturbs membrane organization. Specificity of luminophore binding is always questionable; e.g. cationic lipophilic species widely used to measure membrane potentials also enter membrane-bounded neutral lipid droplet-filled organelles. This appears to be the case in S. vortens, where Coherent Anti-Stokes Raman Scattering (CARS) micro-spectroscopy unequivocally images the latter and simultaneous provides spectral identification at 2840 cm−1. Secondary Harmonic Generation highlights the highly ordered structure of the flagella.

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	Biosciences Chemistry Engineering Physics and Astronomy
Publisher:	Academic Press Inc.
ISSN:	0065-2911
Date of First Compliant Deposit:	4 March 2020
Date of Acceptance:	31 October 2019
Last Modified:	13 Nov 2024 02:45
URI:	https://orca.cardiff.ac.uk/id/eprint/130109

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