nuclear dye for multiphoton imaging

[Ekaterina PAPUSHEVA-2]

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Dear all,

Does someone know a good nuclear chemical dye that can be used in live tissue and perform well with multiphoton excitation?

DAPI gives enormous background

Would be grateful for any advice!
Ekaterina

[Kurt Anderson-3]

we’ve had good luck using hoechst in the intestine: PMID 22745693.

Draq5 did not stain the intestinal epithelium in our hands; maybe it can’t get out of the bloodstream?

there is an appealing new silicone-rhodamine derivative which we haven’t tried yet: PMID 26423723.

party on...


On 18 Dec 2015, at 11:54, Ekaterina PAPUSHEVA <[hidden email]<mailto:[hidden email]>> wrote:

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Dear all,

Does someone know a good nuclear chemical dye that can be used in live tissue and perform well with multiphoton excitation?

DAPI gives enormous background

Would be grateful for any advice!
Ekaterina





Prof. Kurt I. Anderson
Tumor Cell Migration Lab and
Beatson Advanced Imaging Resource (BAIR)
The Beatson Institute for Cancer Research
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[Alfred Bahnson]

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Ekaterina, I don't know whether it will work with multi-photon, but the
SiR-DNA far-red DNA probe is working well for live cell imaging with low
toxicity.

http://www.cytoskeleton.com/live-cell-reagents/spirochrome/sir-dna

Al Bahnson
Kairos Instruments, LLC
Pittsburgh PA


On Fri, Dec 18, 2015 at 6:54 AM, Ekaterina PAPUSHEVA <
[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.
> *****
>
> Dear all,
>
> Does someone know a good nuclear chemical dye that can be used in live
> tissue and perform well with multiphoton excitation?
>
> DAPI gives enormous background
>
> Would be grateful for any advice!
> Ekaterina
>

[George McNamara]

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Hi Ekaterina,

Please test Hoechst 33342 at 50ng/mL and 10 ng/mL (dilute into distilled
water ... with modern detectors, 1 ng/mL may be enough). Live cells are
dim, dead cells are bright (but won't hurt the PMTs if scanning fast). I
forget whether 750nm or 780nm is better (2photon ex ... you are welcome
to test 3p at ~1000nm and above). You can also test DAPI at those
concentrations. I think was Mike Ignatius (Molecular Probes at the time)
who turned me on to "go low" concentrations - cited Idziorek 1995 J
Immunol Methods (PubMed 7561136). Note: some cells have MDR pumps that
extrude drugs, if your cells are practically unstained at 50 ng/mL, you
can try MDR inhibitor (Verapamil?).


Why 33342? ... Paul Rigby emailed me that Hoechst 33342 is more live
cell permeable than 33258.

Enjoy,

George

On 12/18/2015 5:54 AM, Ekaterina PAPUSHEVA 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.
> *****
>
> Dear all,
>
> Does someone know a good nuclear chemical dye that can be used in live tissue and perform well with multiphoton excitation?
>
> DAPI gives enormous background
>
> Would be grateful for any advice!
> Ekaterina
>

--



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/42
http://works.bepress.com/gmcnamara/75
https://www.linkedin.com/in/georgemcnamara

[Michael Giacomelli]

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Hi Ekaterina,

I have tested quite a lot of dyes for nuclear use in cultured/live
human tissue under multiphoton excitation.  My experience is that DAPI
is probably one of the most selective agents.  You should have
virtually no background signal whatsoever even at extremely high
concentrations.

How are you preparing the staining solution?  And what filters are you
using?  Keep in mind that at DAPI excitation wavelengths (<820 nm for
TPE), the SHG and NADH signals in many tissue types will be very
large, so choice of collection filters is essential.


Mike

On Fri, Dec 18, 2015 at 6:54 AM, Ekaterina PAPUSHEVA
<[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.
> *****
>
> Dear all,
>
> Does someone know a good nuclear chemical dye that can be used in live tissue and perform well with multiphoton excitation?
>
> DAPI gives enormous background
>
> Would be grateful for any advice!
> Ekaterina