Research project: Use of biospectroscopic imaging and fluorescence to investigate the macromolecular composition and physiological state of algal cells

The responses of higher plants and algae to stress induced by environmental change, such as variations in the availability of nutrients, is aimed at the maintenance of the status quo. Organisms react to change by redistributing resources so that the reproductive and growth potentials are affected as little as possible. This often results in a massive re-organisation of cellular components.

The study of the changes in the pools of macromolecules in cells and their quantification, especially in relation to each other, is therefore essential for understanding the response of organisms to alterations in environmental conditions. Unfortunately, most of the methods used for the assessment of the size of cellular pools of macromolecules and of their variations are invasive and require the disruption of the cell itself. This of course introduces both large experimental errors and major perturbations of the system. Also, work of this kind is technically challenging and time-consuming, and often requires large numbers of cells, which are not always available in microalgal (phytoplankton) populations.

When FT-IR spectroscopy is applied to intact microalgae, the resulting spectra reflect the total biochemical composition of the cells. Furthermore, FT-IR spectra can be used to determine the relative concentration of macromolecules such as proteins, lipids and carbohydrates in the cells.We have experimentally proved, by comparing chemical measurements with then relative intensity of spectral bands attributed to protein, silica, and carbohydrates, that FT-IR is a reliable way to accurately measure the relative concentration of these classes of compounds in the microalga Chaetoceros muelleri. This spectroscopic method has the advantage over conventional methods in that it minimises the disturbance of the intracellular environment by reducing the manipulation of the sample and, consequently, the introduction of experimental artefacts.

FT-IR spectroscopy appears to be a powerful new analytical approach to a number of problems in microbiology and medicine. It has been used to differentiate between eubacteria and archaebacteria or between cyanobacterial strains and has also been applied to the detection of pre-cancerous changes in isolated human cervical cells . FT-IR spectroscopy combined with microscopy permits examination of the larger individual cells (>20 mm) in a population, obviating interference from contamination by bacteria and other organisms. Examining large numbers of cells in a sample provides information on the population heterogeneity in macromolecular composition.Recent work in our laboratory and elsewhere has shown that when N- or P-limited cells are re-supplied with the limiting nutrient, they exhibit marked transient changes in chlorophyll fluorescence output.

These  nutrient-induced fluorescence transients (NIFTs) occur only when cells are exposed to the specific limiting nutrient and are dependent both on the degree of limitation and the amount of limiting nutrient added. As the re-supplied nutrient is used up from the medium, the chlorophyll fluorescence signal returns to the original value.Irrespective of the nature of the fluorescence change, the response is strongly dependent on the type and concentration of the nutrient added. Unpublished data from our laboratory suggest that the NIFT responses are found in a range of microalgae species from a variety of habitats. This has suggested to us that the technique might have application as a tool for the rapid determination of nutrient status of natural populations of phytoplankton.

New advances in fluorescence technology combined with microscopy allow the imaging of fluorescence of individual cells. In combination with the observed NIFT responses in nutrient limited populations, it should prove feasible to determine the extent of NIFT responses in individual cells and thus explore the heterogeneity of nutrient status in mixed species assemblages of phytoplankton.The techniques of fluorescence combined with FTIR will be of immense benefit to workers in the field of phytoplankton ecophysiology and other areas where there is usually insufficient plant material for standard chemical analysis.
Current areas of investigation include:

Use of FTIR spectroscopy to discriminate toxic and non-toxic cyanobacteria and dinoflagellates
Using Raman and FTIR spectroscopy to determine intra-population variation in macromolecular composition of microalgal cells
Imaging of algal cells using synchrotron FTIR
Studies on the distribution of NIFT responses in different microalgal species
Investigations of the mechanisms underlying the NIFT response in algal cells

For further information see:

  • Beardall, J., Berman, T., Heraud, P., Kadiri, M., Light, B., Patterson, G., Roberts, S., Sahan E., Schulzberger B., Urlinger, U, and Wood, B. (2001) A comparison of methods for detection of nutrient limitation in microalgae. Aquatic Science 63 (1), 107-121.
  • Kansiz, M., Heraud, P., Wood, B., Burden, F., Beardall, J. and McNaughton, D. (1999) FTIR spectroscopy as a tool to discriminate between cyanobacterial strains.Phytochemistry, 52: 407-17.
  • Heraud P.,Wood B., Beardall J. and McNaughton D. (1999) Physiological Changes in microalgae monitored using FTIR Spectroscopy. In:Spectroscopy of Biological Molecules: New Directions, Ed. J. Greve, G.J. Puppels and C. Otto eds, Kluwer Academic Publishers, pp. 449-450.
  • Giordano, M., Kansiz M., Heraud P., Beardall J., Wood B. and McNaughton D. (2001). FT-IR spectroscopy as a novel tool to investigate changes in intracellular macromolecular pools in the marine microalga Chaetoceros muellerii. Journal of Phycology, 37 (2):271-279
  • Holland D., Roberts S. and Beardall J. (2004) Assessment of the nutrient status of phytoplankton: a comparison between conventional bioassays and Nutrient-Induced Fluorescence Transients (NIFTS). Ecological Indicators 4(3): 149-159
  • McNaughton D., Romeo M., Kansiz M., Wood B., Heraud P.,.Burden F and Beardall J. (1999). Infrared Spectroscopy and Multivariate Statistics applied to Medical and biological problems. In: Spectroscopy of Biological Molecules: New Directions, Ed.
  • J. Greve, G.J. Puppels and C. Otto, Kluwer Academic Publishers, pp 475-478.
    Young EB and Beardall J (2003) Rapid ammonium- and nitrate-induced perturbations to chlorophyll a fluorescence in nitrogen-stressed Dunaliella tertiolecta (Chlorophyta). Journal of Phycology 39: 332-342.
  • Wood BR, Heraud P, Stojkovic S, Morrison D, Beardall J and McNaughton D. (2005) A portable Raman acoustic levitation spectroscopic system for the identification and environmental monitoring of algal cells. Analytical Chemistry. 77 (15): 4955-4961.
  • Heraud P, Wood BR, Tobin M, Beardall J and McNaughton, D. (2005) Mapping of nutrient-induced biochemical changes in living algal cells using synchrotron infrared microspectroscopy FEMS Microbiology Letters. 249 : 219–225