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Fura-2 filter question: how much light comes out of a microscope when using a Xenon arc lamp and good 340nm excitation filter?

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George McNamara George McNamara
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Fura-2 filter question: how much light comes out of a microscope when using a Xenon arc lamp and good 340nm excitation filter?

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Dear Confocal listserv members,

Fura-2 filter question: how much light comes out of a microscope when
using a Xenon arc lamp and good 340nm excitation filter?

How much for 380 nm filter?

thanks in advance,

George

--



George McNamara, Ph.D.
Single Cells Analyst
L.J.N. Cooper Lab
University of Texas M.D. Anderson Cancer Center
Houston, TX 77054
Tattletales http://works.bepress.com/gmcnamara/26/
Craig Brideau Craig Brideau
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Re: Fura-2 filter question: how much light comes out of a microscope when using a Xenon arc lamp and good 340nm excitation filter?

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UV power meters are not very common. Has anyone measured this? I'm doing
some confocal imaging with 380 nm and my loss through the system is a
factor of 2000-3000x (measured before scan head and after the objective.

Craig
On 2013-10-26 11:05 AM, "George McNamara" <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/**wa?A0=confocalmicroscopy<http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>
> *****
>
> Dear Confocal listserv members,
>
> Fura-2 filter question: how much light comes out of a microscope when
> using a Xenon arc lamp and good 340nm excitation filter?
>
> How much for 380 nm filter?
>
> thanks in advance,
>
> George
>
> --
>
>
>
> George McNamara, Ph.D.
> Single Cells Analyst
> L.J.N. Cooper Lab
> University of Texas M.D. Anderson Cancer Center
> Houston, TX 77054
> Tattletales http://works.bepress.com/**gmcnamara/26/<http://works.bepress.com/gmcnamara/26/>
>
Johannes Helm Johannes Helm
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Re: Fura-2 filter question: how much light comes out of a microscope when using a Xenon arc lamp and good 340nm excitation filter?

In reply to this post by George McNamara
*****
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Good evening,



as Craig writes, this is not a trivial issue. We had built a CLSM for

Fura-2 measurements, see



P.J. Helm, O. Franksson, and K. Carlsson (1995),

A confocal scanning laser microscope for quantitative ratiometric 3D

measurements of [Ca2+] and Ca2+ diffusions in living cells stained

with Fura-2,

Pflügers Archiv - European Journal of Physiology, Vol. 429, pp. 672-681





and





PJ Helm, A Patwardhan, and EMM Manders (1997),

A study of the precision of confocal, ratiometric, Fura-2 based
measurements,

Cell Calcium 22(4):287-298



The transmission is, as one would expect, strongly dependent on quite a

number of optical elements in the microscope. Specifically, besides the

objective, the tube lens will have to be suitable for the Fura-2

wavelength range. I know that some microscope producers offer special
tube

lenses suitable even for the somewhat deeper UV.



However, transmission at 380nm will normally be considerably better than

transmission at 340nm. In order to get somewhat comparable fluorescence

responses, the 380nm light has to be attenuated.



Also, make sure that Your Xe-arc burner is one with a quartz or fused

silica housing. Some companies offer Xe burners even in strongly UV

absorbing material, which, of course, will result in no 340nm light being

emitted at all. These sometimes are called "Ozon free burners".



Also, if you have a suitable Xe burner, this will produce a considerable

amount of ozon. Make hence sure that there is good ventilation in your

lab!



Best wishes,



Johannes





> *****

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

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

> *****

>

> UV power meters are not very common. Has anyone measured this? I'm
doing

> some confocal imaging with 380 nm and my loss through the system is a

> factor of 2000-3000x (measured before scan head and after the
objective.

>

> Craig

> On 2013-10-26 11:05 AM, "George McNamara"
<[hidden email]>

> wrote:

>

>> *****

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

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

>> *****

>>

>> Dear Confocal listserv members,

>>

>> Fura-2 filter question: how much light comes out of a microscope when

>> using a Xenon arc lamp and good 340nm excitation filter?

>>

>> How much for 380 nm filter?

