TY - JOUR
T1 - A dual compartment cuvette system for correcting scattering in whole-cell absorbance spectroscopy of photosynthetic microorganisms
AU - Hervey, John R. D.
AU - Bombelli, Paolo
AU - Lea-Smith, David J.
AU - Hulme, Alan K.
AU - Hulme, Nathan R.
AU - Rullay, Atvinder K.
AU - Keighley, Robert
AU - Howe, Christopher J.
N1 - 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.
PY - 2022/1
Y1 - 2022/1
N2 - 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.
AB - 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.
KW - Absorption spectroscopy
KW - Chromophores
KW - Cyanobacteria
KW - Light scattering
KW - Microalgae
KW - Whole cell spectra
UR - http://www.scopus.com/inward/record.url?scp=85112573102&partnerID=8YFLogxK
U2 - 10.1007/s11120-021-00866-8
DO - 10.1007/s11120-021-00866-8
M3 - Article
VL - 151
SP - 61
EP - 69
JO - Photosynthesis Research
JF - Photosynthesis Research
SN - 0166-8595
ER -