We report infrared microspectral features of nuclei in a completely inactive and contracted (pyknotic) state, and of nuclei of actively dividing cells. For pyknotic nuclei, the very high local concentration of DNA leads to opaqueness of the chromatin and, consequently, the absence of DNA signals in the IR spectra of very small nuclei. However, these nuclei can be detected by their scattering properties, which can be described by the Mie theory of scattering from dielectric spheres. This scattering depends on the size of the nucleus; consequently, quite different scattering cross-sections are calculated and observed for pyknotic and mitotic nuclei.

Instrumentation used in infrared microspectroscopy (IR-MSP) permits the acquisition of spectra from samples as small as 100 pg (10(-10) g), and as small as 1 pg for Raman microspectroscopy (RA-MSP). This, in turn, allows the acquisition of spectral data from objects as small as fractions of human cells, and of small regions of microtome tissue sections. Since vibrational spectroscopy is exquisitely sensitive to the biochemical composition of the sample, and variations therein, it is possible to monitor metabolic processes in tissue and cells, and to construct spectral maps based on thousands of IR spectra collected from pixels of tissue. These images, in turn, reveal information on tissue structure, distribution of cellular components, metabolic activity and state of health of cells and tissue.

Raman microspectroscopy-based, label-free imaging methods for human cells at sub-micrometre spatial resolution are presented. Since no dyes or labels are used in this imaging modality, the pixel-to-pixel spectral variations are small and multivariate methods of analysis need to be employed to convert the hyperspectral datasets to spectral images. Thus, the main emphasis of this paper is the introduction and comparison of a number of multivariate image reconstruction methods. The resulting Raman spectral imaging methodology directly utilizes the spectral contrast provided by small (bio)chemical compositional changes over the spatial dimension of the sample to construct images that can rival fluorescence images in terms of spatial information, yet without the use of any external dye or label.