Search

Algae are the main primary producers in aquatic environments and therefore of fundamental importance for the global ecosystem. Mid-infrared (IR) microspectroscopy is a non-invasive tool that allows in principle studying chemical composition on a single-cell level. For a long time, however, mid-infrared (IR) imaging of living algal cells in an aqueous environment has been a challenge due to the strong IR absorption of water. In this study, we employed multi-beam synchrotron radiation to measure time-resolved IR hyperspectral images of individual Thalassiosira weissflogii cells in water in the course of acclimation to an abrupt change of CO2 availability (from 390 to 5000 ppm and vice versa) over 75 min. We used a previously developed algorithm to correct sinusoidal interference fringes from IR hyperspectral imaging data. After preprocessing and fringe correction of the hyperspectral data, principal component analysis (PCA) was performed to assess the spatial distribution of organic pools within the algal cells. Through the analysis of 200,000 spectra, we were able to identify compositional modifications associated with CO2 treatment. PCA revealed changes in the carbohydrate pool (1200–950 cm$$^{-1}$$ - 1 ), lipids (1740, 2852, 2922 cm$$^{-1}$$ - 1 ), and nucleic acid (1160 and 1201 cm$$^{-1}$$ - 1 ) as the major response of exposure to elevated CO2 concentrations. Our results show a local metabolism response to this external perturbation.

Short-term acclimation response of individual cells of Thalassiosira weissflogii was monitored by Synchrotron FTIR imaging over the span of 75 minutes. The cells, collected from batch cultures, were maintained in a constant flow of medium, at an irradiance of 120 μmol m−2 s−1 and at 20 °C. Multiple internal reflections due to the micro fluidic channel were modeled, and showed that fringes are additive sinusoids to the pure absorption of the other components of the system. Preprocessing of the hyperspectral cube (x, y, Abs(λ)) included removing spectral fringe using an EMSC approach. Principal component analysis of the time series of hyperspectral cubes showed macromolecular pool variations (carbohydrates, lipids and DNA/RNA) of less than 2% after fringe correction.

A new mid-infrared beamline (IRENI) extracting 320 hor. × 25 vert. mrads2 of radiation providing wide-field illumination for a Bruker Hyperion 3000 IR microscope equipped with an infrared focal plane array (FPA) detector has recently been commissioned at the Synchrotron Radiation Center (SRC) in Stoughton, WI. The swath of radiation from the SRC is extracted as 12 beams and recombined into a 3 × 4 bundle of beams that is refocused onto the sample plane of the infrared microscope illuminating a 40 μm × 60 μm sample area. With this new facility chemical images with high diffraction-limited resolution, for wavelengths across the mid-IR concurrently, can be obtained in minutes. Applications of biomedical, biological and cultural heritage interest show the broad applicability of this new facility. The examples presented here include images of fixed single cells, time dependent in vivo measurements of a single cell and layered organic/inorganic materials.

Extended abstract of a paper presented at Microscopy and Microanalysis 2010 in Portland, Oregon, USA, August 1 – August 5, 2010.