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S W Hell

Publications and source records attributed to S W Hell.

At least 19 recordsLinked to original sources
PubMed2002

Comparison of the axial resolution of practical Nipkow-disk confocal fluorescence microscopy with that of multifocal multiphoton microscopy: theory and experiment.

We compare the axial sectioning capability of multifocal confocal and multifocal multiphoton microscopy in theory and in experiment, with particular emphasis on the background arising from the cross-talk between adjacent imaging channels. We demonstrate that a time-multiplexed non-linear excitation microscope exhibits significantly less background and therefore a superior axial resolution as compared to a multifocal single-photon confocal system. The background becomes irrelevant for thin (< 15 microm) and sparse fluorescent samples, in which case the confocal parallelized system exhibits similar or slightly better sectioning behaviour due to its shorter excitation wavelength. Theoretical and experimental axial responses of practically implemented microscopes are given.

Fluorescence Polarization↗
PubMed2002

A new high-aperture glycerol immersion objective lens and its application to 3D-fluorescence microscopy.

High-resolution light microscopy of glycerol-mounted biological specimens is performed almost exclusively with oil immersion lenses. The reason is that the index of refraction of the oil and the cover slip of approximately 1.51 is close to that of approximately 1.45 of the glycerol mountant, so that refractive index mismatch-induced spherical aberrations are tolerable to some extent. Here we report the application of novel cover glass-corrected glycerol immersion lenses of high numerical aperture (NA) and the avoidance of these aberrations. The new lenses feature a semi-aperture angle of 68.5 degrees, which is slightly larger than that of the diffraction-limited 1.4 NA oil immersion lenses. The glycerol lenses are corrected for a quartz cover glass of 220 microm thickness and for a 80% glycerol-water immersion solution. Featuring an aberration correction collar, the lens can adapt to glycerol concentrations ranging between 72% and 88%, to slight variations of the temperature, and to the cover glass thickness. As the refractive index mismatch-induced aberrations are particularly important to quantitative confocal fluorescence microscopy, we investigated the axial sectioning ability and the axial chromatic aberrations in such a microscope as well as the image brightness as a function of the penetration depth. Whereas there is a significant decrease in image brightness associated with oil immersion, this decrease is absent with the glycerol immersion system. In addition, we show directly the compression of the optic axis in the case of oil immersion and its absence in the glycerol system. The unique advantages of these new lenses in high-resolution microscopy with two coherently used opposing lenses, such as 4 Pi-microscopy, are discussed.

Animals↗
PubMed2001

Breaking Abbe's diffraction resolution limit in fluorescence microscopy with stimulated emission depletion beams of various shapes.

We report on the generation of various hole-centered beams in the focal region of a lens and investigate their effectiveness to break the diffraction barrier in fluorescence microscopy by stimulated emission. Patterning of the phase of the stimulating beam across the entrance pupil of the objective lens produces point-spread-functions with twofold, fourfold, and circular symmetry, which narrow down the focal spot to 65-100 nm. Comparison with high-resolution confocal images exhibits a resolution much beyond the diffraction barrier. Particles that are only 65-nm apart are resolved with focused light.

Journal Article↗
PubMed2001

4Pi-confocal microscopy of live cells.

By coherently adding the spherical wavefronts of two opposing lenses, two-photon excitation 4Pi-confocal fluorescence microscopy has achieved three-dimensional imaging with an axial resolution 3-7 times better than confocal microscopy. So far this improvement was possible only in glycerol-mounted, fixed cells. Here we report 4Pi-confocal microscopy of watery objects and its application to the imaging of live cells. Water immersion of 4Pi-confocal microscopy of membrane stained live Escherichia coli bacteria attains a 4.3-fold better axial resolution as compared to the best water immersion confocal microscope. The resolution enhancement results into a vastly improved three-dimensional representation of the bacteria. The first images of live biological samples with an all-directional resolution in the 190-280 nm range are presented here, thus establishing a new resolution benchmark in live-cell microscopy.

Escherichia coli↗
PubMed2001

Dual-color 4Pi-confocal microscopy with 3D-resolution in the 100 nm range.

We report the development of simultaneous two-color channel recording in 4Pi-confocal microscopy. A marked increase of spatial resolution over confocal microscopy becomes manifested in 4Pi-confocal three-dimensional (3D) data stacks of dual-labeled objects. The fundamentally improved resolution is verified both with densely labeled fluorescence beads as well as with membrane labeled fixed Escherichia coli. The synergistic combination of dual-color 4Pi-confocal recording with image restoration results in dual-color imaging with a 3D resolution in the 100 nm range.

Cell Membrane↗
PubMed2001

KDEL-cargo regulates interactions between proteins involved in COPI vesicle traffic: measurements in living cells using FRET.

