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SHG image of mouse heart

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Shana Watson

SHG image of mouse heart

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Hello,

I am trying to identify the cardiac fibers (myofiber) within an optical section taken from a mouse heart. Can anyone take a look at the image below and confirm what I am actually seeing?

http://imgur.com/ElaYsiR

Craig Brideau

Re: SHG image of mouse heart

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That looks like auto-fluorescence. Can you verify that the signal is
narrow-band (i.e. spectrum should be a copy of your laser)? You can also
check for SHG if you can rotate the polarization of your laser; aligned or
counter-aligned will give you drastically different signal if it is true
SHG.

Craig

On Fri, Aug 18, 2017 at 12:23 PM, Shana Watson <[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.
> *****
>
> Hello,
>
> I am trying to identify the cardiac fibers (myofiber) within an optical
> section taken from a mouse heart. Can anyone take a look at the image below
> and confirm what I am actually seeing?
>
> http://imgur.com/ElaYsiR
>

JOEL B. SHEFFIELD

Re: SHG image of mouse heart

In reply to this post by Shana Watson

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Hi Shana,

Is there a fluorescent label in this sample? If so, what is it? If not,
and you are looking at fluorescence, sometimes formaldehyde will induce a
green fluorescence.

Joel

Joel B. Sheffield, Ph.D
Department of Biology
Temple University
Philadelphia, PA 19122
Voice: 215 204 8839
e-mail: [hidden email]
URL: *https://bio.cst.temple.edu/~jbs/ <https://bio.cst.temple.edu/~jbs/>
<http://tinyurl.com/khbouft>*

On Fri, Aug 18, 2017 at 2:23 PM, Shana Watson <[hidden email]>
wrote:

Arvydas Matiukas

Re: SHG image of mouse heart

In reply to this post by Craig Brideau

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To me it looks like mouse heart myofibers that I imaged with confocal.
It would be helpful to measure fiber diameter that in my images was
10-15um.

Best,
Arvydas

Arvydas Matiukas, Ph.D.
Director of Confocal&Two-Photon Core
SUNY Upstate Medical University
Neuroscience & Physiology Dept
Email: [hidden email]
>>> Craig Brideau <[hidden email]> 8/18/2017 3:55 PM >>>
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*****

That looks like auto-fluorescence. Can you verify that the signal is
narrow-band (i.e. spectrum should be a copy of your laser)? You can also
check for SHG if you can rotate the polarization of your laser; aligned or
counter-aligned will give you drastically different signal if it is true
SHG.

Craig

On Fri, Aug 18, 2017 at 12:23 PM, Shana Watson <[hidden email]>
wrote:

Gary G. Li-2

Re: SHG image of mouse heart

In reply to this post by Shana Watson

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Dear Shana,

I think you have acquired an image of what you were looking for, cardiac
fibers (myofibers).

Firstly, cardiac muscle fiber cells are extensively branched and are
connected to one another at their ends by intercalated discs. Your image
shows such a pattern.

https://upload.wikimedia.org/wikipedia/commons/7/7e/1020_Cardiac_Muscle.jpg

Secondly, biological materials such as collagen and muscle myosin can
produce SHG signals. In muscle tissue, collagen serves as a major
component of the endomysium.

https://en.wikipedia.org/wiki/Endomysium#/media/File:Illu_muscle_structure.jpg

As you know, SHG is a frequency doubling of the excitation light. In order
to confirm further that what you have detected is an SHG image, not from
autofluorescence, you can use a different excitation wavelength and signal
detection range to acquire a test image. Collagen generates high intensity
SHG signal, and in contrast, autofluorescence is usually a broad band
signal of low intensity.

Sincerely

Gary G Li, PhD

On Fri, Aug 18, 2017 at 2:23 PM, Shana Watson <[hidden email]>
wrote:

mcammer

Re: SHG image of mouse heart

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Sorry I cannot post the images at this time, but I can report we've gotten excellent second harmonic imaging of collagen in PFA mouse heart using 940 nm approx 100 fs pulses with a 20X N.A. 1.0 lens.

Michael Cammer, Research Scientist, DART Microscopy Laboratory
NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY 10016
C: 914-309-3270 [hidden email] http://microscopynotes.com/
https://med.nyu.edu/research/research-resources/scientific-cores-shared-resources/microscopy-laboratory

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Gary G. Li
Sent: Friday, August 18, 2017 9:24 PM
To: [hidden email]
Subject: Re: SHG image of mouse heart

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Dear Shana,

I think you have acquired an image of what you were looking for, cardiac fibers (myofibers).

Firstly, cardiac muscle fiber cells are extensively branched and are
connected to one another at their ends by intercalated discs. Your image
shows such a pattern.

https://urldefense.proofpoint.com/v2/url?u=https-3A__upload.wikimedia.org_wikipedia_commons_7_7e_1020-5FCardiac-5FMuscle.jpg&d=DQIBaQ&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=Bi7YtECVx_XfM9rDmGtaq8VZDVn4kIbyDvUfOrTrKPU&s=r26ZeITgSupGiOCgTJGzi8mInH94kp9o2Ml0wFp-R4c&e=

Secondly, biological materials such as collagen and muscle myosin can produce SHG signals. In muscle tissue, collagen serves as a major component of the endomysium.

https://urldefense.proofpoint.com/v2/url?u=https-3A__en.wikipedia.org_wiki_Endomysium-23_media_File-3AIllu-5Fmuscle-5Fstructure.jpg&d=DQIBaQ&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=Bi7YtECVx_XfM9rDmGtaq8VZDVn4kIbyDvUfOrTrKPU&s=3hd_Iwf2hEeeYWSG1ST0sA2Gz1ZhcAgH2vtWd6sdmo4&e=

As you know, SHG is a frequency doubling of the excitation light. In order to confirm further that what you have detected is an SHG image, not from autofluorescence, you can use a different excitation wavelength and signal detection range to acquire a test image. Collagen generates high intensity SHG signal, and in contrast, autofluorescence is usually a broad band signal of low intensity.

Sincerely

Gary G Li, PhD

On Fri, Aug 18, 2017 at 2:23 PM, Shana Watson <[hidden email]>
wrote:

> *****
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> m_Fu9e-WHf2pcCycs&e= Post images on
> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DQIBaQ&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=Bi7YtECVx_XfM9rDmGtaq8VZDVn4kIbyDvUfOrTrKPU&s=uu3Nl4A7pEAU2eEM8sVnZ4nwV9iroQM3x8D9WJLf3gQ&e= and include the link in your posting.
> *****
>
> Hello,
>
> I am trying to identify the cardiac fibers (myofiber) within an
> optical section taken from a mouse heart. Can anyone take a look at
> the image below and confirm what I am actually seeing?
>
> https://urldefense.proofpoint.com/v2/url?u=http-3A__imgur.com_ElaYsiR&
> d=DQIBaQ&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAy
> dlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=Bi7YtECVx_XfM9rDmGtaq8VZDVn4kIbyDvUfO
> rTrKPU&s=I74nAPswVGIIuxPc5RVMEx6VSv8k4-cSz2hD3wsLhHo&e=
>

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