>>

>> thanks in advance,

>>

>> George

>>

>> --

>>

>>

>>

>> George McNamara, Ph.D.

>> Single Cells Analyst

>> L.J.N. Cooper Lab

>> University of Texas M.D. Anderson Cancer Center

>> Houston, TX 77054

>> Tattletales

>>
http://works.bepress.com/**gmcnamara/26/<http://works.bepress.com/g
mcnamara/26/>

>>

>





--

P. Johannes Helm, M.Sc. PhD

Seniorengineer

CMBN

University of Oslo

Institute of Basic Medical Science

Department of Physiology

Postboks 1103 - Blindern

NO-0317 Oslo



Voice: +47 228 51159

Fax: +47 228 51499



WWW: folk.uio.no/jhelm
George McNamara George McNamara
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Re: Fura-2 filter question: how much light comes out of a microscope when using a Xenon arc lamp and good 340nm excitation filter?

*****
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Hi Johannes,

Thanks for the response. My interest is more in BD Sirigen's BUV395
excitation (and in the future its tandem dyes, probably same acceptors
as the BV421 series), page 33 of
http://www.bdbiosciences.com/documents/webinar_071713_multicolor-bv.pdf
which at ~350 nm can be excited separately from BV421 and its tandems
(excitation page 4, emissions page 3), and BV510 (a different polymer,
page 6).
Our flow cytometry core already has on flow cytometer (or sorter?) with
355 nm laser (and probably octagon detector unit) and is getting a
second cytometer with both BUV and BV capability. Now that I work on
making movies of serial killer T-cells and NK cells, having 'my'
microscope compatible with BUV(s) would be useful.
Since I am a big fan of Fura-2, I might want to revisit my youth by
having the scope to also be able to image Fura-2 (starting with
Image-1/FL ~1990, then Image-1/FL and MetaFluor 1992-1997, then
MetaFluor 2000-2005). Lumen Dynamics (XLED1) and ThorLabs
(http://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=3836) offer
~365 nm LEDs (85 mW for ThorLabs). Apparently the ~345 nm LED's are much
less powerful (~1 mW at the lamp), and I have mostly plan apo objective
lenses.

Sincerely,

George
p.s. my thanks to Richard Cole for telling me about ThorLabs LEDs for
microscopes - their imaging division is in Austin, practically next door
neighbors at Texas scale. For my purposes, Lumen's XLED1 would probably
be more useful.