How the occupied KDEL receptor ERD2 is sorted into COPI vesicles for Golgi-to-ER transport is largely unknown. Here, interactions between proteins of the COPI transport machinery occurring during a "wave" of transport of a KDEL ligand were studied in living cells. FRET between CFP and YFP fusion proteins was measured by multifocal multiphoton microscopy and bulk-cell spectrofluorimetry. Ligand binding induces oligomerization of ERD2 and recruitment of ARFGAP to the Golgi, where the (ERD2)n/ARFGAP complex interacts with membrane-bound ARF1. During KDEL ligand transport, interactions of ERD2 with beta-COP and p23 decrease and the proteins segregate. Both p24a and p23 interact with ARF1, but only p24 interacts with ARFGAP. These findings suggest a model for how cargo-induced oligomerization of ERD2 regulates its sorting into COPI-coated buds.

ADP-Ribosylation Factor 1↗
PubMed2001

Space-multiplexed multifocal nonlinear microscopy.

Standard forms of nonlinear microscopy rely on single beam scanning, but the usually weaker signal and the need to image in real-time call for parallelization of the image formation. Since the nonlinear susceptibilities necessitate a comparatively large illumination power, with current laser systems the brightness or field of view of any parallelized nonlinear microscope is limited by the brightness of the laser. For example, by producing an array of high aperture foci, multifocal multiphoton microscopy (MMM) provides real-time, light-efficient three-dimensional fluorescence imaging at high-resolution. The available power limits the degree of parallelization and hence codetermines the field of view. As the utilization of all the laser power is imperative, the focal intensity can be adjusted only through altering the number of foci. This compromises to some extent the flexibility to adjust the focal intensity to benign and effective levels. Here we introduce space-multiplexing (SMX) as a novel option in parallelized nonlinear microscopy, which enables an improved exploitation of the total laser power and facilitates changing the intensity levels in selected regions, without attenuating the total laser power. The basic idea of SMX is to overlap arrays of slightly offset coherent focal fields whose interference modulates the intensity across the sample. For a given degree of parallelization and power, SMX increases the two- and three-photon excited signal of parallelized nonlinear microscopy by a factor of up to 1.5 and 2.5, respectively. To some extent, sensitive regions may be spared out, whereas in regions with weaker nonlinear susceptibilities the intensity is increased. SMX is relevant to all modes of nonlinear microscopy, including parallelized second- and third-harmonic imaging, coherent anti-Stokes Raman scattering, and wide field multiphoton excitation.

Journal Article↗
PubMed2001

Z-polarized confocal microscopy.

In light microscopy the transverse nature of the electromagnetic field precludes a strongly focused longitudinal field component, thus confining polarization spectroscopy and imaging to two dimensions (x,y). Here we describe a simple confocal microscopy arrangement that optimizes for signal from molecules with transition dipoles oriented parallel to the optic axis. In the proposed arrangement, we not only generate a predominant longitudinally (z) polarized focal field, but also engineer the detection scheme in such a way that in a bulk of randomly oriented molecules, the microscope's effective point-spread function is dominated by the contribution of those molecules that are oriented along the optic axis. Our arrangement not only implicitly allows for the determination of the orientation of transition dipoles of single molecules in three dimensions, but also highlights the contribution of z-oriented molecules in three-dimensional imaging.

Microscopy, Confocal↗
PubMed2001

Z-polarized confocal microscopy.

In light microscopy the transverse nature of the electromagnetic field precludes a strongly focused longitudinal field component, thus confining polarization spectroscopy and imaging to two dimensions (x,y). Here we describe a simple confocal microscopy arrangement that optimizes for signal from molecules with transition dipoles oriented parallel to the optic axis. In the proposed arrangement, we not only generate a predominant longitudinally (z) polarized focal field, but also engineer the detection scheme in such a way that in a bulk of randomly oriented molecules, the microscope's effective point-spread function is dominated by the contribution of those molecules that are oriented along the optic axis. Our arrangement not only implicitly allows for the determination of the orientation of transition dipoles of single molecules in three dimensions, but also highlights the contribution of z-oriented molecules in three-dimensional imaging.

Microscopy, Confocal↗
PubMed2001

Coherent use of opposing lenses for axial resolution increase in fluorescence microscopy. I. Comparative study of concepts.

We study the use of coherent counterpropagating interfering waves to increase threefold to sevenfold the optical bandwidth and the resolution of fluorescence microscopy along the optic axis. Systematic comparison of the point-spread function and the optical transfer function (OTF) for the standing-wave microscope (SWM), the incoherent illumination interference image interference microscope (I5M), and the 4Pi confocal microscope reveals essential differences among their resolution capabilities. It is shown that the OTF's of these microscopes differ strongly in contiguity and amplitude within the enlarged range of transferred frequencies, and therefore they also differ in their ability to provide data from which interference artifacts can be removed. We demonstrate that for practical aperture angles the production of an interference pattern is insufficient for improving the axial resolution by the expected factor of 3-7. Conditions of the OTF for unambiguous improvement of axial resolution of arbitrary objects are fulfilled not at all in the SWM, partially in the I5M, and fully in the two-photon 4Pi confocal microscope.

