Hi everybody
I want to know if it is possible to collect Z-stacks from an opaque object using a laser confocal microscopy. In more general sense, Is it possible to construct a 3D image from an opaque object at all?
I can only imagine one way in which it would possible to measure x/y/z-height of the object: affix the object to a glass substrate. Deposit a layer of fluorophore (like a FITC-albumin) over the entirety of the surface, including the top of the object and the substrate. Turn the glass over onto another piece of glass. Image the object "upside down," so that you see the top of the object first, then you see the rest of the glass substrate. The delta between the z-position that you see the top of your object and the glass substrate is the height of the object.
Constructions will be near impossible. You need to image THROUGH an object to reconstruct it. And in most senses of the question, this whole idea would be virtually impossible, to my knowledge.
If your are talking about a biological sample, then you may have to carry out an optical clearing. I am not an expert so I'll rather orient you towards a good review (attached link).
http://www.ncbi.nlm.nih.gov/pubmed/24348874
The biological samples can be cleared by using Murray's clear. They become almost transparent. Before clearing they should be dehydrated in graded isopropanol series (70%-80%-90%-100%-100%, 30 sec each) and subsequently placed in Murray's clear mixture (1 part benzyl alcohol : 2 parts benzyl benzoate). Better if you change the solution three times (1 min each). Murray's clear is also used for mounting the sample.
It is possible if you use the confocal in reflection mode without filtering the reflected light.
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