excite/ emit 300/350 nm with zeiss lsm510?

[Glyn Nelson]

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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hi,

Can anyone tell me if a Zeiss LSM510 on an Axiovert200M with a standard
HBO103 lamphousing has a filter in to cut out UV around 250-300nm?

I've a user who would like to look at tryptophan fluorescence in their IHC
slices.  To do so on our confocal, I'd have to utilise the Hg lamp with a
270/50 excitation filter and suitable dichroic in the reflector filter cube
to the meta head PMTs and obtain a non confocal image over the range of
approx 325-360nm.  I know the image won't look terribly pretty, but they
wish to capture the fluorescence to use it to normalise lipofuscin
fluorescence from sample to sample.

Aside from the fact that I would have to make sure they didn't stare down
the eyepieces with this filter in place, I'm worried that the 'scope body or
lamp housing has a piece of glass in to cut out these sort of excitation
wavelengths from the lamp (I know the lamp has a mercury spike at about 250
nm) so that you don't fry your samples/eyeballs.

Anyone who knows this, or knows where I should look to find out, I'd be
grateful to hear from you (I've tried emailing Zeiss with no reply).

Thanks,

Glyn.

[John Oreopoulos]

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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hi Glyn, I can't say much about the Zeiss LSM510, but you'll have to  
make sure your objective also transmits those excitation wavelengths  
you're interested in. Zeiss might show the characteristic curve for  
your objective on their website. Our research group was also  
interested in looking at tryptophan fluorescence in the past and we  
found that the major hurdle in light throughput was the objective.  
The transmission for for deep UV wavelengths in our objective was  
somewhere on the order of 10%. Also, because the Hg lamp source is  
not focused to a single point as is done with the lasers in the  
confocal unit, I would recommend opening up your pinhole as far as it  
can go.

John Oreopoulos

On 14-May-08, at 10:17 AM, Glyn Nelson wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Hi,
>
> Can anyone tell me if a Zeiss LSM510 on an Axiovert200M with a  
> standard
> HBO103 lamphousing has a filter in to cut out UV around 250-300nm?
>
> I've a user who would like to look at tryptophan fluorescence in  
> their IHC
> slices.  To do so on our confocal, I'd have to utilise the Hg lamp  
> with a
> 270/50 excitation filter and suitable dichroic in the reflector  
> filter cube
> to the meta head PMTs and obtain a non confocal image over the  
> range of
> approx 325-360nm.  I know the image won't look terribly pretty, but  
> they
> wish to capture the fluorescence to use it to normalise lipofuscin
> fluorescence from sample to sample.
>
> Aside from the fact that I would have to make sure they didn't  
> stare down
> the eyepieces with this filter in place, I'm worried that the  
> 'scope body or
> lamp housing has a piece of glass in to cut out these sort of  
> excitation
> wavelengths from the lamp (I know the lamp has a mercury spike at  
> about 250
> nm) so that you don't fry your samples/eyeballs.
>
> Anyone who knows this, or knows where I should look to find out,  
> I'd be
> grateful to hear from you (I've tried emailing Zeiss with no reply).
>
> Thanks,
>
> Glyn.

[Michael Weber-4]

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hi Glyn,

I doubt that you will get a reasonable signal. The main limitation might
be the objective and its transmittance in the range of 300nm, which is
typically in the range of 0%. Check their objective database:

https://www.micro-shop.zeiss.com/us/us_en/objektive.php?s=t

Afaik the rest of the optics work more or less for wavelengths above 350nm.

Michael


Glyn Nelson wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Hi,
>
> Can anyone tell me if a Zeiss LSM510 on an Axiovert200M with a standard
> HBO103 lamphousing has a filter in to cut out UV around 250-300nm?
>
> I've a user who would like to look at tryptophan fluorescence in their IHC
> slices.  To do so on our confocal, I'd have to utilise the Hg lamp with a
> 270/50 excitation filter and suitable dichroic in the reflector filter cube
> to the meta head PMTs and obtain a non confocal image over the range of
> approx 325-360nm.  I know the image won't look terribly pretty, but they
> wish to capture the fluorescence to use it to normalise lipofuscin
> fluorescence from sample to sample.
>
> Aside from the fact that I would have to make sure they didn't stare down
> the eyepieces with this filter in place, I'm worried that the 'scope body or
> lamp housing has a piece of glass in to cut out these sort of excitation
> wavelengths from the lamp (I know the lamp has a mercury spike at about 250
> nm) so that you don't fry your samples/eyeballs.
>
> Anyone who knows this, or knows where I should look to find out, I'd be
> grateful to hear from you (I've tried emailing Zeiss with no reply).
>
> Thanks,
>
> Glyn.

[John Oreopoulos]

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Oh yes, and there is one major piece of glass you can't avoid that's  
in front of the light source... the glass substrate! The only way  
around this is to use special quartz coverslips that transmit the UV,  
but I think they are expensive. Not sure who sells these, but we had  
some of these a long time back. Maybe SPI?

John Oreopoulos

On 14-May-08, at 10:39 AM, Michael Weber wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Hi Glyn,
>
> I doubt that you will get a reasonable signal. The main limitation  
> might be the objective and its transmittance in the range of 300nm,  
> which is typically in the range of 0%. Check their objective database:
>
> https://www.micro-shop.zeiss.com/us/us_en/objektive.php?s=t
>
> Afaik the rest of the optics work more or less for wavelengths  
> above 350nm.
>
> Michael
>
>
> Glyn Nelson wrote:
>> Search the CONFOCAL archive at
>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>> Hi,
>> Can anyone tell me if a Zeiss LSM510 on an Axiovert200M with a  
>> standard
>> HBO103 lamphousing has a filter in to cut out UV around 250-300nm?
>> I've a user who would like to look at tryptophan fluorescence in  
>> their IHC
>> slices.  To do so on our confocal, I'd have to utilise the Hg lamp  
>> with a
>> 270/50 excitation filter and suitable dichroic in the reflector  
>> filter cube
>> to the meta head PMTs and obtain a non confocal image over the  
>> range of
>> approx 325-360nm.  I know the image won't look terribly pretty,  
>> but they
>> wish to capture the fluorescence to use it to normalise lipofuscin
>> fluorescence from sample to sample.
>> Aside from the fact that I would have to make sure they didn't  
>> stare down
>> the eyepieces with this filter in place, I'm worried that the  
>> 'scope body or
>> lamp housing has a piece of glass in to cut out these sort of  
>> excitation
>> wavelengths from the lamp (I know the lamp has a mercury spike at  
>> about 250
>> nm) so that you don't fry your samples/eyeballs.
>> Anyone who knows this, or knows where I should look to find out,  
>> I'd be
>> grateful to hear from you (I've tried emailing Zeiss with no reply).
>> Thanks,
>> Glyn.