On 10/26/2013 1:08 PM, Johannes Helm wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Good evening,
>
>
>
> as Craig writes, this is not a trivial issue. We had built a CLSM for
>
> Fura-2 measurements, see
>
>
>
> P.J. Helm, O. Franksson, and K. Carlsson (1995),
>
> A confocal scanning laser microscope for quantitative ratiometric 3D
>
> measurements of [Ca2+] and Ca2+ diffusions in living cells stained
>
> with Fura-2,
>
> Pflügers Archiv - European Journal of Physiology, Vol. 429, pp. 672-681
>
>
>
>
>
> and
>
>
>
>
>
> PJ Helm, A Patwardhan, and EMM Manders (1997),
>
> A study of the precision of confocal, ratiometric, Fura-2 based
> measurements,
>
> Cell Calcium 22(4):287-298
>
>
>
> The transmission is, as one would expect, strongly dependent on quite a
>
> number of optical elements in the microscope. Specifically, besides the
>
> objective, the tube lens will have to be suitable for the Fura-2
>
> wavelength range. I know that some microscope producers offer special
> tube
>
> lenses suitable even for the somewhat deeper UV.
>
>
>
> However, transmission at 380nm will normally be considerably better than
>
> transmission at 340nm. In order to get somewhat comparable fluorescence
>
> responses, the 380nm light has to be attenuated.
>
>
>
> Also, make sure that Your Xe-arc burner is one with a quartz or fused
>
> silica housing. Some companies offer Xe burners even in strongly UV
>
> absorbing material, which, of course, will result in no 340nm light being
>
> emitted at all. These sometimes are called "Ozon free burners".
>
>
>
> Also, if you have a suitable Xe burner, this will produce a considerable
>
> amount of ozon. Make hence sure that there is good ventilation in your
>
> lab!
>
>
>
> Best wishes,
>
>
>
> Johannes
>
>
>
>
>
>    
>> *****
>>      
>    
>> To join, leave or search the confocal microscopy listserv, go to:
>>      
>    
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>      
>    
>> *****
>>      
>    
>>      
>    
>> UV power meters are not very common. Has anyone measured this? I'm
>>      
> doing
>
>    
>> some confocal imaging with 380 nm and my loss through the system is a
>>      
>    
>> factor of 2000-3000x (measured before scan head and after the
>>      
> objective.
>
>    
>>      
>    
>> Craig
>>      
>    
>> On 2013-10-26 11:05 AM, "George McNamara"
>>      
> <[hidden email]>
>
>    
>> wrote:
>>      
>    
>>      
>    
>>> *****
>>>        
>    
>>> To join, leave or search the confocal microscopy listserv, go to:
>>>        
>    
>>> http://lists.umn.edu/cgi-bin/**wa?
>>>        
> A0=confocalmicroscopy<http://lists.umn.edu/cgi-bin/wa?
> A0=confocalmicroscopy>
>
>    
>>> *****
>>>        
>    
>>>        
>    
>>> Dear Confocal listserv members,
>>>        
>    
>>>        
>    
>>> Fura-2 filter question: how much light comes out of a microscope when
>>>        
>    
>>> using a Xenon arc lamp and good 340nm excitation filter?
>>>        
>    
>>>        
>    
>>> How much for 380 nm filter?
>>>        
>    
>>>        
>    
>>> thanks in advance,
>>>        
>    
>>>        
>    
>>> George
>>>        
>    
>>>        
>    
>>> --
>>>        
>    
>>>        
>    
>>>        
>    
>>>        
>    
>>> George McNamara, Ph.D.
>>>        
>    
>>> Single Cells Analyst
>>>        
>    
>>> L.J.N. Cooper Lab
>>>        
>    
>>> University of Texas M.D. Anderson Cancer Center
>>>        
>    
>>> Houston, TX 77054
>>>        
>    
>>> Tattletales
>>>        
>    
>>>        
> http://works.bepress.com/**gmcnamara/26/<http://works.bepress.com/g
> mcnamara/26/>
>
>    
>>>        
>    
>>      
>
>
>
>
>    


--



George McNamara, Ph.D.
Single Cells Analyst
L.J.N. Cooper Lab
University of Texas M.D. Anderson Cancer Center
Houston, TX 77054
Tattletales http://works.bepress.com/gmcnamara/26/
Craig Brideau Craig Brideau
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Re: Fura-2 filter question: how much light comes out of a microscope when using a Xenon arc lamp and good 340nm excitation filter?

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

I actually have a 1.25 NA NUV objective coming next week from Partec. I
will let you know if it works.

