I am working on the culture of tumor spheroids to evaluate the permeability of a polymeric drug delivery system. I would like to take CLSM images of spheroids when they are alive. Is it resonable staining my spheroids with Hohests since they are freindly for live cells? I have try to use Hochest 33342 on the spheroid and find out that just a layer of cells near the surface could be well stained. Could I draw a conclusion that my DDS penetrate into the center when I observe the fluorescent signal of DDS was surounded by the Hochest outline in the certain depth?
The spheroids staining is not only dependent on the penetration of the stain to the core, but also the penetration depth of the excitation laser and the detection of emission signals from the depths without getting scattered. For thick tissues or spheroids, a multi-photon microscope is highly recommended where the IR laser has a very good excitation penetration depth into the tissue besides being less photo-toxic to the live cells. The detectors on the multi-photon system is highly sensitive and there is no pin-hole unlike the confocal microscope and be capable of detecting faint signals (the reason the multi-photon microscopes are located in dark rooms to avoid stray light being detected by the detectors). The other option is to use the light sheet microscopy.
I agree with Sathya Srinivasan about penetration of the excitation and emission light being a problem in spheroids.
If you need a low-tech solution, consider staining then trying to cut your spheroid with a scalpel and orientate it so that you see the cut surface where the middle of the spheroid is not exposed. You can embed the spheroid in 1% agarose before cutting to help keep track of the orientation.
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