Photographing GFP mice

[Jeremy Sanderson-3]

I have to take photographs of GFP newborn and adult m
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Dear Colleagues,

I have to take photographs of GFP newborn and adult mice to support a microscopy paper.This is to show GFP throughout the coat of the newborns, and in the tail tips and feet of the adults.

Normally mice are photographed with a 60mm f# 2.8 lens on a Nikon D70S with sync flash at 1/60th or 1/125th sec with an ISO of 100 or 200 set on the camera CCD. That, however, is for normal room lighting – plenty of photons.

We don’t want to go above ISO 800 because of noise, yet need to freeze the movement of the mice.

Has anyone done this, and can advise on exposure, whether to use sync flash as well as the UV source.

I have been advised to anaesthetise the mice and photograph out of the cage. We'd prefer not to anaesthetise, but there may be no option.

Any advice, or if you can direct me to someone who has done this. All input gratefully received.

Thanks,
Jeremy Sanderson
Bio-Imaging, MRC Harwell.

[mcammer]

Baby mice we've done with a low power fluorescent dissection 'scope.  If you only need to show the feet and tails of adults, this can be done on a dissection scope too.  We found that using a grayscale camera was fine.  We put GFP mouse paws next to normal mice and then pseudocolored (see http://www.flickr.com/photos/mcammer/422255876/ , although these look like baby mice paws).  One trick we use was to run the camera continuously streaming to the computer while two people held the mice in place.  Then we picked the still from the movie instead of trying to get the mice in position and then clicking the picture.
________________________________________________________
Michael Cammer, Assistant Research Scientist
Skirball Institute of Biomolecular Medicine
Lab: [phone]  Cell: [phone]

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Jeremy Sanderson
Sent: Thursday, February 09, 2012 8:27 AM
To: [hidden email]
Subject: Photographing GFP mice

I have to take photographs of GFP newborn and adult m
*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Dear Colleagues,

I have to take photographs of GFP newborn and adult mice to support a microscopy paper.This is to show GFP throughout the coat of the newborns, and in the tail tips and feet of the adults.

Normally mice are photographed with a 60mm f# 2.8 lens on a Nikon D70S with sync flash at 1/60th or 1/125th sec with an ISO of 100 or 200 set on the camera CCD. That, however, is for normal room lighting – plenty of photons.

We don’t want to go above ISO 800 because of noise, yet need to freeze the movement of the mice.

Has anyone done this, and can advise on exposure, whether to use sync flash as well as the UV source.

I have been advised to anaesthetise the mice and photograph out of the cage. We'd prefer not to anaesthetise, but there may be no option.

Any advice, or if you can direct me to someone who has done this. All input gratefully received.

Thanks,
Jeremy Sanderson
Bio-Imaging, MRC Harwell.


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This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email.
=================================

[Julio Vazquez]

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If you have access to a fluorescent scanner for green fluorescence (Typhoon, etc ...), you may also be able to use that. These are slow though, and you probably will need to immobilize the mice through anesthesia, or other means.

--
Julio Vazquez
Fred Hutchinson Cancer Research Center
Seattle, WA

http://www.fhcrc.org
==

On Feb 9, 2012, at 5:27 AM, Jeremy Sanderson wrote:

> I have to take photographs of GFP newborn and adult m
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Colleagues,
>
> I have to take photographs of GFP newborn and adult mice to support a microscopy paper.This is to show GFP throughout the coat of the newborns, and in the tail tips and feet of the adults.
>
> Normally mice are photographed with a 60mm f# 2.8 lens on a Nikon D70S with sync flash at 1/60th or 1/125th sec with an ISO of 100 or 200 set on the camera CCD. That, however, is for normal room lighting – plenty of photons.
>
> We don’t want to go above ISO 800 because of noise, yet need to freeze the movement of the mice.
>
> Has anyone done this, and can advise on exposure, whether to use sync flash as well as the UV source.
>
> I have been advised to anaesthetise the mice and photograph out of the cage. We'd prefer not to anaesthetise, but there may be no option.
>
> Any advice, or if you can direct me to someone who has done this. All input gratefully received.
>
> Thanks,
> Jeremy Sanderson
> Bio-Imaging, MRC Harwell.
>

[Jim Mansfield]

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I'm kind of jumping in late on this, but do you need to be able to actually
visualize the GFP signal? If so, then standard photography will probably not
work - the GFP fluorescence is usually invisible in room light.

To see the actual fluorescence you'd need to use a small animal fluorescence
imaging system, like those sold by PerkinElmer, for instance. Or you'd have
to build yourself one with excitation and emission filters, illumination
lamps, etc.

Nearly everyone who uses those system anaesthetizes the mice as the exposure
times for fluorescence often are long enough to preclude awake (and moving)
imaging.

Jim


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Julio Vazquez
Sent: Thursday, February 09, 2012 12:45 PM
To: [hidden email]
Subject: Re: Photographing GFP mice

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http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

If you have access to a fluorescent scanner for green fluorescence (Typhoon,
etc ...), you may also be able to use that. These are slow though, and you
probably will need to immobilize the mice through anesthesia, or other
means.

