UV-induced spectral shift and protonation of DNA fluorescent dye Hoechst 33258.

DNA-bound Hoechst 33258 is readily excited with UV light and emits blue fluorescence, however, upon exposure to UV, the dye undergoes photobleaching as well as photoconversion to a blue-excited green-emitting form. We demonstrate that the UV-generated green-emitting form of Hoechst 33258 exhibits spectral properties very similar to the form of the dye that can be obtained by subjecting it to an acidic environment (pH 0.5-3.0). We also demonstrate that exposure of Hoechst 33258 to UV light (or hydrogen peroxide) leads to generation of the protonated (1+, 2+, 3+ and possibly the 4+) forms of the dye. Photoconversion of Hoechst 33258 has recently been exploited in single molecule localisation microscopy, thus understanding photophysics of this process can facilitate further development of high resolution optical imaging.

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

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.

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.

pH and temperature dependent relaxation dynamics of Hoechst-33258: a time resolved fluorescence study.

The photophysical behavior of Hoechst 33258 (H33258) in aqueous solution has been studied by steady-state and time-resolved fluorescence measurements. The intriguing intramolecular geometrical orientations of the dye bring out major modulation on its photophysical behavior, especially in the fluorescence emission characteristics with pH. It has been seen that a change in the solution pH from 7 to 4.5 enhances the emission yield by ~20 fold and this change is ~80-fold on changing the pH from 1.5 to 4.5. While a fast flipping motion among the two benzimidazole rings is considered to be one of the most probable mechanisms for the fast fluorescence decay, a more planar structure of the dicationic form at pH 4.5 having a double bond character between the two benzimidazolium groups is suggested to be the most likely fluorescent species. A similar planar structure is in fact considered to be the fluorescent emitting species of H33258 on minor groove binding to DNA. On the basis of temperature dependent fluorescence decay dynamics explored for the dye in solutions at pH 7 and 4.5, it is understood that a nearly isoenergetic double-well excited state potential is possibly involved in the excited state relaxation dynamics of the dye at pH 7. On increasing the temperature, the conversion to the planar structure is facilitated from the non-planar LE state, enhancing the emission probability of the dye.

Donor fluorescence decay analysis for energy transfer in double-helical DNA with various acceptor concentrations.

We studied fluorescence resonance energy transfer between donors and acceptors bound to double-helical DNA. The donor Hoechst 33258 binds to the minor groove of DNA and the acceptor propidium iodide (PI) is an intercalator. The time-resolved donor decays were measured in the frequency domain. The donor decays were consistent with a random 1-dimensional distribution of acceptors. The decays were analyzed in terms of three 1-dimensional models: a random continuous acceptor distribution; acceptors placed on discrete lattice sites; and a cylindrical model with the acceptor in the center, and the donors on a cylinder surface. The data were well described by all three models. Interpretation in terms of continuous distribution of acceptors revealed a minimum donor to acceptor distance of 13 A, which is 3 bp from the center of Hoechst 33252. These results suggest that PI is excluded from the 4 bp covered by Hoechst 33252 when it is bound to the minor groove of DNA.

Photo-bleaching and photon saturation in flow cytometry.

In flow cytometry, small particles travel at a high speed through a bright light spot. The high light intensity at the point of measurement causes measurable photon saturation. This observation indicates that the rate at which individual dye molecules emit photons is close to the maximum emission rate. Despite the short exposure time, individual molecules may go through a few hundred excitation cycles while they are in the light beam. The absorbed light dose causes significant dye destruction. This article presents experimental procedures to determine the extent of photon saturation and photo-bleaching of dyes bound to cell nuclei in a flow cytometer. Measurements of Hoechst and propidium iodide bound to chromatin show that the amount of dye bleached per emitted photon is the same at low and high illumination intensities. This finding indicates that photon emission and dye destruction are both the result of the absorption of single excitation photons. The experimental observations allow rough estimates of the lifetime of the excited state and the lifetime of the molecule. The lifetime of the Hoechst 33258 bound to DNA is estimated to be 100 excitation-relaxation cycles. The average propidium iodide molecule lasts approximately 200 excitation-relaxation cycles. The theoretical considerations show that the optimal illumination conditions are different for bleaching and nonbleaching dyes. An optical arrangement for high precision measurements of bleaching dyes is presented.

[Photochemical transformation of Hoescht 33258 dye molecules complexed with cell nucleus DNA under the effect of UV-irradiation]. / Fotokhimicheskoe prevrashchenie pod deistviem UF-izlucheniia molekul krasitelia Khekhst 33258 v komplekse s DNK iader kletok.

It is found that with time a decrease of fluorescence intensity of the basic band at 460 nm and appearance of a new band of fluorescence of DNA-specific dye Hoechst 33258 in complex with the cell nucleus DNA under the action of UV emission are observed. It is shown that phototransformation is related to the withdrawal of the nitrogen atom proton of piperazine ring in an excited state of the complex of the dye Hoechst 33258 with the cell nucleus DNA.

UV dose-dependent increase in the Hoechst fluorescence intensity of both normal and BrdU-DNA.

If the DNA nucleoside thymidine is replaced by bromodeoxyuridine, the fluorescence of the nuclei of Hoechst-stained cells is quenched. The decrease of fluorescence intensity determined by flow cytometry and fluorometry is neutralized independent of the degree of BrdU substitution by an UV-exposure with a dose of 5-10 kJ/m2 to the unfiltered spectrum of a 100 W mercury high-pressure lamp. This dose is equivalent to that obtained in fluorescence microscopy after exposure for about 1 s. We suppose that this approximate matching of the intensities both of normal and BrdU in the DNA resulting in no further quenching. However, the fluorescence intensity of normal Hoechst-stained DNA also is increased by a previous exposure to UV light. We explain the time pattern of the Hoechst fluorescence in the course of an exposure with constant dose rate, by the superimposition of the well-known bleaching by an additional increase of the fluorescence intensity. Our results suggest that the UV-exposure of Hoechst dye creates a brightly fluorescing photoproduct which differs spectroscopically from the original dye. This product is stable in the dark and seems to fluorochrome DNA only if it is formed when the Hoechst dye is bound to DNA, thus increasing the nuclear fluorescence. Phosphorescence was not found.