The hazards of DAPI photoconversion: effects of dye, mounting media and fixative, and how to minimize the problem.
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.
Blood Transfusion Centre of Slovenia, Šlajmerjeva 6, 1000 Ljubljana, Slovenia.
Mojca
M
Jež
Tuba
T
Bas
Matija
M
Veber
Andrej
A
Košir
Tanja
T
Dominko
Raymond
R
Page
Primož
P
Rožman
D001562:Benzimidazoles / Q000737:chemistry / Q000528:radiation effects; D006690:Bisbenzimidazole / Q000737:chemistry / Q000528:radiation effects; D018236:Carcinoma, Embryonal / Q000378:metabolism; D045744:Cell Line, Tumor; D005189:False Positive Reactions; D005404:Fixatives / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry / Q000528:radiation effects*; D005990:Glycerol / Q000737:chemistry; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D007211:Indoles / Q000737:chemistry / Q000528:radiation effects*; D008297:Male; D008856:Microscopy, Fluorescence; D055668:Photochemical Processes; D015203:Reproducibility of Results; D013736:Testicular Neoplasms / Q000473:pathology; D014466:Ultraviolet Rays
D001562:Benzimidazoles; D005404:Fixatives; D005456:Fluorescent Dyes; D007211:Indoles; C007293:DAPI; D006690:Bisbenzimidazole; C017807:bisbenzimide ethoxide trihydrochloride; D005990:Glycerol