--
Julio Vazquez
Fred Hutchinson Cancer Research Center
Seattle, WA

http://www.fhcrc.org
==

On Feb 9, 2012, at 5:27 AM, Jeremy Sanderson wrote:

> I have to take photographs of GFP newborn and adult m
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Colleagues,
>
> I have to take photographs of GFP newborn and adult mice to support a
microscopy paper.This is to show GFP throughout the coat of the newborns,
and in the tail tips and feet of the adults.
>
> Normally mice are photographed with a 60mm f# 2.8 lens on a Nikon D70S
with sync flash at 1/60th or 1/125th sec with an ISO of 100 or 200 set on
the camera CCD. That, however, is for normal room lighting - plenty of
photons.
>
> We don't want to go above ISO 800 because of noise, yet need to freeze the
movement of the mice.
>
> Has anyone done this, and can advise on exposure, whether to use sync
flash as well as the UV source.
>
> I have been advised to anaesthetise the mice and photograph out of the
cage. We'd prefer not to anaesthetise, but there may be no option.
>
> Any advice, or if you can direct me to someone who has done this. All
input gratefully received.
>
> Thanks,
> Jeremy Sanderson
> Bio-Imaging, MRC Harwell.
>

[Peter Gabriel Pitrone]

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Hello Jeremy,

I would recommend a faster lens (as fast as you can get, 1.8 or 1.4 if
possible) and bigger chip (I doubt you have access to a Hasselblad, but I
would see about a 35 mm chip at least), other wise you might not get
enough light...

my 0.02 EUR,

Pete

On Thu, February 9, 2012 2:27 pm, Jeremy Sanderson wrote:
| I have to take photographs of GFP newborn and adult m
| *****
| To join, leave or search the confocal microscopy listserv, go to:
| http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
| *****
|
| Dear Colleagues,
|
| I have to take photographs of GFP newborn and adult mice to support a
| microscopy paper.This is to show GFP throughout the coat of the newborns,
| and in the tail tips and feet of the adults.
|
| Normally mice are photographed with a 60mm f# 2.8 lens on a Nikon D70S
| with sync flash at 1/60th or 1/125th sec with an ISO of 100 or 200 set on
| the camera CCD. That, however, is for normal room lighting – plenty of
| photons.
|
| We don’t want to go above ISO 800 because of noise, yet need to freeze
| the movement of the mice.
|
| Has anyone done this, and can advise on exposure, whether to use sync
| flash as well as the UV source.
|
| I have been advised to anaesthetise the mice and photograph out of the
| cage. We'd prefer not to anaesthetise, but there may be no option.
|
| Any advice, or if you can direct me to someone who has done this. All
| input gratefully received.
|
| Thanks,
| Jeremy Sanderson
| Bio-Imaging, MRC Harwell.
|
|


--
Peter Gabriel Pitrone - TechRMS
Microscopy/Imaging Specialist
Prof. Dr. Pavel Tomancak group
Max Planck Institute for
Molecular Biology and Genetics
Pfotenhauerstr. 108
01307 Dresden

"If a straight line fit is required, obtain only two data points." - Anon.

[Steffen Dietzel]

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*****

I am jumping in on this thread with a similar problem: For us it is not
photographing but seeing the GFP.

We need to distinguish fluorescent from non-fluorescent littermaids.
This works well under a fluorescence stereo microscope but we would like
to do the screening with a handheld lamp (in a different room). I
imagine a blue LED flashlight and a cheap long-pass filter.
So far I came across two products, for ~ US$ 200 or ~ € 500,
respectively (http://www.nightsea.com/gfp.htm and
http://www.clarechemical.com/lamps.htm , the latter company also offers
camera filters which might be helpful for Jeremy).

I was hoping for something cheaper. Maybe someone found a 'normal'
bright blue LED flashlight that works for excitation or yet another
solution?

Steffen


On 09.02.2012 18:44, Julio Vazquez wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> If you have access to a fluorescent scanner for green fluorescence (Typhoon, etc ...), you may also be able to use that. These are slow though, and you probably will need to immobilize the mice through anesthesia, or other means.
>
> --
> Julio Vazquez
> Fred Hutchinson Cancer Research Center
> Seattle, WA
>
> http://www.fhcrc.org
> ==
>
> On Feb 9, 2012, at 5:27 AM, Jeremy Sanderson wrote:
>
>> I have to take photographs of GFP newborn and adult m
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Dear Colleagues,
>>
>> I have to take photographs of GFP newborn and adult mice to support a microscopy paper.This is to show GFP throughout the coat of the newborns, and in the tail tips and feet of the adults.
>>
>> Normally mice are photographed with a 60mm f# 2.8 lens on a Nikon D70S with sync flash at 1/60th or 1/125th sec with an ISO of 100 or 200 set on the camera CCD. That, however, is for normal room lighting – plenty of photons.
>>
>> We don’t want to go above ISO 800 because of noise, yet need to freeze the movement of the mice.
>>
>> Has anyone done this, and can advise on exposure, whether to use sync flash as well as the UV source.
>>
>> I have been advised to anaesthetise the mice and photograph out of the cage. We'd prefer not to anaesthetise, but there may be no option.
>>
>> Any advice, or if you can direct me to someone who has done this. All input gratefully received.
>>
>> Thanks,
>> Jeremy Sanderson
>> Bio-Imaging, MRC Harwell.
>>
>

