Multiphoton excitation of the DNA stains DAPI and Hoechst

Abstract

The DNA stains 40′ ,6‐diamidino‐2‐phenylindole hydrochloride (DAPI) and Hoechst 33342 were found to display two‐ or three‐photon excitation from 810 to 910 nm. We examined the effect of excitation wavelength on the mode of excitation for DAPI and Hoechst 33342 in the solvent isobutanol and when bound to double helical DNA. For DAPI and Hoechst 33342 in isobutanol the mode of excitation changed from two‐ to three‐photon excitation over this wavelength range, with apparent transition wavelengths of 855 and 880 nm, respectively. However, when bound to DNA, the transition 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 the mode of excitation was a mixture of two‐ and three‐photon excitation to the longest excitation wavelength of 910 nm, and we were unable to observe pure three‐photon excitation for Hoechst 33342‐DNA. In the transition region the anisotropy of DAPI was dependent on laser power, illustrating that the mode of excitation and transition wavelengths will depend on the precise experimental conditions. Higher spatial resolution was observed for three‐photon excitation of DAPI than for two‐photon excitation of Hoechst 33342. These results suggest that these probes can be used for either two‐ or three‐photon imaging of DNA or chromosomes.

Quantitative comparison of 3D third harmonic generation and fluorescence microscopy images

“…During imaging, the brain slice was mechanically stabilized by a "harp" ( Figure 1B) and perfused by the carbogenated ACSF at 37 8C. Hoechst-33342 dye (H-342) was used to label nuclear DNA of mouse brain cells [11,14,53]. H-342 is a cell-permeable nuclear counterstain for live and fixed cells and tissue sections.…”

“…Like other multi-photon techniques [10][11][12][13][14][15][16][17][18], THG not only enables the recording of label-free images of unfixed 3D volumes of tissue [1][2][3][4][5][6][7][8][9][10] free from spatial distortion artifacts inherent to histopathology, but also potentially allows feedback on the nature of the tissue, i. e. whether it is healthy or tumorous, to the surgeon during surgery, as the relative speed of the imaging modalities approaches 'real' time, and no preparation steps of the tissue are required [5,6,[19][20][21][22][23]. THG was shown recently to yield label-free images of ex-vivo human tumor tissue of histopathological quality, in real-time [6].…”

Imaging properties of high aperture multiphoton fluorescence scanning optical microscopes

“…In general, fluorescent emission is partially polarised and the number of parameters that determine the state of the polarisation is extremely large (Soleillet, 1929). The relationship between illumination and emission polarisation direction also depends on a number of parameters such as the fluorescent molecule's mobility (Axelrod, 1979 and its individual characteristics (Gryczynski et al, 1996a). Accounting for the polarisation states in 2-p and 3-p fluorescence is a more involved process (Friedrich, 1981;Callis, 1993, Gryczynski et al, 1996b) but the conclusion is still that the fluorescent emission is partially polarised.…”

“…Gryczynski et al (1996a) obtained expressions for the angular displacement of the emission transition moment from the direction of the polarised excitation for 1-p, 2-p and 3-p fluorescence. In Case C it is assumed that there is zero angular displacement between excitation and emission, which is never actually the case but, rather, a limiting model which is a good approximation to a highly polarised emission.…”

“…The fluorescent molecule will be treated as a harmonically oscillating dipole whose probability of excitation (strength) is proportional to the intensity (modulus square of the electric field) of the illumination to the power of the order of the multiphoton process (Gryczynski et al, 1996a). In general, the fluorescent emission is partially polarised (Perrin, 1929;Soleillet, 1929) and in this paper the two limiting cases of polarised and unpolarised emission are considered.…”