Antonella I Mazur’s research while affiliated with Northeastern University and other places
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Instrumental advances in infrared micro-spectroscopy have made possible the observation of individual human cells and even subcellular structures. The observed spectra represent a snapshot of the biochemical composition of a cell; this composition varies subtly but reproducibly with cellular effects such as progression through the cell cycle, cell maturation and differentiation, and disease. The aim of this summary is to provide a synopsis of the progress achieved in infrared spectral cytopathology (SCP) - the combination of infrared micro-spectroscopy and multivariate methods of analysis - for the detection of abnormalities in exfoliated human cells of the upper respiratory and digestive tract, namely the oral and nasopharyngeal cavities, and the esophagus.
Instrumental advances in vibrational microspectroscopy have made possible the observation of individual human cells and even subcellular structures. The observed spectra represent a snapshot of the biochemical composition of a cell; this composition varies subtly but reproducibly with cellular effects such as progression through the cell cycle, cell maturation and differentiation, and disease.The aim of this chapter is to summarize the progress achieved since the last edition of this book in using spectral cytopathology (SCP) – the combination of infrared (IR) microspectroscopy and multivariate methods of analysis – for the detection of abnormalities in exfoliated human cells. This work sets the stage for biomedical and diagnostic applications of this technology.
During the last 15years, vibrational spectroscopic methods have been developed, which can be viewed as molecular pathology methods that depend on sampling the entire genome, proteome, and metabolome of cells and tissues, rather than probing for the presence of selected markers. First, this review introduces the background and fundamentals of the spectroscopies underlying the new methodologies, namely infrared and Raman spectroscopy. Then, results are presented in the context of spectral histopathology of tissues for the detection of metastases in lymph nodes, squamous cell carcinoma, adenocarcinomas, brain tumors, and brain metastases. Results from spectral cytopathology of cells are discussed for screening of oral and cervical mucosa and circulating tumor cells. It is concluded that infrared and Raman spectroscopy can complement histopathology and reveal information that is available in classical methods only by costly and time-consuming steps such as immunohistochemistry, polymerase chain reaction, or gene arrays. Because of the inherent sensitivity toward changes in the biomolecular composition of different cell and tissue types, vibrational spectroscopy can even provide information that is in some cases superior to that obtained by any one of the conventional techniques.
During the last 15years, vibrational spectroscopic methods have been developed, which can be viewed as molecular pathology methods that depend on sampling the entire genome, proteome, and metabolome of cells and tissues, rather than probing for the presence of selected markers. First, this review introduces the background and fundamentals of the spectroscopies underlying the new methodologies, namely infrared and Raman spectroscopy. Then, results are presented in the context of spectral histopathology of tissues for the detection of metastases in lymph nodes, squamous cell carcinoma, adenocarcinomas, brain tumors, and brain metastases. Results from spectral cytopathology of cells are discussed for screening of oral and cervical mucosa and circulating tumor cells. It is concluded that infrared and Raman spectroscopy can complement histopathology and reveal information that is available in classical methods only by costly and time-consuming steps such as immunohistochemistry, polymerase chain reaction, or gene arrays. Because of the inherent sensitivity toward changes in the biomolecular composition of different cell and tissue types, vibrational spectroscopy can even provide information that is in some cases superior to that obtained by any one of the conventional techniques.
This paper presents a short review on the improvements in data processing for spectral cytopathology, the diagnostic method developed for large scale diagnostic analysis of spectral data of individual dried and fixed cells. This review is followed by the analysis of the confounding effects introduced by utilizing reflecting "low-emissivity" (low-e) slides as sample substrates in infrared micro-spectroscopy of biological samples such as individual dried cells or tissue sections. The artifact introduced by these substrates, referred to as the "standing electromagnetic wave" artifact, indeed, distorts the spectra noticeably, as postulated recently by several research groups. An analysis of the standing wave effect reveals that careful data pre-processing can reduce the spurious effects to a level where they are not creating a major problem for spectral cytopathology and spectral histopathology.
