Don McNaughton

FOURIER TRANSFORM INFRARED SPECTROSCOPY AS A NOVEL TOOL TO INVESTIGATE CHANGES IN INTRACELLULAR MACROMOLECULAR POOLS IN THE MARINE MICROALGA CHAETOCEROS MUELLERII (BACILLARIOPHYCEAE)

Fourier Transform Infrared (FT‐IR) spectroscopy was used to study carbon allocation patterns in response to changes in nitrogen availability in the diatom Chaetoceros muellerii Lemmerman. The results of the FT‐IR measurements were compared with those obtained with traditional chemical methods. The data obtained with both FT‐IR and chemical methods showed that nitrogen starvation led to the disappearance of the differences in cell constituents and growth rates existing between cells cultured at either high [NO3−] or high [NH4+]. Irrespective of the nitrogen source supplied before nitrogen starvation, a diversion of carbon away from protein, chlorophyll, and carbohydrates into lipids was observed. Under these conditions, cells that had previously received nitrogen as nitrate appeared to allocate a larger amount of mobilized carbon into lipids than cells that had been cultured in the presence of ammonia. All these changes were reversed by resupplying the cultures with nitrogen. The rate of protein accumulation in the N‐replete cells was slower than the rate of decrease under nitrogen starvation. This study demonstrates that the relative proportions of the major macromolecules contained in microalgal cells and their changes in response to external stimuli can be determined rapidly, simultaneously, and inexpensively using FT‐IR. The technique proved to be equally reliable to and less labor intensive than more traditional chemical methods.

Fourier Transform Infrared microspectroscopy and chemometrics as a tool for the discrimination of cyanobacterial strains

Abstract Fourier Transform Infrared (FTIR) microspectroscopy, in combination with chemometrics, was investigated as a novel method to discriminate between cyanobacterial strains. In total, 810 absorbance spectra were recorded from one eukaryotic and five cyanobacterial taxa spanning three genera and including two strains of one species, Microcystis aeruginosa . Principal Component Analysis (PCA) based classification techniques such as Soft Independent Modelling of Class Analogy (SIMCA) and K-Nearest Neighbours (KNN) were investigated. Different spectral regions using derivative spectra were investigated to find the best combinations for classification. The highest rate of correct classifications (99–100%) was achieved using first derivative spectra with a spectral region of 1800–950 cm −1 for both the SIMCA and KNN. A dendrogram constructed using averaged spectra of the six taxa studied showed that the two strains of Microcystis aeruginosa exhibited the highest degree of similarity, while the eukaryotic taxon was the most dissimilar from the prokaryotic taxa.

FTIR microspectroscopic study of cell types and potential confounding variables in screening for cervical malignancies.

FTIR microscopy was applied to the analysis of cell types and other variables present in Pap smears to ascertain the limitations of infrared spectroscopy in the diagnosis of cervical cancer and dysplasia. It was found that leukocytes, and in particular lymphocytes, have spectral features in the phosphodiester region (1300-900 cm[-1]) suggestive of what has previously been described as changes indicative of malignancy. Endocervical cells and fibroblasts have similar spectral features to HeLa cells and consequently could also confound diagnosis. The use of ethanol as a fixative and dehydrating agent results in retention of glycogen in cervical cell types and thus minimizes spectral changes in the glycogen region due to sampling technique. Spectra of seminal fluids exhibit strong bands in the phosphodiester/carbohydrate region; however, sperm contamination should be easily detectable by the presence of a distinctive doublet at 981/968 cm(-1). Erythrocyte spectra exhibit a reduction in glycogen band intensity, but can be discerned by a relatively low-intensity nu(s) PO2- band. Endocervical mucin spectra exhibit a reduction in glycogen bands and a very pronounced nu(s) PO2- band, which is similar in intensity to the corresponding band in HeLa cells. Thrombocytes have strong bands in the phosphodiester region, but thrombocytes can be discerned from other cell types by the presence of two small broad bands at 980 and 935 cm(-1). Candida albicans is characterized by strong bands in the polysaccharide region which could potentially obscure diagnostic bands if C. albicans is present in large numbers. Spectra of bacteria common to the female genital tract, in general, also have strong absorptions in the polysaccharide region; however, bacterial contamination is usually minimal and would not be expected to obscure cervical cell spectra. Nylon threads and bristles from cervical sampling implements produce characteristic IR profiles which allow for easy identification. Given the number of potential confounding variables associated with cervical cytology, a multivariate statistical or neural network analysis would appear to be necessary before the implementation of FTIR technology in clinical laboratories.

