Interferometric analysis of intrasection and intersection thickness variability associated with cryostat microtomy

Mach-Zehnder interferometric measurements were used to assess the extent of section thickness variability (inter- and intrasection) associated with cryostat microtomy of adrenal sections over a typical working range of 10-20 micron. Sections were obtained using a Bright's Cambridge rocking type and a Damon rotary type cryostat microtome to allow comparative analyses. The effective thickness of tissue sections after being mounted onto slides by flash drying was reduced by 90% relative to microtome section thickness setting. A linear relationship between measured thickness and microtome setting was obtained with both instruments. Thickness variability between replicate sections over the range of microtome settings approximated 11% for the rocking microtome and 5% with the rotary microtome. Average intrasection variability was found to be 7% for rocking microtome sections and 4% for sections obtained with the rotary microtome. However, this variability is a negligible source of error in cytophotometric analyses, providing replicate sections are used and an adequate number of measurements are made on mask-delimited individual cells or tissue specimen areas.