--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Walter-Brendel-Zentrum für experimentelle Medizin (WBex)
Head of light microscopy

Mail room:
Marchioninistr. 15, D-81377 München

Building location:
Marchioninistr. 27,  München-Großhadern

[Jim Mansfield]

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*****

Hi Steffen,

I know you said you would like an inexpensive way to get rid of the interference from autofluorescence, and that is a laudable goal. Unfortunately it is not very likely! The problem is that autofluorescence emits at the same wavelengths as the GFP (or any other fluor, for that matter), and there is no simple filter combination that will enable you to separate the GFP photons from the autofluorescence photons, as both have peak emissions at the same wavelength.

The only way to separate the autofluorescence from the signal of interest is to use spectral imaging. That is true of any autofluorescence on a microscope, a macroscope or in a whole animal imaging system.

There are two main commercial whole animal fluorescence imaging systems that do this (the Maestro and the IVIS Spectrum, both now sold by PerkinElmer). For a macroscope, you can use a Nuance multispectral imaging system (PerkinElmer) that fits on the C-mount of any microscope (macroscope or microscope). But none of these options is anywhere near less than 1000 euro!

Here are some references for you to look at, though:

Autofluorescence removal, multiplexing, and automated analysis methods for in-vivo fluorescence imaging. Mansfield JR, Gossage KW, Hoyt CC, Levenson RM. J Biomed Opt. 2005 Jul-Aug;10(4):41207.

Distinguished photons: a review of in vivo spectral fluorescence imaging in small animals. Mansfield JR. Curr Pharm Biotechnol. 2010 Sep 1;11(6):628-38. Review.

Jim



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Steffen Dietzel
Sent: Friday, February 10, 2012 4:53 AM
To: [hidden email]
Subject: Re: Photographing and seeing GFP mice

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

I am jumping in on this thread with a similar problem: For us it is not
photographing but seeing the GFP.

We need to distinguish fluorescent from non-fluorescent littermaids.
This works well under a fluorescence stereo microscope but we would like
to do the screening with a handheld lamp (in a different room). I
imagine a blue LED flashlight and a cheap long-pass filter.
So far I came across two products, for ~ US$ 200 or ~ € 500,
respectively (http://www.nightsea.com/gfp.htm and
http://www.clarechemical.com/lamps.htm , the latter company also offers
camera filters which might be helpful for Jeremy).

I was hoping for something cheaper. Maybe someone found a 'normal'
bright blue LED flashlight that works for excitation or yet another
solution?

Steffen


On 09.02.2012 18:44, Julio Vazquez wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> If you have access to a fluorescent scanner for green fluorescence (Typhoon, etc ...), you may also be able to use that. These are slow though, and you probably will need to immobilize the mice through anesthesia, or other means.
>
> --
> Julio Vazquez
> Fred Hutchinson Cancer Research Center
> Seattle, WA
>
> http://www.fhcrc.org
> ==
>
> On Feb 9, 2012, at 5:27 AM, Jeremy Sanderson wrote:
>
>> I have to take photographs of GFP newborn and adult m
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Dear Colleagues,
>>
>> I have to take photographs of GFP newborn and adult mice to support a microscopy paper.This is to show GFP throughout the coat of the newborns, and in the tail tips and feet of the adults.
>>
>> Normally mice are photographed with a 60mm f# 2.8 lens on a Nikon D70S with sync flash at 1/60th or 1/125th sec with an ISO of 100 or 200 set on the camera CCD. That, however, is for normal room lighting – plenty of photons.
>>
>> We don’t want to go above ISO 800 because of noise, yet need to freeze the movement of the mice.
>>
>> Has anyone done this, and can advise on exposure, whether to use sync flash as well as the UV source.
>>
>> I have been advised to anaesthetise the mice and photograph out of the cage. We'd prefer not to anaesthetise, but there may be no option.
>>
>> Any advice, or if you can direct me to someone who has done this. All input gratefully received.
>>
>> Thanks,
>> Jeremy Sanderson
>> Bio-Imaging, MRC Harwell.
>>
>

--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Walter-Brendel-Zentrum für experimentelle Medizin (WBex)
Head of light microscopy

Mail room:
Marchioninistr. 15, D-81377 München

Building location:
Marchioninistr. 27,  München-Großhadern

[mcammer]

A few years ago (2008?) when looking for a cheap UV source I found a LED light advertised at UV but really with bright light in the 460-480 nm range.  I taped an old FITC filter on the front of a snapshot camera and this combo worked fairly well for imaging GFP except that the camera overexposed the blue light.  




