Multiple Oblique Illumination and High-Definition Microscopy for Breast Fine-Needle Aspirates

Abstract

The ability of multiple oblique illumination (MOI) and high-definition microscopy (Edge R-400 3-D microscope) to improve resolution of cellular detail in the evaluation of cytopathological specimens of Pap smears and thyroid fine-needle aspirates (FNAs) has been demonstrated. However, previous experiments showed that the advantages of MOI and high-definition stereo microscopy were less certain for the breast FNAs. We hypothesized that these findings were due to the lack of sample thickness for the breast FNA specimens. To test this hypothesis, we analyzed breast FNA specimens that were significantly thicker (10.5 microm). The number of lights (1, 2, 3, 4) and the angle of light (+1.5, 0, -3) were varied independently, creating 12 groups. Three images at each combination of settings were digitally captured and analyzed to obtain a histogram. The coefficient of resolution (Cr) was calculated to mathematically evaluate the grayscale histograms for intensities (0-255), where Cr = [¿IM - IN¿ x (N)] (IM, median pixel intensity; IN, measured pixel intensity; and N, number of pixels at given intensity). Mean Cr values demonstrated that the angle of light obliquity was not a factor in altering the resolution and contrast (p = .9). However, there was a significant increase in resolution, as measured by mean Cr values, as the number of lights was successively reduced from four lights to one light. Thus, the thicker specimen did show that increases in resolution were a significant function of the number of lights utilized.