Skeletal muscle SHG imaging

[Cheryl San Emeterio]

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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

[Craig Brideau]

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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:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> 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
>