-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Steffen Dietzel
Sent: Friday, February 10, 2012 4:53 AM
To: [hidden email]
Subject: Re: Photographing and seeing GFP mice

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

I am jumping in on this thread with a similar problem: For us it is not
photographing but seeing the GFP.

We need to distinguish fluorescent from non-fluorescent littermaids.
This works well under a fluorescence stereo microscope but we would like
to do the screening with a handheld lamp (in a different room). I
imagine a blue LED flashlight and a cheap long-pass filter.
So far I came across two products, for ~ US$ 200 or ~ € 500,
respectively (http://www.nightsea.com/gfp.htm and
http://www.clarechemical.com/lamps.htm , the latter company also offers
camera filters which might be helpful for Jeremy).

I was hoping for something cheaper. Maybe someone found a 'normal'
bright blue LED flashlight that works for excitation or yet another
solution?

Steffen


On 09.02.2012 18:44, Julio Vazquez wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> If you have access to a fluorescent scanner for green fluorescence (Typhoon, etc ...), you may also be able to use that. These are slow though, and you probably will need to immobilize the mice through anesthesia, or other means.
>
> --
> Julio Vazquez
> Fred Hutchinson Cancer Research Center
> Seattle, WA
>
> http://www.fhcrc.org
> ==
>
> On Feb 9, 2012, at 5:27 AM, Jeremy Sanderson wrote:
>
>> I have to take photographs of GFP newborn and adult m
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Dear Colleagues,
>>
>> I have to take photographs of GFP newborn and adult mice to support a microscopy paper.This is to show GFP throughout the coat of the newborns, and in the tail tips and feet of the adults.
>>
>> Normally mice are photographed with a 60mm f# 2.8 lens on a Nikon D70S with sync flash at 1/60th or 1/125th sec with an ISO of 100 or 200 set on the camera CCD. That, however, is for normal room lighting – plenty of photons.
>>
>> We don’t want to go above ISO 800 because of noise, yet need to freeze the movement of the mice.
>>
>> Has anyone done this, and can advise on exposure, whether to use sync flash as well as the UV source.
>>
>> I have been advised to anaesthetise the mice and photograph out of the cage. We'd prefer not to anaesthetise, but there may be no option.
>>
>> Any advice, or if you can direct me to someone who has done this. All input gratefully received.
>>
>> Thanks,
>> Jeremy Sanderson
>> Bio-Imaging, MRC Harwell.
>>
>

--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Walter-Brendel-Zentrum für experimentelle Medizin (WBex)
Head of light microscopy

Mail room:
Marchioninistr. 15, D-81377 München

Building location:
Marchioninistr. 27,  München-Großhadern

------------------------------------------------------------
This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email.
=================================

[MJ Tomaszewski]

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To join, leave or search the confocal microscopy listserv, go to:
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*****

Not sure if this would work, but thinkgeek.com has an LED lightbulb that
you can choose colors for.  It's pretty cheap.
http://www.thinkgeek.com/gadgets/lights/831e/?srp=41#tabs

Monica

On Fri, Feb 10, 2012 at 8:10 AM, Cammer, Michael <[hidden email]
> wrote:

