Miloš Miljković

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.

Picosecond spectral coherent anti-Stokes Raman scattering imaging with principal component analysis of meibomian glands

The lipid distribution in the mouse meibomian gland was examined with picosecond spectral anti-Stokes Raman scattering (CARS) imaging. Spectral CARS data sets were generated by imaging specific localized regions of the gland within tissue sections at consecutive Raman shifts in the CH(2) stretching vibrational range. Spectral differences between the location specific CARS spectra obtained in the lipid-rich regions of the acinus and the central duct were observed, which were confirmed with a Raman microspectroscopic analysis, and attributed to meibum lipid modifications within the gland. A principal component analysis of the spectral data set reveals changes in the CARS spectrum when transitioning from the acini to the central duct. These results demonstrate the utility of picosecond spectral CARS imaging combined with multivariate analysis for assessing differences in the distribution and composition of lipids in tissues.

Two step resonant Mie scattering correction of infrared micro-spectral data: human lymph node tissue

In this manuscript, we report the application of EMSC to correct infrared micro-spectral data recorded from tissue that describe resonant Mie scattering contributions. Small breast micro-metastases previously undetectable using the raw measured spectra were provided clear contrast from the surrounding tissue after signal correction. The technique also proved transferrable, successfully correcting imaging data sets recorded from multiple patients. It is envisaged more robust methods of supervised analysis can now be constructed to automatically classify and diagnose tissue spectra.

Applications of Infrared and Raman Microspectroscopy of Cells and Tissue in Medical Diagnostics: Present Status and Future Promises

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.

Spectral Cytopathology of Cervical Samples: Detecting Cellular Abnormalities in Cytologically Normal Cells

Spectral cytopathology (SCP) is a novel spectroscopic method for objective and unsupervised classification of individual exfoliated cells. The limitations of conventional cytopathology are well recognized within the pathology community. In SCP, cellular differentiation is made by observing molecular changes in the nucleus and the cytoplasm, which may or may not produce morphological changes detectable by conventional cytopathology. This proof of concept study shows SCPs potential as an enhancing tool for cytopathologists by aiding in the accurate and reproducible diagnosis of cells in all states of disease. Infrared spectra are collected from cervical cells deposited onto reflectively coated glass slides. Each cell has a corresponding infrared spectrum that describes its unique biochemical composition. Spectral data are processed and analyzed by an unsupervised chemometric algorithm, principal component analysis. In this blind study, cervical samples are classified by analyzing the spectra of morphologically normal looking squamous cells from normal samples and samples diagnosed by conventional cytopathology with low-grade squamous intraepithelial lesions. SCP discriminated cytopathological diagnoses amongst 12 different cervical samples with a high degree of specificity and sensitivity. SCP also correlated two samples with abnormal spectral changes: these samples had a normal cytopathological diagnosis but had a history of abnormal cervical cytology. The spectral changes observed in the morphologically normal looking cells are most likely because of an infection with human papillomavirus (HPV). HPV DNA testing was conducted on five additional samples, and SCP accurately differentiated these samples by their HPV status. SCP tracks biochemical variations in cells that are consistent with the onset of disease. HPV has been implicated as the cause of these changes detected spectroscopically. SCP does not depend on identifying the sparse number of morphologically abnormal cells within a large sample to make an accurate classification, as does conventional cytopathology. These findings suggest that the detection of cellular biochemical variations by SCP can serve as a new enhancing screening method that can identify earlier stages of disease.

Cytopathology by Optical Methods: Spectral Cytopathology of the Oral Mucosa

Spectral cytopathology (SCP) is a novel approach for diagnostic differentiation of disease in individual exfoliated cells. SCP is carried out by collecting information on each cells biochemical composition through an infrared micro-spectral measurement, followed by multivariate data analysis. Deviations from a cells natural composition produce specific spectral patterns that are exclusive to the cause of the deviation or disease. These unique spectral patterns are reproducible and can be identified and used through multivariate statistical methods to detect cells compromised at the molecular level by dysplasia, neoplasia, or viral infection. In this proof of concept study, a benchmark for the sensitivity of SCP is established by classifying healthy oral squamous cells according to their anatomical origin in the oral cavity. Classification is achieved by spectrally detecting cells with unique protein expressions: for example, the squamous cells of the tongue are the only cell type in the oral cavity that have significant amounts of intracytoplasmic keratin, which allows them to be spectrally differentiated from other oral mucosa cells. Furthermore, thousands of cells from a number of clinical specimens were examined, among them were squamous cell carcinoma, malignancy-associated changes including reactive atypia, and infection by the herpes simplex virus. Owing to its sensitivity to molecular changes, SCP often can detect the onset of disease earlier than is currently possible by cytopathology visualization. As SCP is based on automated instrumentation and unsupervised software, it constitutes a diagnostic workup of medical samples devoid of bias and inconsistency. Therefore, SCP shows potential as a complementary tool in medical cytopathology.

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,

Vibrational spectroscopic changes of B‐lymphocytes upon activation

The first study interpreting B-lymphocyte activation in normal lymph nodes using vibrational micro-spectral imaging is reported. Lymphocyte activation indicates the presence and response against a pathogen, regardless of the inciting pathogens etiology, whether a benign, reactive or malignant process. Understanding the biochemical makeup of lymphocyte activation during early stages of disease and immune response may offer significant aid in determining a tumors origin without the presence of malignant metastatic cells but within lymph nodes that are reactive and displaying regions of hyperplasia. Infrared and Raman data scrutinized via unsupervised multivariate methods may provide a physical and reproducible method to determine the biochemical components and variances therein of activated lymph nodes with distinguishing characteristics depending on the malignancy present in the region or elsewhere in the body. The results reported here provide a proof-of-concept study that reveal a potential to screen lymph nodes for disease without the presence of metastatic cells.