Biomedical subjects

S W Hell

Publications and source records attributed to S W Hell.

23 records · Page 2Linked to original sources

PubMed1998

4Pi-confocal imaging in fixed biological specimens.

By combining the wavefronts produced by two high-aperture lenses, two-photon 4Pi-confocal microscopy allows three-dimensional imaging of transparent biological specimens with axial resolution in the 100-140-nm range. We reveal the imaging properties of a two-photon 4Pi-confocal microscope as applied to a fixed cell. We demonstrate that a fast, linear point deconvolution suffices to achieve axially superresolved 3D images in the cytoskeleton. Furthermore, we describe stringent algorithms for alignment and control of the two lenses. We also show how to compensate for the effects of a potential refractive index mismatch of the mounting medium with respect to the immersion system.

Actins↗

PubMed1998

Continuous wave excitation two-photon fluorescence microscopy exemplified with the 647-nm ArKr laser line.

We report on efficient two-photon fluorescence imaging in beam scanning microscopy by exciting UV dyes at the 647-nm line of a continuous wave ArKr mixed gas laser. For a numerical aperture of 1.4 (oil), we used an illumination power of up to 210 mW at the sample. High-resolution images were obtained for DAPI-labelled cell nuclei within 4-60 s. Our method is a simple two-photon alternative to UV confocal imaging with the potential of becoming a very useful feature of laser scanning microscopy.

Animals↗

PubMed1997

Light quenching of pyridine2 fluorescence with time-delayed pulses.

We describe the effects of time-delayed long-wavelength pulses on the intensity and anisotropy decays of pyridine2. The sample was exposed to a continuous train of 360 nm excitation pulses and time-delayed 720 nm pulses. The long-wavelength pulses, which overlapped the emission spectrum of pyridine2, resulted in a spatially localized decrease in intensity at the point of beam overlap. The time-delayed quenching pulses caused a stepwise decrease in the intensity and anisotropy decays, as seen by oscillations in the frequency-domain data. The time-resolved anisotropy was shown to decrease below zero (-0.2) following the vertically polarized quenching pulse. The extent of light quenching depended on the time delay between the excitation and quenching pulses, and can be used to measure the decay time. Light quenching and/or multipulse methods may provide a new class of experiments for fluorescence spectroscopy and imaging.

Fluorescence Polarization↗

PubMed1997

Time-resolved fluorescence spectroscopy and imaging of DNA labeled with DAPI and Hoechst 33342 using three-photon excitation.

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.

Benzimidazoles↗

PubMed1997

Far-field fluorescence microscopy with three-dimensional resolution in the 100-nm range.

We report three-dimensional (3D) microscopy with nearly isotropic resolution in the lambda/5-lambda/10 range. Our approach combines 4PI-confocal two-photon fluorescence microscopy with image restoration. The 3D resolution is demonstrated with densely clustered beads as well as with F-actin fibers in mouse fibroblast cells. A comparison with unrestored two-photon confocal images reveals a total reduction of the uncertainty volume up to a factor of 15.

Animals↗