Imaging with immersion objectives
Due to aberrations caused by refractive index mismatch between air and glass presenting absolute limits on resolution, immersion objectives were developed to reduce scattering and noise caused from refractive aberrations. Immersion objectives are well-suited to deep tissue imaging due to the resultant reduction of refractive index mismatch between sample and objective, as the lens is immersed directly in the medium, eliminating mismatch between the liquid medium and air typically encountered for air lenses.
Oil immersion lenses work very well with Visikol HISTO cleared specimens, however the working distance on oil immersion lenses is typically 250 microns or less, which presents a 500 micron limit on the thickness of tissue to image.
Since some objectives may be corroded or damaged from immersion in Visikol HISTO-2, you should utilize the double chambered cuvette that comes in our Starter Kit. The cuvette allows the sample to be mounted in Visikol HISTO-2, and the immersion objective to be immersed in an alternate medium (water or glycerol).
Water immersion lenses allow for 2-3 mm depth of imaging, however some refractive index mismatch will still occur, since water has a refractive index (1.33) significantly different than Visikol HISTO-2 (1.53).
A further improvement in imaging depth can be obtained by using glycerol immersion objectives. Some refractive mismatch will still occur, however substantially less aberration will result due to the closer match in refractive index between glycerol (1.47) and Visikol HISTO-2 (1.53). Double chambered cuvettes should be used.
For optimum results when imaging very thick tissues (> 1 mm) or whole organs, one should use multi-immersion objectives containing a correction collar. However, a correction collar isn’t required for immersion objectives as long as proper optical design has been done to allow imaging over a range of RI. These objectives (shown in the table above) offer the deepest tissue imaging, allowing for imaging of complete, intact organ systems. The only limitation to depth with these objectives is the effective working distance, since a sample approximately twice the thickness of the working distance length can be imaged.
For mounting specimens for imaging, we suggest using ClearWells. The sample is placed in the well, covered with Visikol HISTO-2 and then a cover slip is placed on top of the slide.