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

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.

High-Dynamic-Range Absorption Spectroscopy by Generating a Wide Path-Length Distribution with Scatterers

“…This study features absorbance measurements utilizing scatterers. As demonstrated in previous studies, many of these approaches aim to alleviate the problems of light scattering and replicate the effects observed in an integrating sphere [34][35][36], as well as to enhance the sensitivity of absorption spectroscopy. In particular, the latter is achieved through a method known as multiscattering-enhanced absorption spectroscopy (MEAS), which involves dispersing gold nanoparticles in the sample solution [37] or surrounding the sample with a scattering cavity [38] to increase the PL and thereby enhance detection sensitivity.…”