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My basic experience is that optical clearing mostly acts by refractive index
matching, but there is some evidence that agents like glycerol also disrupt
collagen fibril structure that helps to reduce diffraction and I would imagine
autofluorescence (Bui et al. Lasers in Surgery and Medicine 41:142148
(2009)).
I have been trying a wide variety of clearing agents for skeletal muscle. From
my experience with clearing agents, 2,2 - Thiodiethanol 97% works very well to
clear and match refractive index for immersion lenses, and allows imaging up to
~200 microns using traditional confocal techniques. Potentially using
multiphoton methods would get you substantially deeper, if that is available.
However, even with multiphoton you have to consider the scattering coming
back up through the tissue, which similarly reduces the optical resolution.
Because of the nature of my experiments, intact scale is important and I have
been hesitant to use ScaleA2 clearing during to the significant swelling
exhibited by the tissues despite the ability to resolve deep into the tissue
(~22% increase in volume). As an alternative to ScaleA2, it may be useful to
try FocusClear (available from CedarLane Labs), which from my experiences
makes tissues incredibly transparent (more so than TDE, and much more so
than glycerol clearing, but it does not have the ideal 1.515/8 RI of oil). Using
FocusClear has allowed me to see much deeper in the tissue (>500 microns,
versus ~80 microns in 50/50 glycerol:pbs clearing). One issue to consider
though is that it seems that far red fluorophore signal (DRAQ5, nuclear stain in
my protocols) appears to be somewhat quenched with MountClear and TDE (so
some increased background fluorescence)
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