IR spectroscopy is an excellent method for biological analyses. It enables the nonperturbative, label-free extraction of biochemical information and images toward diagnosis and the assessment of cell functionality. Although not strictly microscopy in the conventional sense, it allows the construction of images of tissue or cell architecture by the passing of spectral data through a variety of computational algorithms. Because such images are constructed from fingerprint spectra, the notion is that they can be an objective reflection of the underlying health status of the analyzed sample. One of the major difficulties in the field has been determining a consensus on spectral pre-processing and data analysis. This manuscript brings together as coauthors some of the leaders in this field to allow the standardization of methods and procedures for adapting a multistage approach to a methodology that can be applied to a variety of cell biological questions or used within a clinical setting for disease screening or diagnosis. We describe a protocol for collecting IR spectra and images from biological samples (e.g., fixed cytology and tissue sections, live cells or biofluids) that assesses the instrumental options available, appropriate sample preparation, different sampling modes as well as important advances in spectral data acquisition. After acquisition, data processing consists of a sequence of steps including quality control, spectral pre-processing, feature extraction and classification of the supervised or unsupervised type. A typical experiment can be completed and analyzed within hours. Example results are presented on the use of IR spectra combined with multivariate data processing.

Vibrational spectroscopy can provide rapid, label-free, and objective analysis for the clinical domain. Spectroscopic analysis of biofluids such as blood components (e.g. serum and plasma) and others in the proximity of the diseased tissue or cell (e.g. bile, urine, and sputum) offers non-invasive diagnostic/monitoring possibilities for future healthcare that are capable of rapid diagnosis of diseases via specific spectral markers or signatures. Biofluids offer an ideal diagnostic medium due to their ease and low cost of collection and daily use in clinical biology. Due to the low risk and invasiveness of their collection they are widely welcomed by patients as a diagnostic medium. This review underscores recent research within the field of biofluid spectroscopy and its use in myriad pathologies such as cancer and infectious diseases. It highlights current progresses, advents, and pitfalls within the field and discusses future spectroscopic clinical potentials for diagnostics. The requirements and issues surrounding clinical translation are also considered.

We present an approach for estimating and correcting Mie scattering occurring in infrared spectra of single cells, at diffraction limited probe size, as in synchrotron based microscopy. The Mie scattering is modeled by extended multiplicative signal correction (EMSC) and subtracted from the vibrational absorption. Because the Mie scattering depends non-linearly on alpha, the product of the radius and the refractive index of the medium/sphere causing it, a new method was developed for estimating the Mie scattering by EMSC for unknown radius and refractive index of the Mie scatterer. The theoretically expected Mie contributions for a range of different alpha values were computed according to the formulae developed by Van de Hulst (1957). The many simulated spectra were then summarized by a six-dimensional subspace model by principal component analysis (PCA). This subspace model was used in EMSC to estimate and correct for Mie scattering, as well as other additive and multiplicative interference effects. The approach was applied to a set of Fourier transform infrared (FT-IR) absorbance spectra measured for individual lung cancer cells in order to remove unwanted interferences and to estimate ranges of important alpha values for each spectrum. The results indicate that several cell components may contribute to the Mie scattering.

A combination of Fourier-Transform (FT) resonance Raman spectroscopy and site-directed mutagenesis has been used to examine the function of two highly conserved aromatic residues, alpha-Tyr-44 and alpha-Tyr-45, in the light-harvesting 2 (LH2) complex of the photosynthetic bacterium Rhodobacter sphaeroides. In LH2 complexes, aromatic residues located at positions alpha-44 and alpha-45 are thought to be located near the putative binding site for bacteriochlorophyll, and alterations at these positions are known to produce blue shifts in bacteriochlorophyll absorbance. In the present work, mutant LH2 complexes carrying the alterations alpha-Tyr-44-->Phe, alpha-Tyr-45-->Phe and alpha-Tyr-44,-45-->Phe,Leu were examined. FT resonance Raman spectroscopy of the resulting complexes shows the breakage of a hydrogen bond to the 2-acetyl carbonyl group of one of the B850 bacteriochlorophylls in the LH2 complex; in the double mutant, breakage of a second bond is probable. These results suggest that one of these hydrogen bonds is to alpha-Tyr-44, placing this residue in close proximity to ring I of one of the B850 bacteriochlorophyll a pigments. The breakage of one, then two, 2-acetyl carbonyl hydrogen bonds correlates well with the shift in the absorbance of the B850 pigments of 11 nm then 26 nm at 77 K. Thus a consistency between literature theoretical calculations and the observations from both absorption and FT resonance Raman spectroscopy is demonstrated.

