2P standard

[Boswell, Carl A - (cboswell)]

Hi all,
I'm looking for some trustworthy, permanent standard(s) to use in a MP system to monitor the condition of detectors and the system in general.   The goal is a fluorescent source that is immutable.  This could done either by having unrestricted replacement of bleached label with unbleached label (i.e. a solution), or a uniform and very large solid, so that it is unlikely that any one tiny volume will be imaged repeatedly.   We've tried several iterations of an aqueous solution of label, but sealing the prep does not prevent eventual evaporation.  Plus, there seems to be some modification of FITC, for example,  in solution over time (a month) regardless of exposure to light.  I'm not sure I can trust the consistency of colored plastic slides, since they are not manufactured with this role in mind.  One possibility is uranyl glass slides, but Corning says they don't make these any more.  Does anyone know a source?  We're also considering quantum dots in something like immersion oil , in a sealed chamber but I don't have any experience with these labels in a hydrophobic environment.  

Thanks for your insights.
C


Carl A. Boswell, Ph.D.
Molecular and Cellular Biology
Univ. of Arizona
520-954-7053
FAX [phone]

[Mark Cannell]

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How about Fluorescent plastic slides -I got some free from Chroma.

Cheers


On 15/01/2011, at 12:34 PM, Boswell, Carl A - (cboswell) wrote:

> Hi all,
> I'm looking for some trustworthy, permanent standard(s) to use in a  
> MP system to monitor the condition of detectors and the system in  
> general.   The goal is a fluorescent source that is immutable.  This  
> could done either by having unrestricted replacement of bleached  
> label with unbleached label (i.e. a solution), or a uniform and very  
> large solid, so that it is unlikely that any one tiny volume will be  
> imaged repeatedly.   We've tried several iterations of an aqueous  
> solution of label, but sealing the prep does not prevent eventual  
> evaporation.  Plus, there seems to be some modification of FITC, for  
> example,  in solution over time (a month) regardless of exposure to  
> light.  I'm not sure I can trust the consistency of colored plastic  
> slides, since they are not manufactured with this role in mind.  One  
> possibility is uranyl glass slides, but Corning says they don't make  
> these any more.  Does anyone know a source?  We're also considering  
> quantum dots in something like immersion oil , in a sealed chamber  
> but I don't have any experience with these labels in a hydrophobic  
> environment.
>
> Thanks for your insights.
> C
>
>
> Carl A. Boswell, Ph.D.
> Molecular and Cellular Biology
> Univ. of Arizona
> 520-954-7053
> FAX [phone]
>
>

[Craig Brideau]

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I use the Chroma slides and fluorescein solution at known concentrations.
 Use a water dipping lens with the solution and image away from either the
bottom of the dish or the surface of the water.  You want to be about middle
depth.  It won't bleach and as long as you weight it out carefully before
putting it into solution you can repeatably create it.

Craig


On Fri, Jan 14, 2011 at 5:39 PM, Mark Cannell <[hidden email]>wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> How about Fluorescent plastic slides -I got some free from Chroma.
>
> Cheers
>
>
>
> On 15/01/2011, at 12:34 PM, Boswell, Carl A - (cboswell) wrote:
>
>  Hi all,
>> I'm looking for some trustworthy, permanent standard(s) to use in a MP
>> system to monitor the condition of detectors and the system in general.
>> The goal is a fluorescent source that is immutable.  This could done either
>> by having unrestricted replacement of bleached label with unbleached label
>> (i.e. a solution), or a uniform and very large solid, so that it is unlikely
>> that any one tiny volume will be imaged repeatedly.   We've tried several
>> iterations of an aqueous solution of label, but sealing the prep does not
>> prevent eventual evaporation.  Plus, there seems to be some modification of
>> FITC, for example,  in solution over time (a month) regardless of exposure
>> to light.  I'm not sure I can trust the consistency of colored plastic
>> slides, since they are not manufactured with this role in mind.  One
>> possibility is uranyl glass slides, but Corning says they don't make these
>> any more.  Does anyone know a source?  We're also considering quantum dots
>> in something like immersion oil , in a sealed chamber but I don't have any
>> experience with these labels in a hydrophobic environment.
>>
>> Thanks for your insights.
>> C
>>
>>
>> Carl A. Boswell, Ph.D.
>> Molecular and Cellular Biology
>> Univ. of Arizona
>> 520-954-7053
>> FAX [phone]
>>
>>
>>

[mcammer]

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Fluorescent plastic slides are not stabile.  Depending on their exposure to light they bleach or change color.  Using a pulsed laser is a good way to make little burn or other chemical reaction spots in them.  Here are two old web pages with some of the problems (and benefits!) explained:  
http://www.einstein.yu.edu/aif/instructions/fluor-ref-slides/01.htm 
http://www.einstein.yu.edu/aif/instructions/zeiss/liveduo/Zaxis_bleachingtest/index.htm
_______________________________________
Michael Cammer, Assistant Research Scientist
Skirball Institute of Biomolecular Medicine
Lab: [phone]  Cell: [phone]

________________________________________
From: Confocal Microscopy List [[hidden email]] On Behalf Of Mark Cannell [[hidden email]]
Sent: Friday, January 14, 2011 7:39 PM
To: [hidden email]
Subject: Re: 2P standard

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How about Fluorescent plastic slides -I got some free from Chroma.