Spectral cytopathology (SCP) is a robust and reproducible diagnostic technique that employs infrared spectroscopy and multivariate statistical methods, such as principal component analysis to interrogate unstained cellular samples and discriminate changes on the biochemical level. In the past decade, SCP has taken considerable strides in its application for disease diagnosis. Cultured cell lines have proven to be useful model systems to provide detailed biological information to this field; however, the effects of sample fixation and storage of cultured cells are still not entirely understood in SCP. Conventional cytopathology utilizes fixation and staining methods that have been established and widely accepted for nearly a century and are focused on maintaining the morphology of a cell. Conversely, SCP practices must implement fixation protocols that preserve the sample's biochemical composition and maintain its spectral integrity so not to introduce spectral changes that may mask variance significant to disease. It is not only necessary to evaluate the effects on fixed exfoliated cells but also fixed cultured cells because although they are similar systems, they exhibit distinct differences. We report efforts to study the effects of fixation methodologies commonly used in traditional cytopathology and SCP including both fixed and unfixed routines applied to cultured HeLa cells, an adherent cervical cancer cell line. Data suggest parallel results to findings in Part 1 of this series for exfoliated cells, where the exposure time in fixative and duration of sample storage via desiccation contribute to minor spectral changes only. The results presented here reinforce observations from Part 1 indicating that changes induced by disease are much greater than changes observed as a result of alternate fixation methodologies. Principal component analysis of HeLa cells fixed via the same conditions and protocols as exfoliated cells (Part 1) yield nearly identical results. More importantly, the overall conclusion is that it is necessary that all samples subjected to comparative analysis should be prepared identically because although changes are minute, they are present. F or the past decade, infrared (IR) microspectroscopy has climbed its way to being considered a competitive alternative to conventional cytopathology practices. Traditional cytopathology includes the inspection of stained cells, visually measuring predetermined parameters, such as nucleus-to-cytoplasm (N/C) ratio, staining patterns, morphology of nuclear membrane, etc., and assigning a diagnosis based on these parameters. 1,2 IR microspectroscopy is at the forefront of new methods being developed because it is a label free and reproducible method that evaluates a physical measurement, the biochemical composition, of each unstained cell; the term " spectral cytopathology (SCP) " has been coined to describe the combination of microscopic infrared data acquisition and analysis of the spectral data via multivariate methods. 3−5 After IR acquisition, samples can then be subjected to traditional staining protocols and evaluated via conventional cytopathol-ogy means to compare results from both techniques. Since the early successes of SCP, many groups have begun investigating cultured cell lines to provide additional information regarding disease diagnosis and biological information. 6−8 Cultured cells serve several purposes ranging from distinguishing between different cell lines to their behavior in understanding disease and evaluation of drug effects and uptake. 8−10 Cultured cell lines offer a microscopic model system to explore and probe mechanisms, pathways, drug interactions, etc. Most importantly, diseased cells can be potentially biopsied from a patient's organ and propagated in cell culture conditions to be investigated thoroughly. 8,11 Often fixation procedures are applied to preserve cells for extended periods; however, the spectral effects of fixation on cultured cells are not entirely understood. 12 Previous reports claim fixation protocols introduce large spectral changes and obstruct proper analyses, speculating fixation methods as obstacles to be avoided. 13−15 This is the second paper in a series aimed at addressing the effects of fixation and storage conditions on spectral data of cellular samples. In the first paper, we described the influence of these factors to exfoliated oral (buccal) mucosa cells and demonstrated that exceedingly small variances occurred upon various fixation methods that were negligible in comparison to
This paper summarizes the progress achieved over the past fifteen years in applying vibrational (Raman and IR) spectroscopy to problems of medical diagnostics and cellular biology. During this time, a number of research groups have verified the enormous information content of vibrational spectra; in fact, genomic, proteomic, and metabolomic information can be deduced by decoding the observed vibrational spectra. This decoding process is aided enormously by the availability of high-power computer workstations and advanced algorithms for data analysis. Furthermore, commercial instrumentation for the fast collection of both Raman and infrared microspectral data has rendered practical the collection of images based solely on spectral data. The progress in the field has been manifested by a steady increase in the number and quality of publications submitted by established and new research groups in vibrational biological and biomedical arenas.
We have optimized an imaging methodology capable of monitoring individual live HeLa cells using non-synchrotron FTIR in an aqueous environment. This methodology, in combination with MATLAB based pre-processing techniques, allows fast and efficient collection of data with high signal-to-noise ratio in comparison with previous methods using point mode data collection, which required manual operation and more collection time. Also, presented are early results that illustrate interpretable spectral differences from live cells treated with chemotherapeutic drugs, demonstrating the potential of this methodology to develop more desirable modes of treatment for patients in their diagnoses and treatments for disease.
