Spectral deconvolution on DAPI

[weber]

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Dear confocal list,

I would have your advice about this !
Is-it possible to do spectral deconvolution on a dapi staining ?

Regards

Pascal

[Mark Cannell-2]

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Yes, but YMMV.

Regards
On 29/06/2015, at 11:32 am, Pascal Weber <[hidden email]> wrote:

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> *****
>
> Dear confocal list,
>
> I would have your advice about this !
> Is-it possible to do spectral deconvolution on a dapi staining ?
>
> Regards
>
> Pascal

Mark  B. Cannell Ph.D. FRSNZ
Professor of Cardiac Cell Biology
School of Physiology &  Pharmacology
Medical Sciences Building
University of Bristol
Bristol
BS8 1TD UK

[hidden email]

[weber]

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Sorry,

What means YMMV ?

[Zdenek Svindrych-2]

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Your mileage may vary...
z.



---------- Původní zpráva ----------
Od: Pascal Weber <[hidden email]>
Komu: [hidden email]
Datum: 29. 6. 2015 7:51:24
Předmět: Re: Spectral deconvolution on DAPI

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Sorry,

What means YMMV ?"

[Tim Feinstein]

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Yes and we have had good results doing it, but you have two potential
problems.  DAPI has an extremely broad emission spectrum, so it could get
in the way of your 488 label and possibly the yellow-red  as well if they
are not very bright.  Use a spectrum viewer like the ones from
MolProbes/LifeTech/ThermoFisher, Chroma, Zeiss Light Lab etc to check how
well your other emission peaks stand apart from DAPI.

The other problem is kind of the inverse of the first one - 405 nm lasers
only just touch the edge of DAPI's excitation spectrum (again check those
spectra viewers), so imaging DAPI on a confocal can be quite inefficient
compared with a widefield scope.  As a result DAPI intensity can be hard
to control between tissue types, preparations and even one experiment to
the next.  This is always the case but especially problematic when trying
to balance your fluors for a spectral experiment.

As a result we try to use far red stains like To-Pro-3 instead when
staining DNA for confocal.  In our experience To-Pro-3 is VERY bright in
many tissues, so you have to titrate it down well below the recommended
dosage to balance its intensity with other fluors.

All the best,


Tim

Timothy Feinstein, Ph.D.
Research Scientist
University of Pittsburgh Department of Developmental Biology





On 6/29/15, 6:32 AM, "Pascal Weber" <[hidden email]> wrote:

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>*****
>
>Dear confocal list,
>
>I would have your advice about this !
>Is-it possible to do spectral deconvolution on a dapi staining ?
>
>Regards
>
>Pascal

[weber]

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Infact i have lot of different bacteria and they will not have the same spectra.
I want to discriminate them only this method. I have not different staining just
this one but i'm not sure that is the wright way to proceed. My goal is to reach
a good an simple method to verify if the sample is pure or not. Before to do
DNA sequencing.

[rjpalmer]

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I think I missed a reply along the way but let me see if I understand what
you want to do.  You have several samples of dapi-stained bacteria and you
want to detect differences in staining between the samples as a way to
decide whether the bacteria are phylogenetically different?  In other
words, if the staining of A is different from B, then you want to sequence
the two organisms, but if the staining between A and C is similar, you
will consider them to be similar and not worth sequencing?

On 6/29/15, 11:01 AM, "Pascal Weber" <[hidden email]> wrote:

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>*****
>
>Infact i have lot of different bacteria and they will not have the same
>spectra.
>I want to discriminate them only this method. I have not different
>staining just
>this one but i'm not sure that is the wright way to proceed. My goal is
>to reach
>a good an simple method to verify if the sample is pure or not. Before to
>do
>DNA sequencing.

[weber]

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Yes that's it !

[rjpalmer]

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Here is the opinion of a microbiologist.  The signal you get from any
stain other than a 16S rDNA FISH probe (or maybe a very specific antibody)
is not valuable as a taxonomic indicator.  I have not done this
experiment, but I would expect that two completely different bacterial
species could have similar DAPI signals, just as two bacteria with similar
DAPI signals can be very different organisms.  I think DAPI doesn¹t tell
you anything about DNA other than where it is in a cell and how much of it
is there (intensity).  It may be theoretically possible to distinguish
"high GC" organisms from others, but this parameter alone is not a very
good indicator of what bacterium you have.

