Melissa J. Romeo

Raman and Infrared Microspectral Imaging of Mitotic Cells

We report the first ever Raman and infrared microspectroscopic images of human cells at different stages of mitosis. These spectroscopic methods monitor the distribution of condensed nuclear chromatin, and other biochemical components, utilizing inherent protein and DNA spectral markers, and, therefore, do not require the use of any stains. In conjunction with previously reported data from the G1, S, and G2 phases of the cell cycle, the complete cell division cycle has now been mapped by spectroscopic methods. Although the results reported here do not offer new insights into the distribution of biochemical components during mitosis, the recognition of cell division without the use of stains, and the possibility of doing so on living cells, may be useful for an automatic, spectroscopic determination of the proliferation rates of cells and tissues. Spectral images were constructed by plotting spectral intensities of DNA or protein versus the coordinates from which spectra were recorded. We found that both Raman and infrared intensities depend on the overall chromatin density variation among the individual subphases of mitosis.

Infrared micro-spectral imaging: distinction of tissue types in axillary lymph node histology

BackgroundHistopathologic evaluation of surgical specimens is a well established technique for disease identification, and has remained relatively unchanged since its clinical introduction. Although it is essential for clinical investigation, histopathologic identification of tissues remains a time consuming and subjective technique, with unsatisfactory levels of inter- and intra-observer discrepancy. A novel approach for histological recognition is to use Fourier Transform Infrared (FT-IR) micro-spectroscopy. This non-destructive optical technique can provide a rapid measurement of sample biochemistry and identify variations that occur between healthy and diseased tissues. The advantage of this method is that it is objective and provides reproducible diagnosis, independent of fatigue, experience and inter-observer variability.MethodsWe report a method for analysing excised lymph nodes that is based on spectral pathology. In spectral pathology, an unstained (fixed or snap frozen) tissue section is interrogated by a beam of infrared light that samples pixels of 25 μm × 25 μm in size. This beam is rastered over the sample, and up to 100,000 complete infrared spectra are acquired for a given tissue sample. These spectra are subsequently analysed by a diagnostic computer algorithm that is trained by correlating spectral and histopathological features.ResultsWe illustrate the ability of infrared micro-spectral imaging, coupled with completely unsupervised methods of multivariate statistical analysis, to accurately reproduce the histological architecture of axillary lymph nodes. By correlating spectral and histopathological features, a diagnostic algorithm was trained that allowed both accurate and rapid classification of benign and malignant tissues composed within different lymph nodes. This approach was successfully applied to both deparaffinised and frozen tissues and indicates that both intra-operative and more conventional surgical specimens can be diagnosed by this technique.ConclusionThis paper provides strong evidence that automated diagnosis by means of infrared micro-spectral imaging is possible. Recent investigations within the authors laboratory upon lymph nodes have also revealed that cancers from different primary tumours provide distinctly different spectral signatures. Thus poorly differentiated and hard-to-determine cases of metastatic invasion, such as micrometastases, may additionally be identified by this technique. Finally, we differentiate benign and malignant tissues composed within axillary lymph nodes by completely automated methods of spectral analysis.

Chapter 10: Infrared and Raman microscopy in cell biology.

This chapter presents novel microscopic methods to monitor cell biological processes of live or fixed cells without the use of any dye, stains, or other contrast agent. These methods are based on spectral techniques that detect inherent spectroscopic properties of biochemical constituents of cells, or parts thereof. Two different modalities have been developed for this task. One of them is infrared micro-spectroscopy, in which an average snapshot of a cells biochemical composition is collected at a spatial resolution of typically 25 mum. This technique, which is extremely sensitive and can collect such a snapshot in fractions of a second, is particularly suited for studying gross biochemical changes. The other technique, Raman microscopy (also known as Raman micro-spectroscopy), is ideally suited to study variations of cellular composition on the scale of subcellular organelles, since its spatial resolution is as good as that of fluorescence microscopy. Both techniques exhibit the fingerprint sensitivity of vibrational spectroscopy toward biochemical composition, and can be used to follow a variety of cellular processes.

Detection of breast micro-metastases in axillary lymph nodes by infrared micro-spectral imaging

We report the ability of infrared micro-spectral imaging, coupled with completely unsupervised methods of multivariate statistical analysis, to accurately reproduce the histological architecture of axillary lymph nodes and detect metastatic breast cancer cells. The acquisition of spectral data from tissue embedded in paraffin provided spectra free of dispersive artefacts that may be observed for infrared microscopic measurements using a reflection/absorption methodology. As a consequence, superior tissue classification and identification of cellular abnormality unattainable for deparaffinised tissue was achieved.

The infrared spectral signatures of disease: extracting the distinguishing spectral features between normal and diseased states.

O ver the past decade, new medical diagnostic methods have been developed by several research groups worldwide, based on infrared microspectroscopy and microscopic imaging (see, for example, the compiled references in a number of recent books). These methods can be applied both to tissue sections and individual exfoliated cells. The success of these methods in differentiating cancerous from normal tissues, as well as individual cancerous, precancerous, and normal cells, is due to two major factors. First, infrared microspectroscopy monitors, in one measurement, a snapshot of the overall biochemical composition of an individual cell. This composition varies with a number of well-understood cell-biological processes; thus, the cell’s division cycle, its maturation and differentiation, as well as a transition from normal to cancerous states can be monitored via a wellunderstood spectral measurement. This differs significantly from the standard cytopathological methodology, which relies on a visual inspection of cell morphology and tissue architecture and is, therefore, subjective in nature. The second factor for the success of spectral diagnoses is the fact that data can be acquired fairly rapidly: it takes about 500 ms to collect a good infrared micro-spectrum from a voxel of biological material. The size of such a voxel is typically about 12 3 12 3 5 lm in the x, y, and z directions, where the lateral (x,y) dimension is determined by the diffraction limit and the z direction is determined by the thickness of the tissue section or the thickness of a cell. In the case of infrared micro-spectral imaging of human tissues, up to 100 000 individual voxel spectra are collected to create huge hyperspectral data sets, where the term ‘‘hyperspectral’’ implies spatially resolved data with distinct x and y coordinates, and spectral information from each x,y point. The analysis of the hyperspectral dataset is carried out by methods of chemometrics, which detect small, but recurring differences,

Spectral detection of micro-metastases in lymph node histo- pathology

The first detection of breast cancer micrometastases in lymph nodes by infrared spectral imaging and methods of multivariate analysis is reported. Micrometastases are indicators of early spread of cancer from the organ originally affected by disease, and their detection is of prime importance for the staging and treatment of cancer. Infrared spectral imaging, at a spatial resolution of ca. 10-12 mum, can detect small metastases down to the level of a few cancerous cells. The results presented here add to a rapidly growing database of infrared spectral imaging results for cancer diagnostics.

Analysis of microscopic infrared spectra of individual dried and live human cells

The ability of infrared (IR) spectroscopy to distinguish and map cancerous and non-cancerous tissue has opened the question of the origin of spectral differences between normal and cancerous cells. In this contribution, we report IR spectral maps of individual dried cancer cells, some of them in the process of cell division (mitosis), IR spectra of cells suspended in growth medium, and preliminary results of a statistical analysis of thousands of individual dried cancer cells.

Infrared spectral imaging of human cells and tissue: an approach to objective, machine-based histopathology

Human cells from different tissue types, and at different stages of disease, present slight differences in their infrared (IR) absorption spectra. Thus, IR spectroscopy offers the possibility of analyzing individual cells, or biopsy sections of tissues, for the presence of disease, based on totally objective measurements and computer-based analysis of the spectral information.