Multiphoton excitation of the DNA stains DAPI and Hoechst

I. Gryczynski, H. Malak, J. Lakowicz
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引用次数: 38

Abstract

,6-diamidino-2-phenylindole hydrochloride (DAPI) andHoechst 33342 were found to display two- or three-photon excitation from 810 to 910nm. We examined the effect of excitation wavelength on the mode of excitation forDAPI and Hoechst 33342 in the solvent isobutanol and when bound to double helicalDNA. For DAPI and Hoechst 33342 in isobutanol the mode of excitation changed fromtwo- to three-photon excitation over this wavelength range, with apparent transitionwavelengths of 855 and 880 nm, respectively. However, when bound to DNA, thetransition wavelength from two- to three-photon excitation increased for both probes.In the case of DAPI–DNA, the apparent transition wavelength increased to 868 nm,and three-photon excitation occurred above 900 nm. For Hoechst 33342–DNA themode of excitation was a mixture of two- and three-photon excitation to the longestexcitation wavelength of 910 nm, and we were unable to observe pure three-photonexcitation for Hoechst 33342–DNA. In the transition region the anisotropy of DAPIwas dependent on laser power, illustrating that the mode of excitation and transitionwavelengths will depend on the precise experimental conditions. Higher spatialresolution was observed for three-photon excitation of DAPI than for two-photonexcitation of Hoechst 33342. These results suggest that these probes can be used foreither two- or three-photon imaging of DNA or chromosomes.Keywords: fluorescence, spectroscopy, imaging, time-resolved fluorescence,two-photon excitation, three-photon excitation, DAPI, Hoechst 33342, two-photonmicroscopy
DAPI和Hoechst DNA染色的多光子激发
发现6-二氨基-2-苯基吲哚盐酸(DAPI)和hoechst 33342在810 ~ 910nm范围内显示双光子或三光子激发。我们考察了激发波长对forDAPI和Hoechst 33342在异丁醇溶剂中和与双螺旋dna结合时的激发模式的影响。对于异丁醇中的DAPI和Hoechst 33342,在该波长范围内的激发模式由双光子激发变为三光子激发,视过渡波长分别为855 nm和880 nm。然而,当与DNA结合时,两种探针从二光子激发到三光子激发的过渡波长增加。DAPI-DNA的表观跃迁波长增加到868 nm, 900 nm以上发生三光子激发。对于Hoechst 33342-DNA,激发模式为双光子和三光子混合激发,最长激发波长为910 nm,我们无法观察到纯粹的三光子激发。在过渡区,dapi的各向异性取决于激光功率,说明激发模式和过渡波长取决于精确的实验条件。DAPI的三光子激发比Hoechst 33342的双光子激发具有更高的空间分辨率。这些结果表明,这些探针可以用于DNA或染色体的双光子或三光子成像。关键词:荧光,光谱学,成像,时间分辨荧光,双光子激发,三光子激发,DAPI, Hoechst 33342,双光子显微镜
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