Optical disector counting in cryosections and vibratome sections underestimates particle numbers: Effects of tissue quality

Optical disector counting is currently applied most often to cryosections, followed in frequency by resin-embedded tissues, paraffin, and vibratome sections. The preservation quality of these embedding options differs considerably; yet, the effect of tissue morphology on numerical estimates is unknown. We tested whether different embedding media significantly influence numerical estimates in optical disector counting, using the previously calibrated trochlear motor nucleus of hatchling chickens. Animals were perfusion-fixed with paraformaldehyde (PFA) only or in addition with glutaraldehyde (GA), or by Methacarn immersion fixation. Brains were prepared for paraffin, cryo-, vibratome- or celloidin sectioning. Complete penetration of the thionin stain was verified by z-axis analysis. Neuronal nuclei were counted using an unbiased counting rule, numbers were averaged for each group and compared by ANOVA. In paraffin sections, 906 +/- 12 (SEM) neurons were counted, similar to previous calibrated data series, and results obtained from fixation with Methacarn or PFA were statistically indistinguishable. In celloidin sections, 912 +/- 28 neurons were counted-not statistically different from paraffin. In cryosections, 812 12 neurons were counted (underestimate of 10.4%) when fixed with PFA only, but 867 17 neurons were counted when fixed with PFA and GA. Vibratome sections had the most serious aberration with 729 +/- 31 neurons-a deficit of 20%. Thus, our analysis shows that PFA-fixed cryosections and vibratome sections result in a substantial numerical deficit. The addition of GA to the PFA fixative significantly improved counts in cryosections. These results may explain, in part, the significant numerical differences reported from different labs and should help investigators select optimal conditions for quantitative morphological studies.