Monitoring the reversible B to A-like transition of DNA in eukaryotic cells using Fourier transform infrared spectroscopy

The ability to detect DNA conformation in eukaryotic cells is of paramount importance in understanding how some cells retain functionality in response to environmental stress. It is anticipated that the B to A transition might play a role in resistance to DNA damage such as heat, desiccation and toxic damage. To this end, conformational detail about the molecular structure of DNA has been derived primarily from in vitro experiments on extracted or synthetic DNA. Here, we report that a B- to A-like DNA conformational change can occur in the nuclei of intact cells in response to dehydration. This transition is reversible upon rehydration in air-dried cells. By systematically monitoring the dehydration and rehydration of single and double-stranded DNA, RNA, extracted nuclei and three types of eukaryotic cells including chicken erythrocytes, mammalian lymphocytes and cancerous rodent fibroblasts using Fourier transform infrared (FTIR) spectroscopy, we unequivocally assign the important DNA conformation marker bands within these cells. We also demonstrate that by applying FTIR spectroscopy to hydrated samples, the DNA bands become sharper and more intense. This is anticipated to provide a methodology enabling differentiation of cancerous from non-cancerous cells based on the increased DNA content inherent to dysplastic and neoplastic tissue.

Haemozoin (β-haematin) biomineralization occurs by self-assembly near the lipid/water interface

Several blood‐feeding organisms, including the malaria parasite detoxify haem released from host haemoglobin by conversion to the insoluble crystalline ferriprotoporphyrin IX dimer known as haemozoin. To date the mechanism of haemozoin formation has remained unknown, although lipids or proteins have been suggested to catalyse its formation. We have found that β‐haematin (synthetic haemozoin) forms rapidly under physiologically realistic conditions near octanol/water, pentanol/water and lipid/water interfaces. Molecular dynamics simulations show that a precursor of the haemozoin dimer forms spontaneously in the absence of the competing hydrogen bonds of water, demonstrating that this substance probably self‐assembles near a lipid/water interface in vivo.

Tip-Enhanced Raman Scattering (TERS) from Hemozoin Crystals within a Sectioned Erythrocyte

Tip-enhanced Raman scattering (TERS) is a powerful technique to obtain molecular information on a nanometer scale, however, the technique has been limited to cell surfaces, viruses, and isolated molecules. Here we show that TERS can be used to probe hemozoin crystals at less than 20 nm spatial resolution in the digestive vacuole of a sectioned malaria parasite-infected cell. The TERS spectra clearly show characteristic bands of hemozoin that can be correlated to a precise position on the crystal by comparison with the corresponding atomic force microscopy (AFM) image. These are the first recorded AFM images of hemozoin crystals inside malaria-infected cells and clearly show the hemozoin crystals protruding from the embedding medium. TERS spectra recorded of these crystals show spectral features consistent with a five-coordinate high-spin ferric heme complex, which include the electron density marker band ν(4) at 1373 cm(-1) and other porphyrin skeletal and ring breathing modes at approximately 1636, 1557, 1412, 1314, 1123, and 1066 cm(-1). These results demonstrate the potential of the AFM/TERS technique to obtain nanoscale molecular information within a sectioned single cell. We foresee this approach paving the way to a new independent drug screening modality for detection of drugs binding to the hemozoin surface within the digestive vacuole of the malaria trophozoite.

