phenol red vs. hardware autofocus eg Nikon Perfect Focus (PFS) - near IR 870 nm LED.

[Daniel James White]

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Dear All prefect focusers,

We believe we see a correlation between use of phenol red
and Nikon perfect focus (870 nm), hardware autofocus slowly drifting off (over many hours)

Does anyone else have any experience with phenol red causing problems
with hardware autofocus?

cheers

Dan


Dr. Daniel James White BSc. (Hons.) PhD

Leader - Image Processing Facility,
Senior Microscopist,
Light Microscopy Facility.

Max Planck Institute of Molecular Cell Biology and Genetics
Pfotenhauerstrasse 108
01307 DRESDEN
Germany

[phone] (German Mobile)
[phone] (Work phone at MPI-CBG)
[phone] (Fax MPI-CBG LMF)
chalkie666 Skype
http://www.bioimagexd.net  BioImageXD
http://fiji.sc                                        Fiji -  is just ImageJ (Batteries Included)
http://www.chalkie.org.uk                Dan's Homepages
https://ifn.mpi-cbg.de  Biopolis Dresden Imaging Platform (BioDIP)
dan (at) chalkie.org.uk
( white (at) mpi-cbg.de )

[Craig Brideau]

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We discovered this problem a couple weeks ago with Nile Red.  It seems some
dyes that emit into the far red throws off the 870nm tracking system.
Choose your dyes accordingly!

Craig

On Wed, Sep 7, 2011 at 1:35 AM, Daniel James White <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear All prefect focusers,
>
> We believe we see a correlation between use of phenol red
> and Nikon perfect focus (870 nm), hardware autofocus slowly drifting off
> (over many hours)
>
> Does anyone else have any experience with phenol red causing problems
> with hardware autofocus?
>
> cheers
>
> Dan
>
>
> Dr. Daniel James White BSc. (Hons.) PhD
>
> Leader - Image Processing Facility,
> Senior Microscopist,
> Light Microscopy Facility.
>
> Max Planck Institute of Molecular Cell Biology and Genetics
> Pfotenhauerstrasse 108
> 01307 DRESDEN
> Germany
>
> [phone] (German Mobile)
> [phone] (Work phone at MPI-CBG)
> [phone] (Fax MPI-CBG LMF)
> chalkie666                                      Skype
> http://www.bioimagexd.net       BioImageXD
> http://fiji.sc                                  Fiji -  is just ImageJ
> (Batteries Included)
> http://www.chalkie.org.uk               Dan's Homepages
> https://ifn.mpi-cbg.de                  Biopolis Dresden Imaging Platform
> (BioDIP)
> dan (at) chalkie.org.uk
> ( white (at) mpi-cbg.de )
>

[Beat Ludin]

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Interesting finding with Nile Red (not really surprising if you think
about it, though). But does Phenol Red fluoresce in the IR-region? I
ran a fluorescence spectrum on PR many years ago and didn't find any
significant fluorescence. But maybe I didn't look beyond 750nm back
then. Does anybody know more about this?

Beat

At 18:32 07-09-11, you wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>We discovered this problem a couple weeks ago with Nile Red.  It seems some
>dyes that emit into the far red throws off the 870nm tracking system.
>Choose your dyes accordingly!
>
>Craig
>
>On Wed, Sep 7, 2011 at 1:35 AM, Daniel James White <[hidden email]> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Dear All prefect focusers,
> >
> > We believe we see a correlation between use of phenol red
> > and Nikon perfect focus (870 nm), hardware autofocus slowly drifting off
> > (over many hours)
> >
> > Does anyone else have any experience with phenol red causing problems
> > with hardware autofocus?
> >
> > cheers
> >
> > Dan
> >
> >
> > Dr. Daniel James White BSc. (Hons.) PhD
> >
> > Leader - Image Processing Facility,
> > Senior Microscopist,
> > Light Microscopy Facility.
> >
> > Max Planck Institute of Molecular Cell Biology and Genetics
> > Pfotenhauerstrasse 108
> > 01307 DRESDEN
> > Germany
> >
> > [phone] (German Mobile)
> > [phone] (Work phone at MPI-CBG)
> > [phone] (Fax MPI-CBG LMF)
> > chalkie666                                      Skype
> > http://www.bioimagexd.net       BioImageXD
> > http://fiji.sc                                  Fiji -  is just ImageJ
> > (Batteries Included)
> > http://www.chalkie.org.uk               Dan's Homepages
> > https://ifn.mpi-cbg.de                  Biopolis Dresden Imaging Platform
> > (BioDIP)
> > dan (at) chalkie.org.uk
> > ( white (at) mpi-cbg.de )
> >

[Stephen Cody-2]

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G'day Beat,

Ian Johnson (formerly Mol. Probes) in his lectures often mentions that Phenol Red itself is not fluorescent. I found this surprising. However, he thinks the dye used in culture media may often have other red impurities mixed with the phenol red that may be fluorescent. Hence the fluorescence of dyes in media may be highly variable from batch to batch. I haven't tested this myself.

