Cryostat Slice Irregularities May Introduce Bias in Tissue Thickness Estimation: Relevance for Cell Counting Methods

Area of Science:

• Neuroscience
• Stereology
• Microscopy

Background:

• Stereological methods, particularly optical disectors, rely on accurate estimation of section thickness.
• Frozen tissue processing can introduce irregularities that complicate precise thickness measurements.

Purpose of the Study:

• To investigate the impact of frozen tissue irregularities on section thickness estimation in stereology.
• To evaluate the utility of confocal microscopy in assessing these irregularities.

Main Methods:

• Rodent nerve tissues (dorsal root ganglia, spinal cord, brain) were cryosectioned at 16, 40, and 50 μm.
• Sections were digitized using confocal microscopy and visualized with 3D software.
• Geometric section thickness (T geom) and maximal section thickness (T max) were measured and compared.

Main Results:

• Tissue irregularities, primarily from blood vessels and neuronal somas, significantly affected section thickness measurements.
• Significant differences were observed between T max and T geom (p<0.01).
• The T geom/T max ratio varied, indicating substantial deviations from ideal geometric thickness.

Conclusions:

• Frozen tissue irregularities pose a challenge for accurate stereological section thickness estimation.
• Confocal microscopy aids in identifying and quantifying these irregularities.
• Relying solely on maximal thickness can lead to overestimation of tissue volume in stereological analyses.