Craig
On 2013-10-26 12:09 PM, "Johannes Helm" <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Good evening,
>
>
>
> as Craig writes, this is not a trivial issue. We had built a CLSM for
>
> Fura-2 measurements, see
>
>
>
> P.J. Helm, O. Franksson, and K. Carlsson (1995),
>
> A confocal scanning laser microscope for quantitative ratiometric 3D
>
> measurements of [Ca2+] and Ca2+ diffusions in living cells stained
>
> with Fura-2,
>
> Pflügers Archiv - European Journal of Physiology, Vol. 429, pp. 672-681
>
>
>
>
>
> and
>
>
>
>
>
> PJ Helm, A Patwardhan, and EMM Manders (1997),
>
> A study of the precision of confocal, ratiometric, Fura-2 based
> measurements,
>
> Cell Calcium 22(4):287-298
>
>
>
> The transmission is, as one would expect, strongly dependent on quite a
>
> number of optical elements in the microscope. Specifically, besides the
>
> objective, the tube lens will have to be suitable for the Fura-2
>
> wavelength range. I know that some microscope producers offer special
> tube
>
> lenses suitable even for the somewhat deeper UV.
>
>
>
> However, transmission at 380nm will normally be considerably better than
>
> transmission at 340nm. In order to get somewhat comparable fluorescence
>
> responses, the 380nm light has to be attenuated.
>
>
>
> Also, make sure that Your Xe-arc burner is one with a quartz or fused
>
> silica housing. Some companies offer Xe burners even in strongly UV
>
> absorbing material, which, of course, will result in no 340nm light being
>
> emitted at all. These sometimes are called "Ozon free burners".
>
>
>
> Also, if you have a suitable Xe burner, this will produce a considerable
>
> amount of ozon. Make hence sure that there is good ventilation in your
>
> lab!
>
>
>
> Best wishes,
>
>
>
> Johannes
>
>
>
>
>
> > *****
>
> > To join, leave or search the confocal microscopy listserv, go to:
>
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>
> > *****
>
> >
>
> > UV power meters are not very common. Has anyone measured this? I'm
> doing
>
> > some confocal imaging with 380 nm and my loss through the system is a
>
> > factor of 2000-3000x (measured before scan head and after the
> objective.
>
> >
>
> > Craig
>
> > On 2013-10-26 11:05 AM, "George McNamara"
> <[hidden email]>
>
> > wrote:
>
> >
>
> >> *****
>
> >> To join, leave or search the confocal microscopy listserv, go to:
>
> >> http://lists.umn.edu/cgi-bin/**wa?
> A0=confocalmicroscopy<http://lists.umn.edu/cgi-bin/wa?
> A0=confocalmicroscopy>
>
> >> *****
>
> >>
>
> >> Dear Confocal listserv members,
>
> >>
>
> >> Fura-2 filter question: how much light comes out of a microscope when
>
> >> using a Xenon arc lamp and good 340nm excitation filter?
>
> >>
>
> >> How much for 380 nm filter?
>
> >>
>
> >> thanks in advance,
>
> >>
>
> >> George
>
> >>
>
> >> --
>
> >>
>
> >>
>
> >>
>
> >> George McNamara, Ph.D.
>
> >> Single Cells Analyst
>
> >> L.J.N. Cooper Lab
>
> >> University of Texas M.D. Anderson Cancer Center
>
> >> Houston, TX 77054
>
> >> Tattletales
>
> >>
> http://works.bepress.com/**gmcnamara/26/<http://works.bepress.com/g
> mcnamara/26/>
>
> >>
>
> >
>
>
>
>
>
> --
>
> P. Johannes Helm, M.Sc. PhD
>
> Seniorengineer
>
> CMBN
>
> University of Oslo
>
> Institute of Basic Medical Science
>
> Department of Physiology
>
> Postboks 1103 - Blindern
>
> NO-0317 Oslo
>
>
>
> Voice:  +47 228 51159
>
> Fax:    +47 228 51499
>
>
>
> WWW:    folk.uio.no/jhelm
>
Arvydas Matiukas Arvydas Matiukas
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Re: Fura-2 filter question: how much light comes out of a microscope when using a Xenon arc lamp and good 340nm excitation filter?

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

George and Craig,
 
Below are my power measurements for a couple of widefield
Fura-2 systems.  I did not have UV power meter so to estimate
the power just used a visible range sensor at the shortest available
(400 nm) setting (which would provide somewhat underestimated values)
 
Polychrome V output: 83uW (340nm), 1570uW (380nm)
40x/340 objective output:  3.1uW (340nm), 110uW (380nm)
 
Xenon arc lamp output: 120uW (340/10, new filter), 450uW (380/10)
40x/340 objective output:  0.1uW (340nm), 6.7uW (380nm)
 
UV power transmission gets reduced as filter and liquid fiber get old.
It is very  important to use UV transparent objective otherwise
the transmitted UV power is reduced >10-fold.
 
I  hope this info helps. The above power levels are enough to provide
Ca imaging at ~100ms exposure. The power loss through the system
is not as high as Craig reported  but my systems are widefield with fewer
optical elements.
 