Cheers
Steve

Stephen H. Cody

On 19/09/2011, at 5:32 PM, Beat Ludin <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Interesting finding with Nile Red (not really surprising if you think about it, though). But does Phenol Red fluoresce in the IR-region? I ran a fluorescence spectrum on PR many years ago and didn't find any significant fluorescence. But maybe I didn't look beyond 750nm back then. Does anybody know more about this?
>
> Beat
>
> At 18:32 07-09-11, you wrote:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> We discovered this problem a couple weeks ago with Nile Red.  It seems some
>> dyes that emit into the far red throws off the 870nm tracking system.
>> Choose your dyes accordingly!
>>
>> Craig
>>
>> On Wed, Sep 7, 2011 at 1:35 AM, Daniel James White <[hidden email]> wrote:
>>
>> > *****
>> > To join, leave or search the confocal microscopy listserv, go to:
>> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> > *****
>> >
>> > Dear All prefect focusers,
>> >
>> > We believe we see a correlation between use of phenol red
>> > and Nikon perfect focus (870 nm), hardware autofocus slowly drifting off
>> > (over many hours)
>> >
>> > Does anyone else have any experience with phenol red causing problems
>> > with hardware autofocus?
>> >
>> > cheers
>> >
>> > Dan
>> >
>> >
>> > Dr. Daniel James White BSc. (Hons.) PhD
>> >
>> > Leader - Image Processing Facility,
>> > Senior Microscopist,
>> > Light Microscopy Facility.
>> >
>> > Max Planck Institute of Molecular Cell Biology and Genetics
>> > Pfotenhauerstrasse 108
>> > 01307 DRESDEN
>> > Germany
>> >
>> > [phone] (German Mobile)
>> > [phone] (Work phone at MPI-CBG)
>> > [phone] (Fax MPI-CBG LMF)
>> > chalkie666                                      Skype
>> > http://www.bioimagexd.net       BioImageXD
>> > http://fiji.sc                                  Fiji -  is just ImageJ
>> > (Batteries Included)
>> > http://www.chalkie.org.uk               Dan's Homepages
>> > https://ifn.mpi-cbg.de                  Biopolis Dresden Imaging Platform
>> > (BioDIP)
>> > dan (at) chalkie.org.uk
>> > ( white (at) mpi-cbg.de )
>> >

[Iain Johnson]

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The fluorescence of phenol red is very weak (comparable to the intensity of
water Raman scattering with 488 nm excitation; water Raman being the "gold
standard" for a very weak signal in fluorescence detection applications).  I
don't know about other excitation wavelengths.  Based on it's structure, you
would not expect fluorescence of phenol red at >600 nm, but then the purity
of commercial phenol red is probably not better than 80% and the other 20%
could be???.

I have never used Perfect Focus myself, but looking at the technical
descriptions, it is hard to see how optical signals from the specimen are
interfering with the 870 nm reflection from either the top or bottom of the
coverslip in an inverted configuration.  I think this discussion could use
some input on whether the measurements in question are inverted or upright
configuration, what the objective NA is, and whether oil or water immersion
or dry.

Iain

Iain Johnson Consulting
Eugene, OR
[phone]

