Sample mounting

Depending on clearing method and sample type, mounting can be done with:

• clamps
• immersion (dipping) cuvettes
• non-immersion cuvettes

Clamps

DBE, BABB and ECi tend to dissolve several types of plastic. 3D-printed parts made from polyamide (nylon, PA12) are resistant to these solvents. The sample is clamped using the (top) moving jaw and dipped directly into imaging medium (DBE, BABB, or ECi). Samples from 3 mm to 40 mm tall can be imaged this way.

This method also accomodates samples that are embedded in agarose block (suitable, for example, for CUBIC-cleared brains).

Dipping cuvettes

Samples that are prepared with water-based or hydrogel protocol (CLARITY, CUBIC) are typically softer and require a dipping cuvette. The cuvette also separates the outer, stationary chamber (filled with 50-100 ml of matching oil) from the sample mounting medium which is often expensive and subject to evaporation.

Non-dipped cuvettes

For very large samples, such as whole mouse, dipping into a stationary chamber becomes impractical. The sample can be inserted into a large cuvette that is held from the top and imaged difectly, without dipping.

⚠️ This mountig method presents some challenges for imaging because moving the cuvette along X (left-right) shifts the optical ETL settings, while moving in Z shifts the focus plane, due to changing optical path for both illumination and detection. These changes must be compensated in the acquisition software with ETL adjustments for different tiles, and focus adjustment in the start vs the end of Z-stack.

Review Sample Handling Guides for mesoSPIM