Time-Resolved Fluorescence Spectroscopy and Imaging of DNA Labeled with DAPI and Hoechst 33342 Using Three-Photon Excitation

Abstract

We examined the fluorescence spectral properties of the DNA stains DAPI (4',6-diamidino-2-phenylindole, hydrochloride) and Hoechst 33342 (bis-benzimide, or 2,5'-bi'1H-benzimidazole2'-(4-ethoxyphenyl)-5-(4-methyl-1-piperazi nyl)) with two-photon (2h nu) and three-photon (3h nu) excitation using femtosecond pulses from a Ti:sapphire laser from 830 to 885 nm. The mode of excitation of DAPI bound to DNA changed from two-photon at 830 nm to three-photon at 885 nm. In contrast, Hoechst 33342 displayed only two-photon excitation from 830 to 885 nm. DAPI-DNA displayed the same emission spectra and decay times for 2h nu and 3h nu excitation. Hoechst 33342-DNA displayed the same intensity decay for excitation at 830 and 885 nm. Both probes displayed higher anisotropies for multiphoton excitation as compared to one-photon excitation with ultraviolet wavelengths, and DAPI-DNA displays a higher anisotropy for 3h nu at 885 nm than for 2h nu at 830 nm. We used 970-nm excitation of DAPI-stained chromosomes to obtain the first three-dimensional images with three-photon excitation. Three-photon excitation of DAPI-stained chromosomes at 970 nm was demonstrated by the power dependence in the fluorescence microscope.

A molecular theory for two-photon and three-photon fluorescence polarization

“…These limiting case results are in precise agreement with the values that arise specifically when all transition moments are considered parallel, a special case previously reported by Lakowicz et al 62 The correlation serves to verify a limiting case of the present, more general results-but it is also notable that the conditions under which such behavior arises are not only associated with parallel transition moments. The same observations will result, for example if all of the molecular transition tensor elements have similar values.…”

Tracing and ablation of single cells in the mammalian blastocyst using fluorescent DNA staining and multi-photon laser microscopy

“…below and above the focused layer) of the scanned area, whereas infrared excitation makes use of the MP-eVect which is based on the conXuence of the laser energy in the focused plane only, leaving the parts above and below the focus of interest unaVected (cf. Lakowicz et al 1997). This may partly explain the substantial alterations induced by the acquisition of multiple z-layers in the MP protocol which causes an axially extended absorption of energy in a more "SPlike" manner.…”

“…DNA dyes suitable for the multi-photon eVect are excited typically at the ultraviolet (UV) to blue wavelength range (which "disqualiWes" excellent dyes that need higher wavelength excitation such as TO-PRO-3; Bink et al 2001). So far, among the minor groove binders, Hoechst33342 (H342) and DAPI have been most thoroughly studied for their behaviour under IR multiphoton (MP) excitation (Lakowicz et al 1997). Staining protocol complexity, cellular retention time, cytoplasmic background and toxicity appear to vary to some extent amongst these two dyes and another, possibly more water soluble, variety of the Hoechst dye series (Hoechst33528, cf.…”

Advances in Fluorescence Spectroscopy: Multi-Photon Excitation, Engineered Proteins, Modulation Sensing and Microsecond Rhenium Metal-Ligand Complexes

“…Using the femtosecond (fs) pulses from a Ti:sapphire laser, we have observed simultaneous three-photon excitation of a variety of biochemical fluorophores. This includes probes [32], tryptophan [33,34], stained DNA [35], and DPH-labeled membranes [36]. Localization of the excitation for a three-photon process can be improved as compared to two-photon excitation [37].…”