Cheers


On 15/01/2011, at 12:34 PM, Boswell, Carl A - (cboswell) wrote:

> Hi all,
> I'm looking for some trustworthy, permanent standard(s) to use in a
> MP system to monitor the condition of detectors and the system in
> general.   The goal is a fluorescent source that is immutable.  This
> could done either by having unrestricted replacement of bleached
> label with unbleached label (i.e. a solution), or a uniform and very
> large solid, so that it is unlikely that any one tiny volume will be
> imaged repeatedly.   We've tried several iterations of an aqueous
> solution of label, but sealing the prep does not prevent eventual
> evaporation.  Plus, there seems to be some modification of FITC, for
> example,  in solution over time (a month) regardless of exposure to
> light.  I'm not sure I can trust the consistency of colored plastic
> slides, since they are not manufactured with this role in mind.  One
> possibility is uranyl glass slides, but Corning says they don't make
> these any more.  Does anyone know a source?  We're also considering
> quantum dots in something like immersion oil , in a sealed chamber
> but I don't have any experience with these labels in a hydrophobic
> environment.
>
> Thanks for your insights.
> C
>
>
> Carl A. Boswell, Ph.D.
> Molecular and Cellular Biology
> Univ. of Arizona
> 520-954-7053
> FAX [phone]
>
>

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[Sudipta Maiti]

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We use fluorescent solutions. We keep a stock in ethanol in the
refrigerator, and dilute it to water before use. We measure the absorbance
of the final solution in a spectrophotometer evertytime. A droplet on the
cover slip is enough to get counts.
Sudipta

On Fri, 14 Jan 2011 18:05:02 -0700, Craig Brideau wrote

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I use the Chroma slides and fluorescein solution at known concentrations.
>  Use a water dipping lens with the solution and image away from
> either the bottom of the dish or the surface of the water.  You want
> to be about middle depth.  It won't bleach and as long as you weight
> it out carefully before putting it into solution you can repeatably
> create it.
>
> Craig
>
> On Fri, Jan 14, 2011 at 5:39 PM, Mark Cannell

<[hidden email]>wrote:

>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > How about Fluorescent plastic slides -I got some free from Chroma.
> >
> > Cheers
> >
> >
> >
> > On 15/01/2011, at 12:34 PM, Boswell, Carl A - (cboswell) wrote:
> >
> >  Hi all,
> >> I'm looking for some trustworthy, permanent standard(s) to use in a MP
> >> system to monitor the condition of detectors and the system in general.
> >> The goal is a fluorescent source that is immutable.  This could done

either
> >> by having unrestricted replacement of bleached label with unbleached
label
> >> (i.e. a solution), or a uniform and very large solid, so that it is
unlikely
> >> that any one tiny volume will be imaged repeatedly.   We've tried
several
> >> iterations of an aqueous solution of label, but sealing the prep does
not
> >> prevent eventual evaporation.  Plus, there seems to be some
modification of
> >> FITC, for example,  in solution over time (a month) regardless of
exposure
> >> to light.  I'm not sure I can trust the consistency of colored plastic
> >> slides, since they are not manufactured with this role in mind.  One
> >> possibility is uranyl glass slides, but Corning says they don't make
these
> >> any more.  Does anyone know a source?  We're also considering quantum
dots
> >> in something like immersion oil , in a sealed chamber but I don't have
any

> >> experience with these labels in a hydrophobic environment.
> >>
> >> Thanks for your insights.
> >> C
> >>
> >>
> >> Carl A. Boswell, Ph.D.
> >> Molecular and Cellular Biology
> >> Univ. of Arizona
> >> 520-954-7053
> >> FAX [phone]
> >>
> >>
> >>

Dr. Sudipta Maiti
Associate Professor
Dept. of Chemical Sciences
Tata Institute of Fundamental Research
Homi Bhabha Raod, Colaba, Mumbai 400005
Ph. 91-22-2278-2716 / 2539
Fax: [phone]
alternate e-mail: [hidden email]
url: biophotonics.wetpaint.com

[George McNamara]

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Hi Carl,

Instead of using a bleachable fluorescent target, use something that
makes great second harmonic generation (SHG). If you MP laser tunes from
690-1040 nm an SHG specimen will be usable from 395-520 nm (maybe you
can convince NIH's NCRR to give all of us S10 money to add an OPO or OPA
to our MP systems to extend the wavelength range).

Besides sugar crystals, starch granules, collagen fibers (see Rich
Cole's post), Scott Fraser's lab published a PNAS  paper in 2010 using
nanoparticles for multicolor SHG-immunofluorescence (antibody
conjugation procedure developed by a different Caltech lab and cited by
Fraser and colleagues). They used specific types of particles from    
http://www.nanoamor.com/products
Multicolor SHG worked because some types of nanoparticles perferentially
underwent SHG in specific excitation wavelength range, whereas others
had broad excitation range.

