Ex Vivo Fluorescent Bone Stain?

[John Sevick]

*****
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'm very new to microscopy, so I apologize in advance if this is the wrong place to be posing
this question:

I've been struggling to find a fluorescent bone mineral stain that functions ex vivo. In the
literature it seems that most stains (calcein, alizarin complexone, xylenol orange etc.) must
be injected in vivo to work properly – this is also what people in the lab have been telling
me.

Because I'm not familiar with how these stains work, I'm wondering if there is a fluorescent
stain that can be applied to mineralized tissue ex vivo (not necessarily cells, just the
mineralized matrix)?

I am trying to differentiate mineralized tissue from soft tissue of frozen, then thawed
samples using two-photon excitation microscopy.

Any help would be greatly appreciated. Thank you!

Johnathan Sevick, BASc| MSc Candidate
Biomedical Engineering Graduate Program | University of Calgary
Email: [hidden email]

[Steffen Dietzel]

*****
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.
*****

Jonathan,

if you are using multi-photon anyway, have you tried second harmonic
generation microscopy? Bone usually gives a decent signal and you do not
even have to stain. Just hit the sample with twice the wavelength of
whatever your favorite emission filter lets through.

Steffen

Am 14.10.2015 um 18:32 schrieb John Sevick:

> *****
> 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'm very new to microscopy, so I apologize in advance if this is the wrong place to be posing
> this question:
>
> I've been struggling to find a fluorescent bone mineral stain that functions ex vivo. In the
> literature it seems that most stains (calcein, alizarin complexone, xylenol orange etc.) must
> be injected in vivo to work properly – this is also what people in the lab have been telling
> me.
>
> Because I'm not familiar with how these stains work, I'm wondering if there is a fluorescent
> stain that can be applied to mineralized tissue ex vivo (not necessarily cells, just the
> mineralized matrix)?
>
> I am trying to differentiate mineralized tissue from soft tissue of frozen, then thawed
> samples using two-photon excitation microscopy.
>
> Any help would be greatly appreciated. Thank you!
>
> Johnathan Sevick, BASc| MSc Candidate
> Biomedical Engineering Graduate Program | University of Calgary
> Email: [hidden email]
>
>
> --
> ------------------------------------------------------------
> Steffen Dietzel, PD Dr. rer. nat
> Ludwig-Maximilians-Universität München
> Biomedical Center (BMC)
> Head of the Core Facility Bioimaging
>
> Großhaderner Straße 9
> D-82152 Planegg-Martinsried
> Germany

[Tim Feinstein]

*****
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.
*****

Agreed that SHG is the way to go.  Mineralized tissue is generally a real
pain for imaging.  

Is micro-CT out of the question?


Tim

Timothy Feinstein, Ph.D.
Research Scientist
University of Pittsburgh Department of Developmental Biology





On 10/14/15, 1:30 PM, "Confocal Microscopy List on behalf of Steffen
Dietzel" <[hidden email] on behalf of
[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.
>*****
>
>Jonathan,
>
>if you are using multi-photon anyway, have you tried second harmonic
>generation microscopy? Bone usually gives a decent signal and you do not
>even have to stain. Just hit the sample with twice the wavelength of
>whatever your favorite emission filter lets through.
>
>Steffen
>
>Am 14.10.2015 um 18:32 schrieb John Sevick:
>> *****
>> 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'm very new to microscopy, so I apologize in advance if this is the
>>wrong place to be posing
>> this question:
>>
>> I've been struggling to find a fluorescent bone mineral stain that
>>functions ex vivo. In the
>> literature it seems that most stains (calcein, alizarin complexone,
>>xylenol orange etc.) must
>> be injected in vivo to work properly ­ this is also what people in the
>>lab have been telling
>> me.
>>
>> Because I'm not familiar with how these stains work, I'm wondering if
>>there is a fluorescent
>> stain that can be applied to mineralized tissue ex vivo (not
>>necessarily cells, just the
>> mineralized matrix)?
>>
>> I am trying to differentiate mineralized tissue from soft tissue of
>>frozen, then thawed
>> samples using two-photon excitation microscopy.
>>
>> Any help would be greatly appreciated. Thank you!
>>
>> Johnathan Sevick, BASc| MSc Candidate
>> Biomedical Engineering Graduate Program | University of Calgary
>> Email: [hidden email]
>>
>>
>> --
>> ------------------------------------------------------------
>> Steffen Dietzel, PD Dr. rer. nat
>> Ludwig-Maximilians-Universität München
>> Biomedical Center (BMC)
>> Head of the Core Facility Bioimaging
>>
>> Großhaderner Straße 9
>> D-82152 Planegg-Martinsried
>> Germany