Citations (11)
... Da pathologische Anomalien mit Veränderungen in der biochemischen Zusammensetzung und Struktur von Biomolekülen einhergehen, liefert das Raman-Spektrum von Gewebe einen empfindlichen und spezifischen Fingerabdruck der Art und des Zustands von diesem. Dies macht die Raman-Spektroskopie zu einem perfekten Werkzeug für eine markierungsfreie histopathologische Untersuchung von Gewebe [8]. Die Vorteile der Raman-Spektroskopie sind ihre beispiellos hohe molekulare Spezifität und ihre Vielseitigkeit, doch leidet sie unter ihrer geringen Empfindlichkeit. ...
... Huang et al. 68 described the effects of formalin fixa on on RS of cancerous human bronchial ssue, whereas Draux et al. 71 described the influence of formalin and air drying on single cancer cells and a ributed spectral changes to affec on of nucleic acids and proteins. Even though not only a loss of the original chemical composi on but also poten al contamina on due to the process of formalin-fixa on in murine brain ssue was determined by Hacke et al. 76 , several studies proposed formalin fixa on as a sufficient and favorable method for subsequent spectroscopic diagnos c 77,78 . As a proof of concept, Stefanakis et al. 79 demonstrated the feasibility of vibra onal spectroscopy on formalin-fixed malignant brain ssue. ...
... Generally, FTIR-and Raman-SHP are proven, highly accurate label-free methods for tissue classification [6,24,[27][28][29]. Nowadays, vibrational spectra of a thin tissue slice are routinely recorded microscopically with high spatial resolution. ...
... A quality control method for the National certification Program of Breast Centers certification was to ensure that patients received this document within six months after finishing their treatment [8]. Studies revealed that while the SCP was a helpful tool, it did not have the same impact on patient-reported results. ...
... These methods provide precise molecular fingerprints of biological samples, rendering them optimal for the study of lipidomic and metabolomic profiles of organ homogenates. [44][45][46] Vibrational spectroscopy enables the high-sensitivity and high-specificity identification of biochemical alterations. Moreover, these techniques allow for rapid, accurate, and label-free analysis, which is perfectly aligned with the objective of our study. ...
... Trehalose-water-protein interactions have been previously and extensively studied using spectroscopic methods. In particular, Raman spectroscopy has been used because of the advantage of label-free detection in biological samples [25][26][27]. RAMAN spectra analysis was therefore performed on different freeze-dried beads; completely pure trehalose beads, and some of the trehalose-LAMP mixture beads, and the spectra are given (Fig. 3). In the completely pure trehalose bead, the vibrations in the C-O-C skeletal structure produced strong C-C bond stretching peaks in the range of 400 to 1800 cm −1 , which is considered to be a fingerprint region of trehalose. ...
... In biomedical applications, it has been demonstrated that vibrational spectroscopy can differentiate normal and diseased (e.g. cancer, inflammation), and classify early stage diseased states, in tissue, cells, bodily fluids, and the term "spectropathology" has been coined [6][7][8]. ...
... 13,14 These techniques have also been demonstrated to be capable of differentiating between resting and activated lymphocytes, in addition to monocytes and macrophages. [15][16][17][18] These proof-of-concept studies demonstrate that both RS and FTIR are invaluable tools for PBMC analysis. Other studies have shown that RS and FTIR analysis of PBMCs and pure lymphocytes holds great promise for clinical real-world applications in the analysis of malignancies, infectious diseases, immune response, occupational contaminants, drug response, radiotherapeutic response, and for retrospective radiobiological dosimetry. ...
... Cell aggregates are not desirable since they result in the distortion of IR microscopy spectra due to multiple scattering and other effects. 11,45 Cells were cultured for 24 h until they attached onto the substrate. Finally, these asynchronous cells were either fixed as a control group or further synchronized to G1/S or G2/M phases. ...
... ~1 m near the 3400 cm -1 region and ~2 m near the 1640 cm -1 region, and remains within the limit of linear response of the IR detector. Spectral background of culture is likely to cause over compensation in the spectral regions of water absorption where water leading e.g., to artificial underestimation of the apparent absorbance of the amide I band of the cell [249]. IR spectroscopy developed a reasonably matured methodology in issue and cell studies [129,219]. ...