> A few years ago (2008?) when looking for a cheap UV source I found a LED
> light advertised at UV but really with bright light in the 460-480 nm
> range.  I taped an old FITC filter on the front of a snapshot camera and
> this combo worked fairly well for imaging GFP except that the camera
> overexposed the blue light.
>
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Steffen Dietzel
> Sent: Friday, February 10, 2012 4:53 AM
> To: [hidden email]
> Subject: Re: Photographing and seeing GFP mice
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I am jumping in on this thread with a similar problem: For us it is not
> photographing but seeing the GFP.
>
> We need to distinguish fluorescent from non-fluorescent littermaids.
> This works well under a fluorescence stereo microscope but we would like
> to do the screening with a handheld lamp (in a different room). I
> imagine a blue LED flashlight and a cheap long-pass filter.
> So far I came across two products, for ~ US$ 200 or ~ € 500,
> respectively (http://www.nightsea.com/gfp.htm and
> http://www.clarechemical.com/lamps.htm , the latter company also offers
> camera filters which might be helpful for Jeremy).
>
> I was hoping for something cheaper. Maybe someone found a 'normal'
> bright blue LED flashlight that works for excitation or yet another
> solution?
>
> Steffen
>
>
> On 09.02.2012 18:44, Julio Vazquez wrote:
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > If you have access to a fluorescent scanner for green fluorescence
> (Typhoon, etc ...), you may also be able to use that. These are slow
> though, and you probably will need to immobilize the mice through
> anesthesia, or other means.
> >
> > --
> > Julio Vazquez
> > Fred Hutchinson Cancer Research Center
> > Seattle, WA
> >
> > http://www.fhcrc.org
> > ==
> >
> > On Feb 9, 2012, at 5:27 AM, Jeremy Sanderson wrote:
> >
> >> I have to take photographs of GFP newborn and adult m
> >> *****
> >> To join, leave or search the confocal microscopy listserv, go to:
> >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >> *****
> >>
> >> Dear Colleagues,
> >>
> >> I have to take photographs of GFP newborn and adult mice to support a
> microscopy paper.This is to show GFP throughout the coat of the newborns,
> and in the tail tips and feet of the adults.
> >>
> >> Normally mice are photographed with a 60mm f# 2.8 lens on a Nikon D70S
> with sync flash at 1/60th or 1/125th sec with an ISO of 100 or 200 set on
> the camera CCD. That, however, is for normal room lighting – plenty of
> photons.
> >>
> >> We don’t want to go above ISO 800 because of noise, yet need to freeze
> the movement of the mice.
> >>
> >> Has anyone done this, and can advise on exposure, whether to use sync
> flash as well as the UV source.
> >>
> >> I have been advised to anaesthetise the mice and photograph out of the
> cage. We'd prefer not to anaesthetise, but there may be no option.
> >>
> >> Any advice, or if you can direct me to someone who has done this. All
> input gratefully received.
> >>
> >> Thanks,
> >> Jeremy Sanderson
> >> Bio-Imaging, MRC Harwell.
> >>
> >
>
>
> --
> ------------------------------------------------------------
> Steffen Dietzel, PD Dr. rer. nat
> Ludwig-Maximilians-Universität München
> Walter-Brendel-Zentrum für experimentelle Medizin (WBex)
> Head of light microscopy
>
> Mail room:
> Marchioninistr. 15, D-81377 München
>
> Building location:
> Marchioninistr. 27,  München-Großhadern
>
> ------------------------------------------------------------
> This email message, including any attachments, is for the sole use of the
> intended recipient(s) and may contain information that is proprietary,
> confidential, and exempt from disclosure under applicable law. Any
> unauthorized review, use, disclosure, or distribution is prohibited. If you
> have received this email in error please notify the sender by return email
> and delete the original message. Please note, the recipient should check
> this email and any attachments for the presence of viruses. The
> organization accepts no liability for any damage caused by any virus
> transmitted by this email.
> =================================
>

[Chris Baumann]

*****Commercial Response************

Steffen,

Chroma Technology Corp, the filter manufacture, sells filter goggles.  Something like this http://chroma.com/products/catalog/G-Series/G-515GFP coupled with a black light may do the trick if your GFP expression levels are high enough.

Chris

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Steffen Dietzel
Sent: Friday, February 10, 2012 4:53 AM
To: [hidden email]
Subject: Re: Photographing and seeing GFP mice

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

I am jumping in on this thread with a similar problem: For us it is not photographing but seeing the GFP.

We need to distinguish fluorescent from non-fluorescent littermaids.
This works well under a fluorescence stereo microscope but we would like to do the screening with a handheld lamp (in a different room). I imagine a blue LED flashlight and a cheap long-pass filter.
So far I came across two products, for ~ US$ 200 or ~ € 500, respectively (http://www.nightsea.com/gfp.htm and http://www.clarechemical.com/lamps.htm , the latter company also offers camera filters which might be helpful for Jeremy).

I was hoping for something cheaper. Maybe someone found a 'normal'
bright blue LED flashlight that works for excitation or yet another solution?

Steffen


On 09.02.2012 18:44, Julio Vazquez wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> If you have access to a fluorescent scanner for green fluorescence (Typhoon, etc ...), you may also be able to use that. These are slow though, and you probably will need to immobilize the mice through anesthesia, or other means.
>
> --
> Julio Vazquez
> Fred Hutchinson Cancer Research Center Seattle, WA
>
> http://www.fhcrc.org
> ==
>
> On Feb 9, 2012, at 5:27 AM, Jeremy Sanderson wrote:
>
>> I have to take photographs of GFP newborn and adult m
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Dear Colleagues,
>>
>> I have to take photographs of GFP newborn and adult mice to support a microscopy paper.This is to show GFP throughout the coat of the newborns, and in the tail tips and feet of the adults.
>>
>> Normally mice are photographed with a 60mm f# 2.8 lens on a Nikon D70S with sync flash at 1/60th or 1/125th sec with an ISO of 100 or 200 set on the camera CCD. That, however, is for normal room lighting – plenty of photons.
>>
>> We don’t want to go above ISO 800 because of noise, yet need to freeze the movement of the mice.
>>
>> Has anyone done this, and can advise on exposure, whether to use sync flash as well as the UV source.
>>
>> I have been advised to anaesthetise the mice and photograph out of the cage. We'd prefer not to anaesthetise, but there may be no option.
>>
>> Any advice, or if you can direct me to someone who has done this. All input gratefully received.
>>
>> Thanks,
>> Jeremy Sanderson
>> Bio-Imaging, MRC Harwell.
>>
>

--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Walter-Brendel-Zentrum für experimentelle Medizin (WBex) Head of light microscopy

