what fluorescent proteins are working best for precision localization microscopy (nanoscopy) - in your and your users hands?

[Jurek Dobrucki]

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Dear Colleagues,

in the contect of UV-induced photoconversion of Hoechst dyes and DAPI, this
recent paper from my Lab may be of interest as well:

UV-activated conversion of Hoechst 33258, DAPI, and Vybrant DyeCycle
fluorescent dyes into blue-excited, green-emitting protonated forms.

Zurek-Biesiada D, Kędracka-Krok S, Dobrucki JW.

Cytometry A. 2013 Feb 15. doi: 10.1002/cyto.a.22260. [Epub ahead of print]

Abstract:
Hoechst 33258, DAPI and Vybrant DyeCycle are commonly known DNA fluorescent
dyes that are excited by UV and emit in the blue region of the spectrum of
visible light. Conveniently, they leave the reminder of the spectrum for
microscopy detection of other cellular targets labeled with probes emitting
in green, yellow or red. However, an exposure of these dyes to UV induces
their photoconversion and results in production of the forms of these dyes
that are excited by blue light and show fluoresce maxima in green and a
detectable fluorescence in yellow and orange regions of the spectrum.
Photoconversion of Hoechst 33258 and DAPI is
reversible and independent of the dye concentration or the presence of DNA.
Spectrofluorimetry and mass spectrometry analyses indicate that exposure to
UV induces protonation of Hoechst 33258 and DAPI.

Regards,

Jurek Dobrucki
--
Jerzy Dobrucki, Ph.D., D.Sc.
Professor of Biophysics
Head, Division of Cell Biophysics
Faculty of Biochemistry, Biophysics and
    Biotechnology
Jagiellonian University
ul. Gronostajowa 7
30-387 Krakow, Poland
tel. [phone]; fax. [phone]
[hidden email]
http://www.wbbib.uj.edu.pl/helios

***************************************************************************************
Fluorescence Microscopy. From Principles to Biological Applications
Viley-VCH; Edition April 2013
http://www.wiley-vch.de/publish/en/books/forthcomingTitles/PH00/3-527-32922-6/?sID=v3pdgfe82f6af627ajo8b6o2c7
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On Sat, Apr 13, 2013 at 3:42 PM, George McNamara
<[hidden email]>wrote:

