Nuclear volume estimation using different sampling, measurement and calculation methods

OBJECTIVE: To study the reliability of volume parameter measured on tissue sections through different sampling, measurement and calculation methods. STUDY DESIGN: The largest nuclear profile image under a 100x, NA 1.30 oil i mmersion objective of primary spermatocytes and spherical spermatoblasts on 11-mu m-thick seminiferous tubule sections and tissue images, under a 20x objective, on 4-mu m sections were captured. Their volumes were measured an d calculated by the five methods provided by the Technology for Image and G raphics Engineering Research cell image analysis system. RESULTS: The nuclear volumes obtained by nucleator and area equivalent diam eter on the largest nuclear profile image were almost the same, including b inary images by automated and manual interactive nucleator and grey scale i mages only by the latter. Nuclear volumes, calculated by random Feret diame ter and equivalent diameter of the perimeter, the minimal circumference of the largest nuclear profile binary image, were obviously larger than those of the nucleator and area equivalent diameter. Due to different-sized nucle ar slices entrapped in the same section, those nuclear volumes from the sem iniferous tubule tissue images were strikingly lower than that of the large st nuclei profile image. The shape factors of primary spermatocytes and sph erical spermatoblast nuclei under 100x and 20x objectives were approximatel y the same. CONCLUSION: The sample preparation, sampling methods and calculation formul as suitable to nuclear form are necessary to obtain reproducible volume par ameters.