Bioimaging最新文献

Two-photon laser scanning microscopy is discussed from a fluorophore and specimen point of view. Issues to be considered in the selection of the fluorophore and in specimen preparation for multiphoton microscopy are described. The transverse resolution is estimated, based on a photobleaching method. We also show that the performance of a multiphoton laser scanning microscope is significantly enhanced by the use of a confocal aperture.

Image formation in three- and two-photon confocal fluorescence scanning microscopy is analysed under the paraxial approximation. The analysis incorporates the effect of a finite-sized detector and the effect of the fluorescence wavelength. The resolution in the transverse and axial directions is examined for edge and layer objects, respectively. For a given excitation energy gap, three-photon confocal and non-confocal fluorescence microscopes feature a resolution close to that of their two-photon counterparts. However, the resolution of the three-photon microscopes is improved by up to 40-50% when the same excitation wavelength is used. This may prove advantageous in practical three-photon fluorescence microscopy.

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.

Fluorescence correlation spectroscopy (FCS) can be used to investigate the photobleaching properties of fluorophores in solution. The advantage with this method is that in addition to the photobleaching rate the formation and decay rates of the triplet state can be measured. In this way, it is possible to calculate the photodestruction quantum yield and relate the photostability of a fluorescent compound in a certain environment to the photodynamical behaviour of the singlet-triplet transitions. This is likely to contribute to a better understanding of the mechanisms of photobleaching given the central importance of dye triplet states in photobleaching processes. The approach was applied to the measurement and characterization of the photobleaching of Rh6G in aqueous solution and FITC in 1 mM sodium carbonate buffer (pH 9). The photobleaching yields measured are discussed in view of the simultaneous triplet properties at different excitation intensities, oxygen concentrations as well as in the presence or absence of quencher molecules. This study suggests that FCS is likely to provide a valuable tool for the elucidation of the mechanisms of photobleaching, which are far from understood in all their details.

Nonlinear excitation of fluorophores through molecular absorption of two or three near-infra-red photons from the tightly focused femtosecond pulses of a mode-locked laser offers the cellular biologist an unprecedented panoply of biomolecular indicators for microscopic imaging and cellular analysis. Measurements of the two-photon excitation spectra of 25 ultra-violet and visible absorbing fluorophores from 690 to 1050 nm reveal useful cross sections for near infra-red excitation, providing an artist's palette of emission markers, chemical indicators, and native cellular absorbers for living biological preparations. Measurements of three-photon fluorophore excitation spectra now suggest relatively benign wavelengths to excite deeper UV fluorophores. The inherent optical sectioning capabilities of focused nonlinear excitation provides three-dimensional resolution for imaging and avoids out-of-focus background. Measured nonlinear excitation spectra are described and implications to nonlinear microscopy for biological imaging are defined.

Image formation in three-photon fluorescence microscopy is analysed. The point spread function, three-dimensional optical transfer function and axial resolution are considered. The results are generalized to multiphoton fluorescence. Three-photon fluorescence is particularly amenable to resolution improvement by use of pupil filters.