Mail room:
Marchioninistr. 15, D-81377 München

Building location:
Marchioninistr. 27,  München-Großhadern

[Jamie Hayden]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

I needed to photograph precisely this situation for a paper and cover in Biology of Reproduction in August 2003. (Brinster, et. al., Restoration of Fertility by Germ Cell Transplantation Requires Effective Recipient Preparation, Biology of Reproductuction 2003; 69:412-420.) At the time, I used a Nikon 990 consumer camera (the original style that swiveled in the middle). It did not have particular good noise levels above 200 ISO, so the eventual exposure was 1/8 sec, f2.6 ISO 200 (from the metadata). A yellow "viewing filter" from an Illumitool imaging system was placed in front of the lens - this is approximately the same as an old Kodak #15 deep yellow filter. With the same conditions, you would probably be able to boost your ISO, and thereby your shutter speed, a few stops due to the better sensitivity of a more modern camera. The metadata says I used a daylight white balance, but I seem to remember making a custom setting taken from a piece of white paper under the UV light - with the yellow filter in place.

The major issues to deal with were the lighting and controlling the mice. For the light, we used a rather strong, hand-held UV light source. I'm sorry, but I have no specs on that, other than remembering that it looked like a large round searchlight with a handle. One person held the UV source at a distance that gave us an even illumination and the initial exposure was determined based on those conditions. This part was done in relatively dark conditions, with a closed door in the room we were working with. To add enough light to see the fur of the adult mouse and the existence of some litter mates that were not GFP positive, we opened the door to let in ambient light (probably fluorescent lights) and moved the table with the mice closer and closer to the open door until we got a good light balance. Flash did not work well at the time because I had no external control - meaning I only had the on-camera flash and that could not be reduced enough to avoid blowing out the GFP. Even if I could, the directional light would have not looked very good. Today, I could see using a studio light, very low power, and bounced, but have not actually tried that.

As for the mice, the good news is that the pups won't move much. They tend to huddle together to keep warm and stay put. The adult, however, was rather jumpy. I made a boxed-in area lined with black velvet to keep everybody in one place and had helpers to watch and corral the adult (it was a male because the paper was about male germ-line stem cells). The main challenge was to anticipate when the mouse would be in the right spot and stop moving for 1/8 sec - kind of like timing a photograph of a bouncing ball to get it at the top of the bounce where the movement is minimized. I took about 50 images before all the conditions came together.

A useful bit of information about photographing live, small lab animals (mice, rats, guinea pigs and small rabbits) If they are placed on a stand that is more than 3 times higher their body length, they will not jump down, so you can make a small platform to keep them in a limited area. The only time this ever failed on me was with young mice that started jumping like jumping beans as soon as we put them on top.

I hope this helps. If you have any questions, feel free to contact me offline

Jamie Hayden

*********************************
James Hayden, RBP, FBCA
Manager, Imaging Core Facility
The Wistar Institute
3601 Spruce St.
Philadelphia, PA   19104
[phone]
[hidden email]<mailto:[hidden email]>







On Feb 10, 2012, at 9:43 AM, Chris Baumann wrote:

*****Commercial Response************

Steffen,

Chroma Technology Corp, the filter manufacture, sells filter goggles.  Something like this http://chroma.com/products/catalog/G-Series/G-515GFP coupled with a black light may do the trick if your GFP expression levels are high enough.

Chris

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Steffen Dietzel
Sent: Friday, February 10, 2012 4:53 AM
To: [hidden email]<mailto:[hidden email]>
Subject: Re: Photographing and seeing GFP mice

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

I am jumping in on this thread with a similar problem: For us it is not photographing but seeing the GFP.

We need to distinguish fluorescent from non-fluorescent littermaids.
This works well under a fluorescence stereo microscope but we would like to do the screening with a handheld lamp (in a different room). I imagine a blue LED flashlight and a cheap long-pass filter.
So far I came across two products, for ~ US$ 200 or ~ € 500, respectively (http://www.nightsea.com/gfp.htm and http://www.clarechemical.com/lamps.htm , the latter company also offers camera filters which might be helpful for Jeremy).

I was hoping for something cheaper. Maybe someone found a 'normal'
bright blue LED flashlight that works for excitation or yet another solution?

Steffen


On 09.02.2012 18:44, Julio Vazquez wrote:
*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

If you have access to a fluorescent scanner for green fluorescence (Typhoon, etc ...), you may also be able to use that. These are slow though, and you probably will need to immobilize the mice through anesthesia, or other means.

--
Julio Vazquez
Fred Hutchinson Cancer Research Center Seattle, WA

http://www.fhcrc.org
==

On Feb 9, 2012, at 5:27 AM, Jeremy Sanderson wrote:

I have to take photographs of GFP newborn and adult m
*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Dear Colleagues,

I have to take photographs of GFP newborn and adult mice to support a microscopy paper.This is to show GFP throughout the coat of the newborns, and in the tail tips and feet of the adults.