Arvydas
~~~~~~~~~~~~~~~
 

 
 
Arvydas Matiukas, Ph.D.
Director of Confocal&Two-Photon Core
Department of Neurosci& Physiology
SUNY Upstate Medical University
766 Irving Ave., WH 3167
Syracuse, NY 13210
tel.: 315-464-7997
fax: 315-464-8014
email: [hidden email]
>>> Craig Brideau <[hidden email]> 10/26/2013 12:14 PM >>>
*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

UV power meters are not very common. Has anyone measured this? I'm doing
some confocal imaging with 380 nm and my loss through the system is a
factor of 2000-3000x (measured before scan head and after the objective.

Craig
On 2013-10-26 11:05 AM, "George McNamara" <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/**wa?A0=confocalmicroscopy<http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>
> *****
>
> Dear Confocal listserv members,
>
> Fura-2 filter question: how much light comes out of a microscope when
> using a Xenon arc lamp and good 340nm excitation filter?
>
> How much for 380 nm filter?
>
> thanks in advance,
>
> George
>
> --
>
>
>
> George McNamara, Ph.D.
> Single Cells Analyst
> L.J.N. Cooper Lab
> University of Texas M.D. Anderson Cancer Center
> Houston, TX 77054
> Tattletales http://works.bepress.com/**gmcnamara/26/<http://works.bepress.com/gmcnamara/26/>
>
Craig Brideau Craig Brideau
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Re: Fura-2 filter question: how much light comes out of a microscope when using a Xenon arc lamp and good 340nm excitation filter?

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

Thanks for the statistics, Arvydas. Your numbers are very helpful!  My own
system has a really big scan lens internally, so I suspect that is why I am
getting so much more loss than your system.  It will be interesting to see
how much my numbers change when I try it with a proper UV objective!

Craig


On Mon, Oct 28, 2013 at 2:56 PM, Arvydas Matiukas <[hidden email]>wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> George and Craig,
>
> Below are my power measurements for a couple of widefield
> Fura-2 systems.  I did not have UV power meter so to estimate
> the power just used a visible range sensor at the shortest available
> (400 nm) setting (which would provide somewhat underestimated values)
>
> Polychrome V output: 83uW (340nm), 1570uW (380nm)
> 40x/340 objective output:  3.1uW (340nm), 110uW (380nm)
>
> Xenon arc lamp output: 120uW (340/10, new filter), 450uW (380/10)
> 40x/340 objective output:  0.1uW (340nm), 6.7uW (380nm)
>
> UV power transmission gets reduced as filter and liquid fiber get old.
> It is very  important to use UV transparent objective otherwise
> the transmitted UV power is reduced >10-fold.
>
> I  hope this info helps. The above power levels are enough to provide
> Ca imaging at ~100ms exposure. The power loss through the system
> is not as high as Craig reported  but my systems are widefield with fewer
> optical elements.
>
> Arvydas
> ~~~~~~~~~~~~~~~
>
>
>
>
> Arvydas Matiukas, Ph.D.
> Director of Confocal&Two-Photon Core
> Department of Neurosci& Physiology
> SUNY Upstate Medical University
> 766 Irving Ave., WH 3167
> Syracuse, NY 13210
> tel.: 315-464-7997
> fax: 315-464-8014
> email: [hidden email]
> >>> Craig Brideau <[hidden email]> 10/26/2013 12:14 PM >>>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> UV power meters are not very common. Has anyone measured this? I'm doing
> some confocal imaging with 380 nm and my loss through the system is a
> factor of 2000-3000x (measured before scan head and after the objective.
>
> Craig
> On 2013-10-26 11:05 AM, "George McNamara" <[hidden email]>
> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/**wa?A0=confocalmicroscopy<
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>
> > *****
> >
> > Dear Confocal listserv members,
> >
> > Fura-2 filter question: how much light comes out of a microscope when
> > using a Xenon arc lamp and good 340nm excitation filter?
> >
> > How much for 380 nm filter?
> >
> > thanks in advance,
> >
> > George
> >
> > --
> >
> >
> >
> > George McNamara, Ph.D.
> > Single Cells Analyst
> > L.J.N. Cooper Lab
> > University of Texas M.D. Anderson Cancer Center
> > Houston, TX 77054
> > Tattletales http://works.bepress.com/**gmcnamara/26/<
> http://works.bepress.com/gmcnamara/26/>
> >
>
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