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> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/**wa?A0=confocalmicroscopy<http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>
> *****
>
> Hi Adrian,
>
> Thanks for pointing out the Jez et al 2013 HCB paper (I had read the
> Piterburg 2012 JM paper, thanks for the reminder of it as well).
>
> I see Jez et al's abstract included,
>
> "Hoechst 33342 exhibited the lowest photoconversion while that for DAPI
> and Hoechst 33258 was much stronger. Different fixation methods did not
> substantially affect the strength of photoconversion. We also suggest
> avoiding the use of mounting medium with high glycerol concentrations since
> glycerol showed the strongest impact on photoconversion."
>
> I'll have to read the paper to see if H33342 is worth buying. Until I read
> the paper, I won't know whether the authors claim that " glycerol showed
> the strongest impact " is correct or (my guess) that "anti-fade" reagent(s)
> added to commercial glycerol based mounting media are at fault.
>
> for that matter, it is possible that an additive in the Cytoseal is
> responsible for the observation I mentioned - MSDS for Cytoseal 60 is:
>
> Toluene CAS# 108-88-3 -65%
> Acrylic Resin CAS# 28262-63-7 -35%
> Antioxidant CAS# 128-37-0
> Butyl Benzyl Phthalate CAS# 85-68-7
>
> The Antioxidant CAS# 128-37-0 is (according to various google search
> results), 2-6-di-tert-butyl_p-cresol
> also known as Butylhydroxytoluene;BHT
> http://www.caslab.com/2-6-di-**tert-butyl_p-cresol_CAS_128-**37-0/<http://www.caslab.com/2-6-di-tert-butyl_p-cresol_CAS_128-37-0/>
> I believe BHT is a food additive.
>
>
> Sincerely,
>
> George
>
>
> On 4/13/2013 5:25 AM, Adrian Smith wrote:
>
>> *****
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>> http://lists.umn.edu/cgi-bin/**wa?A0=confocalmicroscopy<http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>
>> *****
>>
>> On 12/04/2013, at 10:59 PM, George McNamara<geomcnamara@**EARTHLINK.NET<[hidden email]>>
>>  wrote:
>>
>>
>>
>>>   Michael's DAPI in CytoSeal photoconverted to green fluorescence after
>>> prolonged excitation with 405 nm laser (several of us have seen, and
>>> mentioned on listserv, this with Hoechst ... I suppose it is possible the
>>> slide was mislabeled and was actually Hoechst???).
>>>
>>>
>>
>> Hi George,
>>
>> I'm not 100% sure what you mean by "photoconverted to green fluorescence"
>> but we have had issues with DAPI photoconversion after 405nm excitation on
>> a couple of occasions in the last few years.
>>
>> There have been a couple of publications in that last year that describe
>> exactly what we have seen:-
>>
>>
>>
>>> J Microsc. 2012 Apr;246(1):89-95. doi: 10.1111/j.1365-2818.2011.**03591.x.
>>> Epub 2012 Jan 31.
>>> Photoconversion of DAPI following UV or violet excitation can cause DAPI
>>> to fluoresce with blue or cyan excitation.
>>> Piterburg M, Panet H, Weiss A.
>>> Abstract
>>> 4'-6-Diamidino-2-phenylindole is a fluorescent dye commonly used to
>>> visualize deoxyribonucleic acid or cell nuclei in fixed cell preparations,
>>> and is often used together with fluorescein or green fluorescent protein,
>>> which can be excited without exciting 4'-6-Diamidino-2-phenylindole. It is
>>> assumed that when using typical fluorescein or green fluorescent protein
>>> filter cubes, 4'-6-Diamidino-2-phenylindole will not be observed. In this
>>> paper, we show that following observation of 4'-6-Diamidino-2-phenylindole
>>> using UV or violet excitation, it may become sensitive to the blue/cyan
>>> excitation used in fluorescein/green fluorescent protein filter cubes. This
>>> has serious implications for the use of 4'-6-Diamidino-2-phenylindole
>>> together with widely used green fluorophores in double labelling
>>> experiments.
>>>
>>>
>>>
>>>
>>
>>
>>
>>
>>
>>> Histochem Cell Biol. 2013 Jan;139(1):195-204. doi:
>>> 10.1007/s00418-012-1039-8. Epub 2012 Oct 14.
>>> The hazards of DAPI photoconversion: effects of dye, mounting media and
>>> fixative, and how to minimize the problem.
>>> Jež M, Bas T, Veber M, Košir A, Dominko T, Page R, Rožman P.
>>> Abstract
>>> Immunocytochemistry is a powerful tool for detection and visualization
>>> of specific molecules in living or fixed cells, their localization and
>>> their relative abundance. One of the most commonly used fluorescent DNA
>>> dyes in immunocytochemistry applications is 4',6-diamidino-2-phenylindole
>>> dihydrochloride, known as DAPI. DAPI binds strongly to DNA and is used
>>> extensively for visualizing cell nuclei. It is excited by UV light and
>>> emits characteristic blue fluorescence. Here, we report a phenomenon based
>>> on an apparent photoconversion of DAPI that results in detection of a DAPI
>>> signal using a standard filter set for detection of green emission due to
>>> blue excitation. When a sample stained with DAPI only was first imaged with
>>> the green filter set (FITC/GFP), only a weak cytoplasmic autofluorescence
>>> was observed. Next, we imaged the sample with a DAPI filter set, obtaining
>>> a strong nuclear DAPI signal as expected. Upon reimaging the same samples
>>> with a FITC/GFP filter set, robust nuclear fluorescence was observed. We
>>> conclude that excitation with UV results in a photoconversion of DAPI that
>>> leads to detection of DAPI due to excitation and emission in the FITC/GFP
>>> channel. This phenomenon can affect data interpretation and lead to
>>> false-positive results when used together with fluorochrome-labeled nuclear
>>> proteins detected with blue excitation and green emission. In order to
>>> avoid misinterpretations, extra precaution should be taken to prepare
>>> staining solutions with low DAPI concentration and DAPI (UV excitation)
>>> images should be acquired after all other higher wavelength images. Of
>>> various DNA dyes tested, Hoechst 33342 exhibited the lowest photoconversion
>>> while that for DAPI and Hoechst 33258 was much stronger. Different fixation
>>> methods did not substantially affect the strength of photoconversion. We
>>> also suggest avoiding the use of mounting medium with high glycerol
>>> concentrations since glycerol showed the strongest impact on
>>> photoconversion. This photoconversion effect cannot be avoided even when
>>> using narrow bandpass filter sets.
>>>
>>>
>>
>>
>> Regards,
>>
>> Adrian Smith
>> Centenary Institute, Sydney, Australia
>>
>>
>>
>

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