Re: Skeletal muscle SHG imaging

Posted by Craig Brideau on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Skeletal-muscle-SHG-imaging-tp7586725p7586728.html

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First pass I can think of is that the higher NA of the 40x is increasing
the amount of SHG that scatters backward. Basically you are generating SHG
over a wider 'family' of angles so more of it is likely to scatter
backwards. Conversely, the lower-NA 10x will strongly favor forward
scattering as it has a narrower cone of angles. Also note the higher NA 40x
is more likely to catch back scattered light overall.

Craig

On Mon, Apr 10, 2017 at 9:16 AM, Cheryl San Emeterio <[hidden email]>
wrote:

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>
> Hi all,
>
> I hope you are well. I'm doing some forward and backward SHG imaging on
> whole mount muscle samples and had a question on why the F/B SHG looks
> differently on 10x vs 40x. On 10x, with the master gains similar, the
> forward SHG signal is very bright, probably brighter than the backward SHG,
> which I've seen published in various papers. However, when I switch over to
> a 40x oil objective (obviously I had to change the settings, decrease laser
> power, etc) - the backwards SHG is now very bright, much brighter than the
> forward SHG. Can anyone shed some light on the practical reason why this
> would be?
>
> Also, I'm picking up a "ghostly" SHG signal in the muscle fibers in the
> backward SHG on 40x (I've read that the myosin within fibers should be
> mostly within the forward SHG signal in the characteristic "striped"
> pattern). I can see the characteristic wavy collagen (backward SHG) between
> the fibers, but then the fibers are showing up in backward SHG with some
> faint striated pattern (whereas with the low laser power I'm using on 40x
> I'm hardly picking up the striated myosi pattern in forward SHG) . I'm
> unsure as to whether I should be collecting this "ghostly" signal within
> the fibers ob backward SHG, or whether I should decrease gain/offset until
> that signal is no longer detected. I'm unable to attach an image here but
> would be happy to correspond via email ([hidden email]).
>
> Finally, would you expect newly deposited collagen (as in the case of
> granulation tissue) to have a fainter SHG signal compared to collagen in a
> resting muscle?
>
> Thank you all in advance for any advice you are able to share any advice.
>
> Best,
> Cheryl
>