A decade of vibrational micro-spectroscopy of human cells and tissue (1994-2004)

Abstract

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.

Fig. 1

Photomicrograph (A) and early tissue map (ca. 1997, B) of a section of cervical epithelium, collected manually. Data acquisition: 80 μm spatial resolution, >24 hours, ca. 350 pixels. Data processing using a pattern recognition algorithm.

Fig. 2

(A) Photomicrograph of stained cervical tissue section containing the squamous-columnar junction (SCJ). Section was stained after spectral data acquisition. (B) Spectral map of the section shown in (A). Data acquisition: ca. 20 μm spatial resolution, 6 h, ca. 3,000 pixels. Data processing via hierarchical cluster analysis. (C) Means cluster spectra of three distinct tissue types from the map in (B)

Fig. 3

(A) Photomicrograph of stained lymph node section, 5 × 1.5 mm². (B) Infrared spectral image of section adjacent to that shown in (A). Data acquisition: 25 μm spatial resolution, 40 min, ca. 12,000 pixels. Data processing via hierarchical cluster analysis. (C) Dendrogram of clustering process for the map shown in (B). (D) Mean 2nd derivative cluster spectra extracted from the data set depicted in (B) for the follicular and paracortical areas (lymphocytes), and different regions of connective tissue.

Fig. 4

Infrared and Raman spectral images of a HeLa cell in the metaphase of mitosis. (A) Visual image taken through infrared microscope (B) infrared spectral map, based on amide I intensity (6.25 μm nominal spatial resolution), (C) visual image taken through Raman microscope, (D) Raman spectral image based on amide I intensity, (E) Raman spectral image based on nucleic acid intensity. Spatial resolution ca. 1 μm.