I am currently doing an experiment to image thick (~100 micron) cut pieces of aorta and am having a few problems. To imagine the shape of my tissue, I am cutting rings or bands of aorta and positioning them upright on slides. Because I am imaging down into the cylindrical aortic pieces to collect a 3D z-stack, the penetration power is quite low and it is impossible to see the whole height of the sample. Is there any sort of tissue prep that can help improve the quality of the laser penetration and increase both resolution and depth of the image? Unfortunately scanning electron and multiphoton microscopy are not available to me at this time.
You can use tissue clearing methods like iDISCO, CLARITY, PACT, BABB, etc. to help render your tissue more transparent. Granted, there may be some tissue shrinkage (or swelling) depending on which clearing method you choose.
It does also depend on whether you are staining with antibodies, in which case antibody penetration may also be an issue. As suggested by David Hughes Burk Clearing reagents are now quite accessible, like ScaleA2 (urea) and Ce3D are relatively fast-acting, have minimal shrinkage/swelling effect and preserve most fluorochromes. People also develop modified versions of these reagents to suit their tissue of interest.
Dear Bradley,
while definitely less effective than tissue clearing, using a more appropriate objective could help you achieve deeper imaging. In particular, if you are using oil immersion, consider switching to a water immersion objective. If you are already using a water immersion/dipping objective, consider using one with lower numerical aperture.
Both this changes will reduce the resolution of your images, but could greatly improve tissue penetration, in case what you are experiencing is a phase aberration or scattering.
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