I assume you have plenty of sample material, and that these bacteria can
be grown again for nucleic acid extraction/analysis.  If that is the case,
and you do not have hundreds of samples to process, you are much better
off simply sequencing the 16s ribosomal gene in each sample.  Extraction
is with a kit, you amplify the gene using a standard primer set to produce
amplicons of around 500 bases.  Sequence these, submit them to BLAST and
you will get a reasonable idea of what you have.  This procedure is being
done by high-school students nowadays.

Maybe I am missing something here.  Happy to discuss in greater detail if
you¹d like to take the discussion off the listserv.

Robert J. Palmer Jr., PhD
Bldg 30, Room 207
NIDCR/NIH
Bethesda MD 20892
301-594-0025




On 6/29/15, 12:42 PM, "Pascal Weber" <[hidden email]> wrote:

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>
>Yes that's it !

[Craig Brideau]

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If your spectral acquisition is very broad, you will catch most/all of the
DAPI curve. The more of the curve shape you can catch, the better the unmix
will be. As mentioned by Tim, 405nm is non-optimal for excitation, but it
tends to be the most common laser for confocal of blue dyes. In my own
experience it works fairly well, just watch out for scatter from the laser
contaminating your spectra.

Craig Brideau

On Mon, Jun 29, 2015 at 10:42 AM, Pascal Weber <[hidden email]>
wrote:

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> *****
>
> Yes that's it !
>

[Tim Feinstein]

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Hi Pascal,

If these are photosynthetic bacteria then their autofluorescence spectra
may help identify them.  In a past life I tried that with dinoflagellates
in a spectrofluorimeter.  I do not think DAPI will be useful unless you
have already ruled out every other possible way of classifying them.

Of course it depends on your experimental design.  If every dish can only
be one out of four or five possible strains then you might get away with
identifying a distinct spectrum for each strain, as long as you show that
spectrum variabilities do not also come from life stage, nutrient
conditions, stress level, ambient  environment etc.  If the list of
possible strains is long or open-ended then you should try something else.
 

Best,


Tim

Timothy Feinstein, Ph.D.
Research Scientist
University of Pittsburgh Department of Developmental Biology





On 6/29/15, 11:01 AM, "Pascal Weber" <[hidden email]> wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>Post images on http://www.imgur.com and include the link in your posting.
>*****
>
>Infact i have lot of different bacteria and they will not have the same
>spectra.
>I want to discriminate them only this method. I have not different
>staining just
>this one but i'm not sure that is the wright way to proceed. My goal is
>to reach
>a good an simple method to verify if the sample is pure or not. Before to
>do
>DNA sequencing.

[Jurek Dobrucki]

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Pascal,
another issue to keep in mind when detecting DAPI, especially if the
bacteria are also stained with some green dye or GFP, is
photoconversion of this dye to a green emitting form (Zurek-Biesiada
et al. Photoconversion of Hoechst 33258, DAPI, and Vybrant DyeCycle...
Cytometry A. 2013 May;83(5):441-51).
Hope this helps,
Jurek Dobrucki

On Mon, Jun 29, 2015 at 8:33 PM, Feinstein, Timothy N <[hidden email]> wrote:

> *****
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> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hi Pascal,
>
> If these are photosynthetic bacteria then their autofluorescence spectra
> may help identify them.  In a past life I tried that with dinoflagellates
> in a spectrofluorimeter.  I do not think DAPI will be useful unless you
> have already ruled out every other possible way of classifying them.
>
> Of course it depends on your experimental design.  If every dish can only
> be one out of four or five possible strains then you might get away with
> identifying a distinct spectrum for each strain, as long as you show that
> spectrum variabilities do not also come from life stage, nutrient
> conditions, stress level, ambient  environment etc.  If the list of
> possible strains is long or open-ended then you should try something else.
>
>
> Best,
>
>
> Tim
>
> Timothy Feinstein, Ph.D.
> Research Scientist
> University of Pittsburgh Department of Developmental Biology
>
>
>
>
>
> On 6/29/15, 11:01 AM, "Pascal Weber" <[hidden email]> wrote:
>
>>*****
>>To join, leave or search the confocal microscopy listserv, go to:
>>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>Post images on http://www.imgur.com and include the link in your posting.
>>*****
>>
>>Infact i have lot of different bacteria and they will not have the same
>>spectra.
>>I want to discriminate them only this method. I have not different
>>staining just
>>this one but i'm not sure that is the wright way to proceed. My goal is
>>to reach
>>a good an simple method to verify if the sample is pure or not. Before to
>>do
>>DNA sequencing.