Posted by
George McNamara on
URL: http://confocal-microscopy-list.275.s1.nabble.com/what-fluorescent-proteins-are-working-best-for-precision-localization-microscopy-nanoscopy-in-your-a-tp7580157p7580187.html
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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/I believe BHT is a food additive.
Sincerely,
George
On 4/13/2013 5:25 AM, Adrian Smith wrote:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> *****
>
> On 12/04/2013, at 10:59 PM, George McNamara<
[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
>
>