Normally mice are photographed with a 60mm f# 2.8 lens on a Nikon D70S with sync flash at 1/60th or 1/125th sec with an ISO of 100 or 200 set on the camera CCD. That, however, is for normal room lighting – plenty of photons.

We don’t want to go above ISO 800 because of noise, yet need to freeze the movement of the mice.

Has anyone done this, and can advise on exposure, whether to use sync flash as well as the UV source.

I have been advised to anaesthetise the mice and photograph out of the cage. We'd prefer not to anaesthetise, but there may be no option.

Any advice, or if you can direct me to someone who has done this. All input gratefully received.

Thanks,
Jeremy Sanderson
Bio-Imaging, MRC Harwell.




--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Walter-Brendel-Zentrum für experimentelle Medizin (WBex) Head of light microscopy

Mail room:
Marchioninistr. 15, D-81377 München

Building location:
Marchioninistr. 27,  München-Großhadern

[Christian-103]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

"UV flashlights" range from $3 to a few hundred dollars, they're used for black light hunting for scorpions.  There is no reason to spend hundreds of dollars on an LED rig if you can get enough out of a hand held flashlight.  Of course those range from 385 to 405 nm, and you probably would prefer something closer to 488 nm.  A quick google search does turn up "GFP Flashlights".  If you do use something as simple as this, please let us know.

Christian


--- On Fri, 2/10/12, James Hayden <[hidden email]> wrote:

From: James Hayden <[hidden email]>
Subject: Re: Photographing and seeing GFP mice
To: [hidden email]
Date: Friday, February 10, 2012, 10:29 AM

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

I needed to photograph precisely this situation for a paper and cover in Biology of Reproduction in August 2003. (Brinster, et. al., Restoration of Fertility by Germ Cell Transplantation Requires Effective Recipient Preparation, Biology of Reproductuction 2003; 69:412-420.) At the time, I used a Nikon 990 consumer camera (the original style that swiveled in the middle). It did not have particular good noise levels above 200 ISO, so the eventual exposure was 1/8 sec, f2.6 ISO 200 (from the metadata). A yellow "viewing filter" from an Illumitool imaging system was placed in front of the lens - this is approximately the same as an old Kodak #15 deep yellow filter.

[Michael Herron]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

I have done this in the distant past.  For adult mice it helps a great
deal to remove the fur which autofluors .  Back then we used
"Softsheen-Carson Magic Shaving Powder Gold Fragrant" available at
drugstores.
You make a slurry out of it and paint a lightly anesthetized mouse
with it and let it stand for a couple minutes, then the fur just
rinses off under warm gently flowing tap water. Not sure is this would
pass your IRB these days, so ask!  The fur is completely removed and
does not grow back for a week or more.

To hold the mice still a 12x12 inch piece of plastic wrap stretched
over the mice and held in place by one person as another person
handles the imaging. The mice seem comfortable during this an I was
able to image 5 pups at a time which gave me a chance to include
negative controls.  This also works for RFP imaging.

For illumination I usually used a fiberoptic gooseneck lamp with a
filter slot that is in the unit before the fiber outlet (Schott).  Be
sure there is an infrared filter too.  To keep the mice warm during
this imaging I had a rubberized mat that is heated to ~37 degrees That
I did the imaging upon.  The camera I used was a SPOT and my exposures
were as long as 5 minutes but usually much less. A more sensitive
camera would be even better.

As for LED illumination of litter mates, I used a blue LED flashlight
and any long wave glasses would work fine.  Both the stuff from
Nightsea and Clare work too.  The Clare "Dark Reader" Transilluminator
is also great for gels so it has a dual use.

_Again, I must stress that all these methods should be approved by
your IRB committee._





On Fri, Feb 10, 2012 at 10:29 AM, James Hayden <[hidden email]> wrote:

