First, to ensure the sample retains endogenous fluorescence, protein antigenicity, and structural integrity throughout the clearing process, the tissue needs to be preserved beyond simple PFA fixation. There are many preservation techniques, but SHIELD is the most robust technique currently available and can be easily applied via either a perfusion or post-fixation step.

Second, the tissue needs to be delipidated – this is generally referred to as the clearing step. Light-scattering lipid membranes in the tissue prevent deep penetration of light as they create refractive index mismatches that limit imaging depth. Delipidation allows for imaging of whole organs without sectioning, and is achieved by introducing the detergent SDS to the sample at elevated temperatures. Clearing can be done passively or can be sped up significantly using electrophoretic methods, like those used in our SmartClear II Pro. The SmartClear II Pro is a batch processing tissue clearing device that allows for significantly faster, easier, and more reliable tissue clearing vs. passive methods, all with higher throughput.

Next, specific targets of interest in the sample can be labeled with molecular probes, allowing for targeted analysis of specific proteins, structural markers, cell populations, and more. Small samples can be stained with passive methods, but larger, whole organ samples are more difficult to label homogeneously and take a long time to label (several weeks). Our SmartLabel device uses stochastic electrotransport technology (Kim, PNAS, 2015) combined with SWITCH (Murray, Cell, 2015) to increase the speed of labeling to just 24 hours. For more information, please visit our Technology page.

After delipidation and optional labeling, the sample must then be index matched. Even after removing the lipids, the sample will appear translucent – not transparent – due to the refractive index mismatch between the sample and surrounding fluid. A simple incubation in EasyIndex with gentle shaking will match the refractive index and render the sample optically transparent. For new users of EasyIndex, it is important to shake the bottle well before use.

Once the sample has been properly index matched it can be imaged intact. Some examples of appropriate imaging modalities are confocal, two-photon, and light-sheet microscopy. In general, light-sheets are better suited for large format, whole-organ imaging. LifeCanvas’s SmartSPIM is optimized for resolution, speed, and flexibility for large, cleared samples and can image a mouse brain hemisphere at high resolution in just ~20 minutes per imaging channel.