[Use of the OTO-method in electron microscopic studies]

Area of Science:

• Electron microscopy techniques
• Biological tissue preparation
• Histology

Context:

• Standard biological tissue preparation for electron microscopy involves multiple fixation and staining steps.
• Conventional contrasting methods often use heavy metals like lead and uranium, raising safety and disposal concerns.
• Achieving high contrast in biological samples is crucial for visualizing ultrastructural details.

Purpose:

• To present a modified OTO (Osmium-Thiocarbohydrazide-Osmium) staining protocol.
• To improve the contrast of biological tissue structures during the fixation process.
• To eliminate the requirement for post-fixation staining with lead citrate and uranyl acetate.

Summary:

• The modified OTO method involves intermittent treatment of prefixed tissues with osmium tetroxide and thiocarbohydrazide.
• This intermittent application leads to the formation of an osmium-thiocarbohydrazide copolymer, appearing as "osmium black".
• The resulting "osmium black" provides high intrinsic tissue contrast, reflecting true osmiophilia.

Impact:

• Simplifies the electron microscopy sample preparation workflow.
• Reduces the use of hazardous heavy metal stains like lead citrate and uranyl acetate.
• Enhances the visualization of biological ultrastructure through superior intrinsic contrast.