>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I needed to photograph precisely this situation for a paper and cover in Biology of Reproduction in August 2003. (Brinster, et. al., Restoration of Fertility by Germ Cell Transplantation Requires Effective Recipient Preparation, Biology of Reproductuction 2003; 69:412-420.) At the time, I used a Nikon 990 consumer camera (the original style that swiveled in the middle). It did not have particular good noise levels above 200 ISO, so the eventual exposure was 1/8 sec, f2.6 ISO 200 (from the metadata). A yellow "viewing filter" from an Illumitool imaging system was placed in front of the lens - this is approximately the same as an old Kodak #15 deep yellow filter. With the same conditions, you would probably be able to boost your ISO, and thereby your shutter speed, a few stops due to the better sensitivity of a more modern camera. The metadata says I used a daylight white balance, but I seem to remember making a custom setting taken from a piece of white paper under the UV light - with the yellow filter in place.
>
> The major issues to deal with were the lighting and controlling the mice. For the light, we used a rather strong, hand-held UV light source. I'm sorry, but I have no specs on that, other than remembering that it looked like a large round searchlight with a handle. One person held the UV source at a distance that gave us an even illumination and the initial exposure was determined based on those conditions. This part was done in relatively dark conditions, with a closed door in the room we were working with. To add enough light to see the fur of the adult mouse and the existence of some litter mates that were not GFP positive, we opened the door to let in ambient light (probably fluorescent lights) and moved the table with the mice closer and closer to the open door until we got a good light balance. Flash did not work well at the time because I had no external control - meaning I only had the on-camera flash and that could not be reduced enough to avoid blowing out the GFP. Even if I could, the directional light would have not looked very good. Today, I could see using a studio light, very low power, and bounced, but have not actually tried that.
>
> As for the mice, the good news is that the pups won't move much. They tend to huddle together to keep warm and stay put. The adult, however, was rather jumpy. I made a boxed-in area lined with black velvet to keep everybody in one place and had helpers to watch and corral the adult (it was a male because the paper was about male germ-line stem cells). The main challenge was to anticipate when the mouse would be in the right spot and stop moving for 1/8 sec - kind of like timing a photograph of a bouncing ball to get it at the top of the bounce where the movement is minimized. I took about 50 images before all the conditions came together.
>
> A useful bit of information about photographing live, small lab animals (mice, rats, guinea pigs and small rabbits) If they are placed on a stand that is more than 3 times higher their body length, they will not jump down, so you can make a small platform to keep them in a limited area. The only time this ever failed on me was with young mice that started jumping like jumping beans as soon as we put them on top.
>
> I hope this helps. If you have any questions, feel free to contact me offline
>
> Jamie Hayden
>
> *********************************
> James Hayden, RBP, FBCA
> Manager, Imaging Core Facility
> The Wistar Institute
> 3601 Spruce St.
> Philadelphia, PA   19104
> [phone]
> [hidden email]<mailto:[hidden email]>
>
>
>
>
>
>
>
> On Feb 10, 2012, at 9:43 AM, Chris Baumann wrote:
>
> *****Commercial Response************
>
> Steffen,
>
> Chroma Technology Corp, the filter manufacture, sells filter goggles.  Something like this http://chroma.com/products/catalog/G-Series/G-515GFP coupled with a black light may do the trick if your GFP expression levels are high enough.
>
> Chris
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Steffen Dietzel
> Sent: Friday, February 10, 2012 4:53 AM
> To: [hidden email]<mailto:[hidden email]>
> Subject: Re: Photographing and seeing GFP mice
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I am jumping in on this thread with a similar problem: For us it is not photographing but seeing the GFP.
>
> We need to distinguish fluorescent from non-fluorescent littermaids.
> This works well under a fluorescence stereo microscope but we would like to do the screening with a handheld lamp (in a different room). I imagine a blue LED flashlight and a cheap long-pass filter.
> So far I came across two products, for ~ US$ 200 or ~ € 500, respectively (http://www.nightsea.com/gfp.htm and http://www.clarechemical.com/lamps.htm , the latter company also offers camera filters which might be helpful for Jeremy).
>
> I was hoping for something cheaper. Maybe someone found a 'normal'
> bright blue LED flashlight that works for excitation or yet another solution?
>
> Steffen
>
>
> On 09.02.2012 18:44, Julio Vazquez wrote:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> If you have access to a fluorescent scanner for green fluorescence (Typhoon, etc ...), you may also be able to use that. These are slow though, and you probably will need to immobilize the mice through anesthesia, or other means.
>
> --
> Julio Vazquez
> Fred Hutchinson Cancer Research Center Seattle, WA
>
> http://www.fhcrc.org
> ==
>
> On Feb 9, 2012, at 5:27 AM, Jeremy Sanderson wrote:
>
> I have to take photographs of GFP newborn and adult m
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Colleagues,
>
> I have to take photographs of GFP newborn and adult mice to support a microscopy paper.This is to show GFP throughout the coat of the newborns, and in the tail tips and feet of the adults.
>
> Normally mice are photographed with a 60mm f# 2.8 lens on a Nikon D70S with sync flash at 1/60th or 1/125th sec with an ISO of 100 or 200 set on the camera CCD. That, however, is for normal room lighting – plenty of photons.
>
> We don’t want to go above ISO 800 because of noise, yet need to freeze the movement of the mice.
>
> Has anyone done this, and can advise on exposure, whether to use sync flash as well as the UV source.
>
> I have been advised to anaesthetise the mice and photograph out of the cage. We'd prefer not to anaesthetise, but there may be no option.
>
> Any advice, or if you can direct me to someone who has done this. All input gratefully received.
>
> Thanks,
> Jeremy Sanderson
> Bio-Imaging, MRC Harwell.
>
>
>
>
> --
> ------------------------------------------------------------
> Steffen Dietzel, PD Dr. rer. nat
> Ludwig-Maximilians-Universität München
> Walter-Brendel-Zentrum für experimentelle Medizin (WBex) Head of light microscopy
>
> Mail room:
> Marchioninistr. 15, D-81377 München
>
> Building location:
> Marchioninistr. 27,  München-Großhadern

--
Michael J. Herron,  U of MN, Dept. of Entomology
  [hidden email]
     612-624-3688 (office) 612-625-5299 (FAX)