Enjoy,

George



On 1/14/2011 6:34 PM, Boswell, Carl A - (cboswell) wrote:

> Hi all,
> I'm looking for some trustworthy, permanent standard(s) to use in a MP system to monitor the condition of detectors and the system in general.   The goal is a fluorescent source that is immutable.  This could done either by having unrestricted replacement of bleached label with unbleached label (i.e. a solution), or a uniform and very large solid, so that it is unlikely that any one tiny volume will be imaged repeatedly.   We've tried several iterations of an aqueous solution of label, but sealing the prep does not prevent eventual evaporation.  Plus, there seems to be some modification of FITC, for example,  in solution over time (a month) regardless of exposure to light.  I'm not sure I can trust the consistency of colored plastic slides, since they are not manufactured with this role in mind.  One possibility is uranyl glass slides, but Corning says they don't make these any more.  Does anyone know a source?  We're also considering quantum dots in something like immersion oil , in a sealed chamber but I don't have any experience with these labels in a hydrophobic environment.
>
> Thanks for your insights.
> C
>
>
> Carl A. Boswell, Ph.D.
> Molecular and Cellular Biology
> Univ. of Arizona
> 520-954-7053
> FAX [phone]
>
>
>    

[Mark Cannell]

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To join, leave or search the confocal microscopy listserv, go to:
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Since SHG is highly dependent on phase matching how do you ensure that  
the sample has exactly the same orientation and control the  
backscatter needed to test the PMTs?

Further to an earlier post,  my 4 year old plastic plastic slides look  
as good as new -but then I've looked after them and not exposed them  
to sunlight.

Cheers

On 17/01/2011, at 3:38 AM, George McNamara wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Carl,
>
> Instead of using a bleachable fluorescent target, use something that  
> makes great second harmonic generation (SHG). If you MP laser tunes  
> from 690-1040 nm an SHG specimen will be usable from 395-520 nm  
> (maybe you can convince NIH's NCRR to give all of us S10 money to  
> add an OPO or OPA to our MP systems to extend the wavelength range).
>
> Besides sugar crystals, starch granules, collagen fibers (see Rich  
> Cole's post), Scott Fraser's lab published a PNAS  paper in 2010  
> using nanoparticles for multicolor SHG-immunofluorescence (antibody  
> conjugation procedure developed by a different Caltech lab and cited  
> by Fraser and colleagues). They used specific types of particles  
> from     http://www.nanoamor.com/products
> Multicolor SHG worked because some types of nanoparticles  
> perferentially underwent SHG in specific excitation wavelength  
> range, whereas others had broad excitation range.
>
> Enjoy,
>
> George
>
>
>
> On 1/14/2011 6:34 PM, Boswell, Carl A - (cboswell) wrote:
>> Hi all,
>> I'm looking for some trustworthy, permanent standard(s) to use in a  
>> MP system to monitor the condition of detectors and the system in  
>> general.   The goal is a fluorescent source that is immutable.  
>> This could done either by having unrestricted replacement of  
>> bleached label with unbleached label (i.e. a solution), or a  
>> uniform and very large solid, so that it is unlikely that any one  
>> tiny volume will be imaged repeatedly.   We've tried several  
>> iterations of an aqueous solution of label, but sealing the prep  
>> does not prevent eventual evaporation.  Plus, there seems to be  
>> some modification of FITC, for example,  in solution over time (a  
>> month) regardless of exposure to light.  I'm not sure I can trust  
>> the consistency of colored plastic slides, since they are not  
>> manufactured with this role in mind.  One possibility is uranyl  
>> glass slides, but Corning says they don't make these any more.  
>> Does anyone know a source?  We're also considering quantum dots in  
>> something like immersion oil , in a sealed chamber but I don't have  
>> any experience with these labels in a hydrophobic environment.
>>
>> Thanks for your insights.
>> C
>>
>>
>> Carl A. Boswell, Ph.D.
>> Molecular and Cellular Biology
>> Univ. of Arizona
>> 520-954-7053
>> FAX [phone]
>>
>>
>>

[Steffen Dietzel]

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On 16.01.2011 15:38, George McNamara wrote:
> Hi Carl,
>
> Instead of using a bleachable fluorescent target, use something that
> makes great second harmonic generation (SHG). If you MP laser tunes from
> 690-1040 nm an SHG specimen will be usable from 395-520 nm (maybe you
> can convince NIH's NCRR to give all of us S10 money to add an OPO or OPA
> to our MP systems to extend the wavelength range).

If you succeed in getting that OPO, you also can use it as a source for
THG (Third harmonic generation). A water-glass interface (water on glass
slide) will give you a bright and uniform signal if the system is well
aligned. It still will work only with your blue detectors, though (e.g.
1270 nm / 3 = 423 nm)

Steffen


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