We have applied Fourier transform infrared (FTIR) spectroscopic imaging, coupling a mercury cadmium telluride (MCT) focal plane array detector (FPA) and a Michelson step scan interferometer, to the investigation of various states of malignant human prostate tissue. The MCT FPA used consists of 64x64 pixels, each 61 micrometers 2, and has a spectral range of 2-10.5 microns. Each imaging data set was collected at 16-1 resolution, resulting in 512 image planes and a total of 4096 interferograms. In this article we describe a method for separating different tissue types contained within FTIR spectroscopic imaging data sets of human prostate tissue biopsies. We present images, generated by the Fuzzy C-Means clustering algorithm, which demonstrate the successful partitioning of distinct tissue type domains. Additionally, analysis of differences in the centroid spectra corresponding to different tissue types provides an insight into their biochemical composition. Lastly, we demonstrate the ability to partition tissue type regions in a different data set using centroid spectra calculated from the original data set. This has implications for the use of the Fuzzy C-Means algorithm as an automated technique for the separation and examination of tissue domains in biopsy samples.
We describe a non-invasive, in vivo hyperspectral imaging method for visualizing the spatial distribution of dermal tissue oxygenation. Real-time images of the dermis are acquired both at multiple, contiguous wavelengths and at relatively narrow spectral bandwidths to generate a data cube consisting of one spectral and two spatial dimensions. For data collection, the sample area is illuminated by radiation, which is delivered by liquid light guides from a quartz tungsten halogen source. Reflected light from the sample is first passed through a liquid crystal tunable filter and then imaged onto a silicon charged coupled device detector. The subsequently digitized data are presented in terms of spectral images reflecting multivariate least squares analyses based upon linear combinations of oxy- and deoxyhemoglobin reference spectra. The generated gray scale images directly represent the varying spatial distributions of dermal tissue oxygenation. As an example, imaging data are obtained from normal tissue and induced ischemic tissue for which both the venous and arterial blood flow was artificially occluded.
FT-IR microspectrometry, particularly in combination with digital imaging techniques shows great promise for in-vivo and ex-vivo medical diagnosis. The statement is based on the knowledge that this method delivers information of the chemical structure and composition of a sample and the fact that any disease is linked to changes in the molecular and structural composition of cells and tissues. Typically, these changes are highly specific for a given tissue structure and are therefore potentially detectable by FT-IR microspectrometry. In this paper we present several approaches for the representation of mid-infrared microspectroscopic data acquired with high spatial resolution by the use of a MCT focal plane array detector. The applicability of image reassembling methodologies like functional group analysis, image reconstruction based on factor analysis and artificial neural network analysis to the IR data is discussed.
The utility of Raman microscopy and imaging for the characterization of a variety of chemical and biological systems is discussed. Measurements have been carried out with an optical microscope coupled to a Raman spectrometer that contains light paths for both single point and imaging measurements. Laser irradiation and signal collection are implemented using epi-illumination through a single microscope objective. For point Raman microspectroscopy. In our arrangement, the laser is defocused to provide wide- field illumination. The Raman signal from within the irradiated sample area is directed through a narrow-band liquid crystal tunable filter (LCTF) and imaged onto the CCD. Spectroscopic information is obtained by recording Raman images through the LCTF over successively tuned frequencies. Raman spectra for various point within the sample thus are obtained in parallel by each pixel in the detector array. Microspectra were recorded within various sample, including bacteria. Spectroscopic features of interest were then investigated in greater spatial detail using the LCTF imaging methodology.
Conventional Fourier transform IR spectroscopy has proven to be an invaluable research and diagnostic approach for the study of a wide range of biomedical problems. In this article we describe a new biomedical imaging method which integrates high-resolution IR spectroscopy with high- definition digital imaging. The continuing development and commercialization of long-wavelength IR cameras or focal- plane arrays has been a key enabling technology. These imaging systems are capable of rapidly generating chemically specific images from a variety of unstained biological tissue and cells. Image contrast is intrinsic to the sample and is determined only by its biochemical composition. In addition, data from a single experiment can be digitally manipulated to produce numerous images of the same sample, for which different spatial and biochemical properties are emphasized. We present data demonstrating the potential of the technique to generate spectroscopic signatures and images from single human breast cells.
We describe a Raman imaging microscope that produces high-fidelity, large format Raman images and Raman spectra at close to diffraction-limited spatial resolution. A silicon charge- coupled device is used as a high sensitivity array detector. Wavelength selection of Raman scattered emission is achieved by an acousto-optic tunable filter, which maintains image fidelity and provides either continuous or random wavelength selection. Laser illumination is delivered to the object by means of an infinity corrected microscope objective, either by a galvanometer scanning system or a widefield fiber optic. The laser scanning mechanism has higher power densities and provides Raman microprobe capabilities when stopped at a prescribed point. The fiber optic illumination scheme, however, is useful for delicate samples which might be damaged by the higher power densities generated by the point scanner mechanism and for sample alignment and system focusing. Instrument features, including factors that determine the system's spatial and spectral resolution, are discussed in detail. Images and spectra of test objects and samples that demonstrate the capabilities of this imaging spectrometer are presented. The potential of intrinsic chemical imaging is discussed in terms of its use in the analyses of a variety of chemical and biological samples.
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