Spectral Imaging Microscopy Can Define Hyperchromasia in Small Lymphoid Cells

Abstract

Spectral Imaging Microscopy (SIM) is a technique for performing image‐enabled spectroscopic analysis using a standard light microscope. We employed a prototype to a current, commercially available, spectral imaging system (NuanceTM, CRI, Inc.) that electro‐optically selects narrow bands of light to be collected by a cooled, monochrome CCD camera. For each sample, images were taken every 10 nm from 420 nm to 720 nm to create a spectral image “stack.” Analysis of the images with a prototype to the Nuance TM Analyze software (CRI, Inc.) gave both standard color (RGB) and spectral information at each pixel of the images. A representative 40x field was spectrally imaged from H&E slides of 104 biopsy specimens of mycosis fungoides (MF). Optical densities and spectral properties of small lymphocytes were determined. Hyperchromatic cells were pseudocolored to show their distribution. In addition to the important architectural features of MF, SIM can define whether hyperchromatic or normochromatic small lymphocytes are epidermotropic in the lesion by setting appropriate threshold values. Hyperchromasia can be attributed to increased nuclear eosinophilia or basophilia. These differences are difficult to estimate by eye. Since lymphoblasts have enlarged nuclei with dispersed chromatin, a combination of spectral and spatial data is required for their analysis.