On Mon, Sep 19, 2011 at 2:32 PM, Stephen Cody <[hidden email]>wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> G'day Beat,
>
> Ian Johnson (formerly Mol. Probes) in his lectures often mentions that
> Phenol Red itself is not fluorescent. I found this surprising. However, he
> thinks the dye used in culture media may often have other red impurities
> mixed with the phenol red that may be fluorescent. Hence the fluorescence of
> dyes in media may be highly variable from batch to batch. I haven't tested
> this myself.
>
> Cheers
> Steve
>
> Stephen H. Cody
>
> On 19/09/2011, at 5:32 PM, Beat Ludin <[hidden email]> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Interesting finding with Nile Red (not really surprising if you think
> about it, though). But does Phenol Red fluoresce in the IR-region? I ran a
> fluorescence spectrum on PR many years ago and didn't find any significant
> fluorescence. But maybe I didn't look beyond 750nm back then. Does anybody
> know more about this?
> >
> > Beat
> >
> > At 18:32 07-09-11, you wrote:
> >> *****
> >> To join, leave or search the confocal microscopy listserv, go to:
> >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >> *****
> >>
> >> We discovered this problem a couple weeks ago with Nile Red.  It seems
> some
> >> dyes that emit into the far red throws off the 870nm tracking system.
> >> Choose your dyes accordingly!
> >>
> >> Craig
> >>
> >> On Wed, Sep 7, 2011 at 1:35 AM, Daniel James White <[hidden email]>
> wrote:
> >>
> >> > *****
> >> > To join, leave or search the confocal microscopy listserv, go to:
> >> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >> > *****
> >> >
> >> > Dear All prefect focusers,
> >> >
> >> > We believe we see a correlation between use of phenol red
> >> > and Nikon perfect focus (870 nm), hardware autofocus slowly drifting
> off
> >> > (over many hours)
> >> >
> >> > Does anyone else have any experience with phenol red causing problems
> >> > with hardware autofocus?
> >> >
> >> > cheers
> >> >
> >> > Dan
> >> >
> >> >
> >> > Dr. Daniel James White BSc. (Hons.) PhD
> >> >
> >> > Leader - Image Processing Facility,
> >> > Senior Microscopist,
> >> > Light Microscopy Facility.
> >> >
> >> > Max Planck Institute of Molecular Cell Biology and Genetics
> >> > Pfotenhauerstrasse 108
> >> > 01307 DRESDEN
> >> > Germany
> >> >
> >> > [phone] (German Mobile)
> >> > [phone] (Work phone at MPI-CBG)
> >> > [phone] (Fax MPI-CBG LMF)
> >> > chalkie666                                      Skype
> >> > http://www.bioimagexd.net       BioImageXD
> >> > http://fiji.sc                                  Fiji -  is just
> ImageJ
> >> > (Batteries Included)
> >> > http://www.chalkie.org.uk               Dan's Homepages
> >> > https://ifn.mpi-cbg.de                  Biopolis Dresden Imaging
> Platform
> >> > (BioDIP)
> >> > dan (at) chalkie.org.uk
> >> > ( white (at) mpi-cbg.de )
> >> >
>

[Sylvie Le Guyader-2]

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

Dear all

That's a very interesting thread!

We have had problems with small changes of focus which seem to come up about 1h after the start of our 8h long time lapse acquisitions. More than a focus drift, we get the cells to come slightly in and out of the focus level we have set. Of course the cells are alive so they move across the field but they also move at the start of the movie and this doesn't happen then.
Because we use several xy positions and a 60x oil objective (NA1.4), we thought that this came from the combination of the oil sliding along the objective as well as spreading under the dish. We tried to solve this problem with choosing nearby xy positions and setting the stage speed to minimum but the problem remains.
This doesn't occur in every well though so about 1/3 to 1/2 of the movies are perfectly in focus throughout the 8h.

According to what Dan says, it would be possible that a slight change in pH in e.g. slightly more crowded wells, would interfere with the PFS. I agree with Ian that it doesn't seem logical considering we use an inverted microscope but it is very easy to try the same experiment with phenol red free medium.

I suppose that you tried, Dan and that's why you claim that there might be an interference, is that right?

Med vänlig hälsning / Best regards
 
Sylvie
 
@@@@@@@@@@@@@@@@@@@@@@@@
Sylvie Le Guyader
Live Cell Imaging Unit
Dept of Biosciences and Nutrition
Karolinska Institutet
Novum
14183 Huddinge
Sweden
office: [phone]
LCI room: [phone]
mobile: [phone]

> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[hidden email]] On Behalf Of Iain Johnson
> Sent: 20 September 2011 04:53
> To: [hidden email]
> Subject: Re: phenol red vs. hardware autofocus eg Nikon Perfect Focus (PFS) -
> near IR 870 nm LED.
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> The fluorescence of phenol red is very weak (comparable to the intensity of
> water Raman scattering with 488 nm excitation; water Raman being the "gold
> standard" for a very weak signal in fluorescence detection applications).  I
> don't know about other excitation wavelengths.  Based on it's structure, you
> would not expect fluorescence of phenol red at >600 nm, but then the purity
> of commercial phenol red is probably not better than 80% and the other 20%
> could be???.
>
> I have never used Perfect Focus myself, but looking at the technical
> descriptions, it is hard to see how optical signals from the specimen are
> interfering with the 870 nm reflection from either the top or bottom of the
> coverslip in an inverted configuration.  I think this discussion could use
> some input on whether the measurements in question are inverted or upright
> configuration, what the objective NA is, and whether oil or water immersion
> or dry.
>
> Iain
>
> Iain Johnson Consulting
> Eugene, OR
> [phone]
>
> On Mon, Sep 19, 2011 at 2:32 PM, Stephen Cody
> <[hidden email]>wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > G'day Beat,
> >
> > Ian Johnson (formerly Mol. Probes) in his lectures often mentions that
> > Phenol Red itself is not fluorescent. I found this surprising. However, he
> > thinks the dye used in culture media may often have other red impurities
> > mixed with the phenol red that may be fluorescent. Hence the fluorescence of
> > dyes in media may be highly variable from batch to batch. I haven't tested
> > this myself.
> >
> > Cheers
> > Steve
> >
> > Stephen H. Cody
> >
> > On 19/09/2011, at 5:32 PM, Beat Ludin <[hidden email]> wrote:
> >
> > > *****
> > > To join, leave or search the confocal microscopy listserv, go to:
> > > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > > *****
> > >
> > > Interesting finding with Nile Red (not really surprising if you think
> > about it, though). But does Phenol Red fluoresce in the IR-region? I ran a
> > fluorescence spectrum on PR many years ago and didn't find any significant
> > fluorescence. But maybe I didn't look beyond 750nm back then. Does anybody
> > know more about this?
> > >
> > > Beat
> > >
> > > At 18:32 07-09-11, you wrote:
> > >> *****
> > >> To join, leave or search the confocal microscopy listserv, go to:
> > >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > >> *****
> > >>
> > >> We discovered this problem a couple weeks ago with Nile Red.  It seems
> > some
> > >> dyes that emit into the far red throws off the 870nm tracking system.
> > >> Choose your dyes accordingly!
> > >>
> > >> Craig
> > >>
> > >> On Wed, Sep 7, 2011 at 1:35 AM, Daniel James White <[hidden email]>
> > wrote:
> > >>
> > >> > *****
> > >> > To join, leave or search the confocal microscopy listserv, go to:
> > >> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > >> > *****
> > >> >
> > >> > Dear All prefect focusers,
> > >> >
> > >> > We believe we see a correlation between use of phenol red
> > >> > and Nikon perfect focus (870 nm), hardware autofocus slowly drifting
> > off
> > >> > (over many hours)
> > >> >
> > >> > Does anyone else have any experience with phenol red causing problems
> > >> > with hardware autofocus?
> > >> >
> > >> > cheers
> > >> >
> > >> > Dan
> > >> >
> > >> >
> > >> > Dr. Daniel James White BSc. (Hons.) PhD
> > >> >
> > >> > Leader - Image Processing Facility,
> > >> > Senior Microscopist,
> > >> > Light Microscopy Facility.
> > >> >
> > >> > Max Planck Institute of Molecular Cell Biology and Genetics
> > >> > Pfotenhauerstrasse 108
> > >> > 01307 DRESDEN
> > >> > Germany
> > >> >
> > >> > [phone] (German Mobile)
> > >> > [phone] (Work phone at MPI-CBG)
> > >> > [phone] (Fax MPI-CBG LMF)
> > >> > chalkie666                                      Skype
> > >> > http://www.bioimagexd.net       BioImageXD
> > >> > http://fiji.sc                                  Fiji -  is just
> > ImageJ
> > >> > (Batteries Included)
> > >> > http://www.chalkie.org.uk               Dan's Homepages
> > >> > https://ifn.mpi-cbg.de                  Biopolis Dresden Imaging
> > Platform
> > >> > (BioDIP)
> > >> > dan (at) chalkie.org.uk
> > >> > ( white (at) mpi-cbg.de )
> > >> >
> >

[Tim Feinstein-2]

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

Hmm.  I was taught in my formative years to avoid phenol red on a fluorescence microscope, perhaps more from superstition than because of a specific problem, so I always use either OptiMEM + FBS or another medium without Phenol Red.  We have done up to twenty-four hour imaging of live cells at 37 C without any problem from the PFS focus control.  

cheers,


TF

Timothy Feinstein, PhD
Postdoctoral Fellow
Laboratory for GPCR Biology
Dept. of Pharmacology & Chemical Biology
University of Pittsburgh, School of Medicine
BST W1301, 200 Lothrop St.
Pittsburgh, PA  15261

[Kimberly Young]

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

Hi All,
I don't have a ton of experience here, but from what I know about phenol
red, the compound does not fluoresce on its own, but it does have a fairly
high visible light extinction coefficient. This would mean pretty high
background in fluorescence imaging unless you increase your light source
intensity. For near IR imaging and live cell, this could mean a significant
amount of thermal drift over time.
Good luck with the focus drift,
Kimberly Young

McGill University,
Montreal, Canada

On Tue, Sep 20, 2011 at 10:09 AM, Tim Feinstein <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hmm.  I was taught in my formative years to avoid phenol red on a
> fluorescence microscope, perhaps more from superstition than because of a
> specific problem, so I always use either OptiMEM + FBS or another medium
> without Phenol Red.  We have done up to twenty-four hour imaging of live
> cells at 37 C without any problem from the PFS focus control.
>
> cheers,
>
>
> TF
>
> Timothy Feinstein, PhD
> Postdoctoral Fellow
> Laboratory for GPCR Biology
> Dept. of Pharmacology & Chemical Biology
> University of Pittsburgh, School of Medicine
> BST W1301, 200 Lothrop St.
> Pittsburgh, PA  15261
>