A dual compartment cuvette system for correcting scattering in whole-cell absorbance spectroscopy of photosynthetic microorganisms

Hervey, John R. D., Bombelli, Paolo, Lea-Smith, David J., Hulme, Alan K., Hulme, Nathan R., Rullay, Atvinder K., Keighley, Robert and Howe, Christopher J. (2022) A dual compartment cuvette system for correcting scattering in whole-cell absorbance spectroscopy of photosynthetic microorganisms. Photosynthesis Research, 151. pp. 61-69. ISSN 0166-8595

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Abstract

Absorption spectroscopy is widely used to determine absorption and transmission spectra of chromophores in solution, in addition to suspensions of particles, including micro-organisms. Light scattering, caused by photons deflected from part or all of the cells or other particles in suspension, results in distortions to the absorption spectra, lost information and poor resolution. A spectrophotometer with an integrating sphere may be used to alleviate this problem. However, these instruments are not universally available in biology laboratories, for reasons such as cost. Here, we describe a novel, rapid, and inexpensive technique that minimises the effect of light scattering when performing whole-cell spectroscopy. This method involves using a custom made dual compartment cuvette containing titanium dioxide in one chamber as a scattering agent. Measurements were conducted of a range of different photosynthetic micro-organisms of varying cell size and morphology, including cyanobacteria, eukaryotic microalgae and a purple non-sulphur bacterium. A concentration of 1 mg ml−1 titanium dioxide, using a spectrophotometer with a slit width of 5 nm, produced spectra for cyanobacteria and microalgae similar (1–4% difference) to those obtained using an integrating sphere. The spectrum > 520 nm was similar to that with an integrating sphere with the purple non-sulphur bacterium. This system produced superior results to those obtained using a recently reported method, the application of the diffusing agent, Scotch™ Magic tape, to the side of the cuvette. The protocol can be completed in an equivalent period of time to standard whole-cell absorbance spectroscopy techniques, and is, in principle, suitable for any dual-beam spectrophotometer.

Item Type: Article
Additional Information: Funding Information: John Hervey and David Lea-Smith were funded by the Waste Environmental Education Research Trust. Paolo Bombelli was funded by the Leverhulme Trust (RPG-2015-393) and The Italian Ministry of University and Research (MIUR), within the SIR2014 Grant, project RBSI14JKU3.
Uncontrolled Keywords: absorption spectroscopy,chromophores,cyanobacteria,light scattering,microalgae,whole cell spectra,biochemistry,plant science,cell biology ,/dk/atira/pure/subjectarea/asjc/1300/1303
Faculty \ School: Faculty of Science > School of Biological Sciences
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Depositing User: LivePure Connector
Date Deposited: 19 Aug 2021 00:30
Last Modified: 28 Jan 2022 10:33
URI: https://ueaeprints.uea.ac.uk/id/eprint/81148
DOI: 10.1007/s11120-021-00866-8

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