Problems with single molecule detection

[kahms]

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Dear list members,

though it is not directly a  confocal question I would like to ask for
your help.
We have a microscopic setup  which we want to use for  single molecule
detection. For characterisation of some fluorophores we tried to
immobilize them to the surface of a coverslip.
But we have severe problems to get rid of signals we always observe even
though there is no fluorophore on the coverslip. Especially when
exciting with 488 nm (but also with 633 nm) we see some flickering on
the surface which can be even detected by eye. We tried several cleaning
strategies (aceton, 5 M KOH, piranha, hellmanex etc.) but we could never
get rid of these signals. We also checked if it is the millipore water
we used but we have the same problems when looking at a "bare" cleaned
coverslip. Has anybody made the same experiences or knows some further
things to check? Could it be the glass?

Thanks in advance

Martin


--
Dr. Martin Kahms
Institut für Medizinische Physik und Biophysik
Center for Nanotechnology (CeNTech)
Heisenbergstr. 11
48149 Münster
Tel.:[phone]
Fax: [phone]

[Eli Rothenberg]

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

Hi Martin,
The fluorescence quality (or lack of it) of coverslips varies greatly from brand to brand. I would suggest trying to buy best quality coverslips, or alternatively you can try fused-silica coverslips which may provide you with a standard for purity (though pricey, 12-20 bucks ea, so far I found SPI to offer the best quality).
After cleaning (whichever method you'd like, although sonicating in soap followed by KOH should do the job) trying brief burning of the coverslips with an open flame (you have to do it fast- if you wait too long the coverslip will brake or deform) will usually get rid the organic stuff that fluorescence there.

If you don't have an open flame you can alternatively try backing them for 3-6 hours at 300-500 oC. Make sure your oven is clean and your coverslips are not touching any surface.
You can use aluminum foil to construct a coverslip holder.

  good luck

Eli

---- Original message ----

>Date: Mon, 14 Jan 2008 18:17:53 +0100
>From: kahms <[hidden email]>  
>Subject: Problems with single molecule detection  
>To: [hidden email]
>
>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>Dear list members,
>
>though it is not directly a  confocal question I would like to ask for
>your help.
>We have a microscopic setup  which we want to use for  single molecule
>detection. For characterisation of some fluorophores we tried to
>immobilize them to the surface of a coverslip.
>But we have severe problems to get rid of signals we always observe even
>though there is no fluorophore on the coverslip. Especially when
>exciting with 488 nm (but also with 633 nm) we see some flickering on
>the surface which can be even detected by eye. We tried several cleaning
>strategies (aceton, 5 M KOH, piranha, hellmanex etc.) but we could never
>get rid of these signals. We also checked if it is the millipore water
>we used but we have the same problems when looking at a "bare" cleaned
>coverslip. Has anybody made the same experiences or knows some further
>things to check? Could it be the glass?
>
>Thanks in advance
>
>Martin
>
>
>--
>Dr. Martin Kahms
>Institut für Medizinische Physik und Biophysik
>Center for Nanotechnology (CeNTech)
>Heisenbergstr. 11
>48149 Münster
>Tel.:[phone]
>Fax: [phone]

________________________________
Eli Rothenberg, Ph.D.
Post Doctoral Research Associate,
Howard Hughes Medical Institute,
Department of Physics,
University of Illinois,
Urbana-Champaign. 61801.
Illinois, USA
Tel: [phone];
Fax: [phone];
Email: [hidden email]

[Kevin Braeckmans]

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

Dear Martin,

I assume that you are talking about a widefield setup?

Are you sure that it is not just laser reflection at the cover slip? If you
want to detect single molecule fluorescence you will need to use a laser
notch filter (> OD 6) to suppress any reflected laser photons, possibly in
combination with a second fluorescence emission filter (which also further
suppresses any remaining reflected laser photons).

Using only a single fluorescence emission filter (just as in confocal) will
not be sufficient. Also, maybe your filters are damaged and have to be
replaced.

Hope this helps.

Best regards,

Kevin



Kevin Braeckmans, Ph.D.
Lab. General Biochemistry & Physical Pharmacy
Ghent University
Harelbekestraat 72
9000 Ghent
Belgium
Tel: [phone]
Fax: [phone]
E-mail: [hidden email]

> -----Oorspronkelijk bericht-----
> Van: Confocal Microscopy List [mailto:[hidden email]]
> Namens kahms
> Verzonden: maandag 14 januari 2008 18:18
> Aan: [hidden email]
> Onderwerp: Problems with single molecule detection
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Dear list members,
>
> though it is not directly a  confocal question I would like to ask for
> your help.
> We have a microscopic setup  which we want to use for  single molecule
> detection. For characterisation of some fluorophores we tried to
> immobilize them to the surface of a coverslip.
> But we have severe problems to get rid of signals we always observe
> even
> though there is no fluorophore on the coverslip. Especially when
> exciting with 488 nm (but also with 633 nm) we see some flickering on
> the surface which can be even detected by eye. We tried several
> cleaning
> strategies (aceton, 5 M KOH, piranha, hellmanex etc.) but we could
> never
> get rid of these signals. We also checked if it is the millipore water
> we used but we have the same problems when looking at a "bare" cleaned
> coverslip. Has anybody made the same experiences or knows some further
> things to check? Could it be the glass?
>
> Thanks in advance
>
> Martin
>
>
> --
> Dr. Martin Kahms
> Institut für Medizinische Physik und Biophysik
> Center for Nanotechnology (CeNTech)
> Heisenbergstr. 11
> 48149 Münster
> Tel.:[phone]
> Fax: [phone]

[kahms]

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

Dear Kevin,

thanks a lot for your answer. Yes indeed I was talking about a widefield
setup. We have notch filters with OD>6 in our microscope und putting in
an additional emmision filter does not change anything.

Martin

--
Dr. Martin Kahms
Institut für Medizinische Physik und Biophysik
Center for Nanotechnology (CeNTech)
Heisenbergstr. 11
48149 Münster
Tel.:[phone]
Fax: [phone]

[Eli Rothenberg]

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

If you're using widefield you might
want to do through-objective TIR excitation
which will reduce any background you might have.
Also, since you say your coverslips a problematic
you might want to filter glass autofluorescence
in the red so use either a band-pass for you
fluorophores or a shortpass that cuts off
red emission.
One more thing about single mol. excitation is
that if you excite too much you'll photobleach
your dye molecules, and excite organic impurities in the glass surface.

Hope this helps

Eli

---- Original message ----

>Date: Tue, 15 Jan 2008 09:52:50 +0100
>From: kahms <[hidden email]>  
>Subject: Re: Problems with single molecule detection  
>To: [hidden email]
>
>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>Dear Kevin,
>
>thanks a lot for your answer. Yes indeed I was talking about a widefield
>setup. We have notch filters with OD>6 in our microscope und putting in
>an additional emmision filter does not change anything.
>
>Martin
>
>--
>Dr. Martin Kahms
>Institut für Medizinische Physik und Biophysik
>Center for Nanotechnology (CeNTech)
>Heisenbergstr. 11
>48149 Münster
>Tel.:[phone]
>Fax: [phone]

________________________________
Eli Rothenberg, Ph.D.
Post Doctoral Research Associate,
Howard Hughes Medical Institute,
Department of Physics,
University of Illinois,
Urbana-Champaign. 61801.
Illinois, USA
Tel: [phone];
Fax: [phone];
Email: [hidden email]