Fourier transform infrared and Raman microspectroscopy are currently being developed as new methods for the rapid identification of clinically relevant microorganisms. These methods involve measuring spectra from microcolonies which have been cultured for as little as 6 h, followed by the nonsubjective identification of microorganisms through the use of multivariate statistical analyses. To examine the biological heterogeneity of microorganism growth which is reflected in the spectra, measurements were acquired from various positions within (micro)colonies cultured for 6, 12, and 24 h. The studies reveal that there is little spectral variance in 6-h microcolonies. In contrast, the 12-and 24-h cultures exhibited a significant amount of heterogeneity. Hierarchical cluster analysis of the spectra from the various positions and depths reveals the presence of different layers in the colonies. Further analysis indicates that spectra acquired from the surface of the colonies exhibit higher levels of glycogen than do the deeper layers of the colony. Additionally, the spectra from the deeper layers present with higher RNA levels than the surface layers. Therefore, the 6-h colonies with their limited heterogeneity are more suitable for inclusion in a spectral database to be used for classification purposes. These results also demonstrate that vibrational spectroscopic techniques can be useful tools for studying the nature of colony development and biofilm formation.In recent years, there has been much effort invested into the development of new techniques for the identification of microorganisms. Many of these methods are aimed at providing the clinician with more rapid identification of the microorganism responsible for infection in order to begin the appropriate course of antimicrobial treatment (1,9,15,21,27,31,44,51). The emergence of these novel methods reflects the rise in drug-resistant microorganisms, which requires that antimicrobial treatment be more effectively managed (2, 12, 28, 52). Among the new methods are those based on vibrational spectroscopic techniques, namely Fourier transform infrared (FT-IR) and Raman spectroscopies. Vibrational spectroscopic methods are reagentless procedures in which there is no need to add dyes or labels for spectral measurement. These nondestructive techniques are based on the absorption (FT-IR) or scattering (Raman) of light directed onto a sample. The amount of light absorbed or scattered depends on the molecules found within the sample and the environment in which these molecules are found. With these highly sensitive techniques, the frequency of light in the resulting spectrum provides biochemical information regarding the molecular composition and molecular structure of and molecular interaction in cells and tissues (24,55). Raman and infrared spectroscopies are complementary techniques which together can provide a more complete impression of the biochemical information within a sample. Furthermore, these two methods differ such that each is capable of providing informatio...

Site-directed mutagenesis has been used to examine the function of a highly conserved aromatic residue, darp43, in the light-harvesting 1 antenna ofthe photosynthetic bacterium Rhodobacter sphaeroides. In this antenna dfrp43 is thought to be located near the putative binding site for bacteriochlorophyll; in this work it was changed to both Tyr and The photosynthetic apparatus of the purple bacterium Rhodobacter sphaeroides consists of a membrane protein complex, the reaction center (RC), surrounded by a core light-harvesting complex, LH1, also referred to as B875, in a fixed stoichiometry of -12 LH1 a(3 dimers per RC. An additional peripheral light-harvesting complex, LH2, also referred to as B800-850, occurs in variable amounts in response to environmental levels of light and oxygen (1, 2). The structure of the RC of Rb. sphaeroides is known to atomic resolution (3, 4); however, the structures of the LH1 and LH2 complexes are not known, although diffracting crystals ofvarious LH complexes have been reported (5-10). As yet, these structures have not been solved, so it is necessary to employ other methods such as mutagenesis and spectroscopy to obtain relevant data. As a result of these approaches, more information is becoming available on the interaction of the chromophores of LH1 and LH2 with nearby residues of the antenna polypeptides (11,12). These data can be used to test existing model structures derived from spectroscopic and electrophoretic mobility studies of wild-type (WT) complexes. The models that have been proposed for bacterial LH complexes all follow a similar pattern in that the af8 heterodimer, which in the case of LH1 would bind 2 bacteriochlorophyll (bchl) molecules, is considered to be the minimum possible building block. The topology and conformation of these subunits are thought to be similar, each with a cytoplasmically exposed N terminus, a single transmembrane helix, and a periplasmic C terminus. There are indications of turns at or near the membrane interfaces (13 (20), and Rhodocyclus gelatinosus (21), leading to the speculation that this bchl dimer is the building block of the LH1 complex. Characterization of the B820 form (22) supports the idea that it is an excitonically coupled bchl a dimer. Moreover, resonance Raman measurements of the B820 showed that it differed from the B873 form in the interactions of the C2 acetyl carbonyl; the loss of a H bond was one possible explanation of the Raman data. A more detailed Raman study of the LH1 ofRs. rubrum G9 has shown that upon formation of the B820, the strength of one H bond was increased (23 FT, Fourier transform; WT, wild type; P-OG, n-octyl P-Dglucopyranoside; ODPS, n-octyldipropyl sulfoxide. 7124The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Rapid and accurate identification of enterococci at the species level is an essential task in clinical microbiology since these organisms have emerged as one of the leading causes of nosocomial infections worldwide. Vibrational spectroscopic techniques (infrared [IR] and Raman) could provide potential alternatives to conventional typing methods, because they are fast, easy to perform, and economical. We present a comparative study using phenotypic, genotypic, and vibrational spectroscopic techniques for typing a collection of 18 Enterococcus strains comprising six different species. Classification of the bacteria by Fourier transform (FT)-IR spectroscopy in combination with hierarchical cluster analysis revealed discrepancies for certain strains when compared with results obtained from automated phenotypic systems, such as API and MicroScan. Further diagnostic evaluation using genotypic methods-i.e., PCR of the species-specific ligase and glycopeptide resistance genes, which is limited to the identification of only four Enterococcus species and 16S RNA sequencing, the "gold standard" for identification of enterococci-confirmed the results obtained by the FT-IR classification. These results were later reproduced by three different laboratories, using confocal Raman microspectroscopy, FT-IR attenuated total reflectance spectroscopy, and FT-IR microspectroscopy, demonstrating the discriminative capacity and the reproducibility of the technique. It is concluded that vibrational spectroscopic techniques have great potential as routine methods in clinical microbiology.

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