Quantitative Biomedical Optics Theory, Methods, and Applications

In Chapter 5 we reviewed methods of fluorescence spectroscopy and fluorescence measurements related to bioscience applications that enable monitoring of cellular metabolism, drug distribution, enzymatic processes, etc. In Chapter 6 we covered methods for spectroscopy of vibrational modes, which can yield diagnostic information by identification of the molecular “fingerprints” of biomolecules. These methods are powerful, and they predominantly provide information on the biochemistry of cells and tissue. Nonetheless, when considering potential clinical diagnostic applications, one might ask, “How does a pathologist diagnose cancer, when looking at a histology slide under a microscope?” After all, the pathologist is using an optical detector/camera (the human eye) to examine light coming from a sample of tissue through an optical instrument (the microscope). The term histology (from Greek: histos = tissue; logia = science or study) refers to the study of the microscopic structure of cells and tissue. Thus, the traditional practice of histopathology (the microscopic study of diseased tissue, usually for the purpose of diagnosis), which has been around for more than 120 years, may be regarded as one of the earliest techniques of biomedical optics to be put into practice.

In the common practice of histopathology, a small tissue sample is surgically cut from the organ where disease is suspected in a patient, a procedure called biopsy, and the sample is examined microscopically as part of the clinical diagnosis process. The biopsy sample is first fixed in formalin, which dehydrates it. It is then embedded in paraffin (for mechanical stability), and a very thin (typically 3–10 μm) slice is cut with a microtome and placed on a microscope slide. The thin slice is typically stained, most commonly with hematoxylin and eosin stains (when combined, called H&E stain), which stain for DNA (hence, nuclei) and other dense structures, mostly proteins. It is interesting to note that without such staining, very little cellular structure is visible in conventional bright-field microscopy, due to lack of contrast.

Getting back to the question about what a pathologist looks for when reading a slide and determining a diagnosis for disease (say, cancer), most of the time the pathologist is not performing biochemical assays or conducting genetic analysis: most of the time the pathologist is visually examining the cellular architecture, shapes and structures. Are the nuclei enlarged? What is the ratio of nuclear volume to cell volume? Is the nuclear staining especially dense (hyperchromatic), indicating excessive DNA?