Visikol HISTO Beta Test Feedback - Shallow Imaging Depth
If you image your samples without utilizing refractive index matched immersion lenses (e.g. glycerol immersion lens, CLARITY optimized objective), an objective with a refractive index adjusting collar, or without adapting your air-lens for use with Visikol HISTO to eliminate changes in refractive index in the system, the maximum depth of imaging is approximately 500 µm due to attenuation caused by spherical aberration. This will limit the thickness of the tissues you can image to 1 mm (imaging from both sides). Researchers commonly believe that they are seeing poor immunolabel penetration whereas they are actually experiencing the limitations of their imaging system.
Using a multi-immersion objective or objectives with a refractive index correction collar can correct for this issue. Inverted microscopes are not recommended for use with Visikol HISTO (or other clearing techniques) because they cannot achieve a continuous path of matching refractive index, and will therefore limit the overall depth that can be achieved during imaging to approx. 500 µm. This concern is only for confocal instruments - light sheet microscopes should not have this issue, as most are built to deal with multiple clearing media (e.g. Ultramicroscope II).