Quantitative Determination of the Biodegradable Polymer Poly(β-hydroxybutyrate) in a Recombinant Escherichia coli Strain by Use of Mid-Infrared Spectroscopy and Multivariative Statistics

ABSTRACT Fourier transform infrared (FTIR) spectroscopy in combination with the partial least squares (PLS) multivariative statistical technique was used for quantitative analysis of the poly(β-hydroxybutyrate) (PHB) contents of bacterial cells. A total of 237 replicate spectra from 34 samples were obtained together with gas chromatography-determined reference PHB contents. Using the PLS regression, we were able to relate the infrared spectra to the reference PHB contents, and the correlation coefficient between the measured and predicted values for the optimal model with a standard error of prediction of 1.49% PHB was 0.988. With this technique, there are no solvent requirements, sample preparation is minimal and simple, and analysis time is greatly reduced; our results demonstrate the potential of FTIR spectroscopy as an alternative to the conventional methods used for analysis of PHB in bacterial cells.

Raman microspectroscopy and imaging provides insights into heme aggregation and denaturation within human erythrocytes.

The oxygenation process of a human erythrocyte is monitored using a Raman microimaging technique. Raman images of the 1638 cm(-1) band are recorded in the oxygenated and deoxygenated state using only 120 s of laser exposure and approximately 1 mW of defocused laser power. The images show hemoglobin oxygenating and deoxygenating within the cell. Prolonged laser imaging exposure (<180 s) at low temperatures results in photoinduced and/or thermal degradation. The effect of thermal degradation is investigated by recording spectra of erythrocytes as a function of temperature between 4 and 52 degrees C. Five bands at 1396, 1365, 1248, 972, and 662 cm(-1) are identified as markers for heme aggregation. Raman images recorded of cells after prolonged laser exposure appear to show heme aggregation commencing in the middle and moving toward the periphery of the cell. UV-visible spectra of erythrocytes show the Soret band to be broader and red shifted (approximately 3 nm) at temperatures between 45 and 55 degrees indicative of excitonic interactions. It is postulated that the enhancement of the aggregation marker bands observed at 632.8-nm excitation results primarily from excitonic interactions between the aggregated hemes in response to protein denaturation. The results have important medical implications in detecting and monitoring heme aggregation associated with hemopathies such as sickle cell disease.

Raman imaging of hemozoin within the food vacuole of Plasmodium falciparum trophozoites

Micro‐Raman spectra of hemozoin encapsulated within the food vacuole of a Plasmodium falciparum‐infected erythrocyte are presented. The spectrum of hemozoin is identical to the spectrum of β‐hematin at all applied excitation wavelengths. The unexpected observation of dramatic band enhancement of A1g modes including ν4 (1374 cm−1) observed when applying 780 nm excitation enabled Raman imaging of hemozoin in the food vacuole. This unusual enhancement, resulting from excitonic coupling between linked porphyrin moieties in the extended porphyrin array, enables the investigation of hemozoin within its natural environment for the first time.

Shedding New Light on the Molecular Architecture of Oocytes Using a Combination of Synchrotron Fourier Transform-Infrared and Raman Spectroscopic Mapping

Synchrotron Fourier transform-infrared (FT-IR) and Raman microspectroscopy were applied to investigate changes in the molecular architecture of mouse oocytes and demonstrate the overall morphology of the maturing oocyte. Here we show that differences were identified between immature mouse oocytes at the germinal vesicle (GV) and mature metaphase II (MII) stage when using this technology, without the introduction of any extrinsic markers, labels, or dyes. GV mouse oocytes were found to have a small, centrally located lipid deposit and another larger polar deposit of similar composition. MII oocytes have very large, centrally located lipid deposits. Each lipid deposit for both cell types contains an inner and outer lipid environment that differs in composition. To assess interoocyte variability, line scans were recorded across the diameter of the oocytes and compared from three independent trials (GV, n = 91; MII, n = 172), and the data were analyzed with principal component analysis (PCA). The average spectra and PCA loading plots show distinct and reproducible changes in the CH stretching region that can be used as molecular maturation markers. The method paves the way for developing an independent assay to assess oocyte status during maturation providing new insights into lipid distribution at the single cell level.