Use of three-dimensional microscopy with multiple oblique illumination in evaluating cytopathology preparations

OBJECTIVE: To analyze whether the use of multiple oblique illumination (MOI), in contrast to axial illumination (AI), improves imagery in bright field microscopy. Specimens containing thick cell clusters, such as cervical Pap smears, are often misread because of out-of-focus cell clusters. We hypothesized that visualization of these specimens with MOI would enhance this information as compared with AI. STUDY DESIGN: Micrometer images and Pap smears were analyzed in focus, and 8 and 40 microns out of focus by MOI and AI. All fields were captured to a remote computer, histograms were constructed, and mean light intensity was calculated. Mathematical formulation was used to define the histogram distribution of the micrometer images. Statistical analysis was performed using one-way ANOVA and Newman-Keuls test. RESULTS: K(focus) was improved (P < .01) and at 20 and 40 microns out of focus, mean light intensities were greater (P < .003, P < .05, respectively) with MOI as compared to AI. CONCLUSION: MOI improves out-of-focus information by increasing light penetration through the specimen and enhancing contrast and resolution.