Journal Article↗
PubMed2001

Coherent use of opposing lenses for axial resolution increase. II. Power and limitation of nonlinear image restoration.

We analyze the ability of nonlinear image restoration to remove interference artifacts in microscopes that enlarge the axial optical bandwidth through coherent counterpropagating waves. We calculate the images of an elaborate test object as produced by confocal, standing-wave, incoherent illumination interference image interference, and 4Pi confocal microscopes, and we subsequently investigate the extent to which the initial object can be restored by the information allowed by their optical transfer function. We find that nonlinear restoration is successful only if the transfer function is sufficiently contiguous and has amplitudes well above the noise level, as is mostly the case in a two-photon excitation 4Pi confocal microscope.

Journal Article↗
PubMed2000

EFGP and DsRed expressing cultures of Escherichia coli imaged by confocal, two-photon and fluorescence lifetime microscopy.

The green fluorescent protein (GFP) has become an invaluable marker for monitoring protein localization and gene expression in vivo. Recently a new red fluorescent protein (drFP583 or DsRed), isolated from tropical corals, has been described [Matz, M.V. et al. (1999) Nature Biotech. 17, 969-973]. With emission maxima at 509 and 583 nm respectively, EGFP and DsRed are suited for almost crossover free dual color labeling upon simultaneous excitation. We imaged mixed populations of Escherichia coli expressing either EGFP or DsRed by one-photon confocal and by two-photon microscopy. Both excitation modes proved to be suitable for imaging cells expressing either of the fluorescent proteins. DsRed had an extended maturation time and E. coli expressing this fluorescent protein were significantly smaller than those expressing EGFP. In aging bacterial cultures DsRed appeared to aggregate within the cells, accompanied by a strong reduction in its fluorescence lifetime as determined by fluorescence lifetime imaging microscopy.

Cytoplasm↗
PubMed2000

Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission.

The diffraction barrier responsible for a finite focal spot size and limited resolution in far-field fluorescence microscopy has been fundamentally broken. This is accomplished by quenching excited organic molecules at the rim of the focal spot through stimulated emission. Along the optic axis, the spot size was reduced by up to 6 times beyond the diffraction barrier. The simultaneous 2-fold improvement in the radial direction rendered a nearly spherical fluorescence spot with a diameter of 90-110 nm. The spot volume of down to 0.67 attoliters is 18 times smaller than that of confocal microscopy, thus making our results also relevant to three-dimensional photochemistry and single molecule spectroscopy. Images of live cells reveal greater details.

Escherichia coli↗
PubMed2000

Live cell imaging by multifocal multiphoton microscopy.

Multifocal multiphoton microscopy (MMM) permits parallel multiphoton excitation by scanning an array of high numerical aperture foci across a plane in the sample. MMM is particularly suitable for live cell investigations since it combines advantages of standard multiphoton microscopy such as optical sectioning and suppression of out-of-focus phototoxicity with high recording speeds. Here we describe several applications of MMM to live cell imaging using the neuroendocrine cell line PC12 and bovine chromaffin cells. Stainings were performed with the acidophilic dye acridine orange and the lipophilic dyes FM1-43 and Fast DiA as well as by transfection of the cells with GFP. In both bovine chromaffin and PC12 cells structural elements of nuclear chromatin and the 3-D distribution of acidic organelles inside the cells were visualized. In PC12 cells differentiated by nerve growth factor examples of neurites were monitored. Stainings of membranes were used to reconstruct the morphology of cells and neurites in three dimensions by volume-rendering and by isosurface plots. 3-D reconstructions were composed from stacks of about 50 images each with a diameter of 30-100 microm that were acquired within a few seconds. We conclude that MMM proves to be a technically simple and very effective method for fast 3-D live cell imaging at high resolution.

Acridine Orange↗
PubMed1999

Ca2+ fluorescence imaging with pico- and femtosecond two-photon excitation: signal and photodamage.

The signal and limitations of calcium florescence imaging using nonresonant multiphoton absorption of near-infrared femto- and picosecond laser pulses were examined. The fluorescence changes of various Ca(2+)-indicators induced by transient increases of the intradendritic calcium concentration were evaluated by evoking physiological activity in neocortical neurons in rat brain slices. Photodamage was noticeable as irreversible changes in the parameters describing the calcium fluorescence transients. At higher two-photon excitation rates, a great variety of irregular functional and structural alterations occurred. Thus, signal and observation time were limited by phototoxic effects. At lower excitation rates, photodamage accumulated linearly with exposure time. Femtosecond and picosecond laser pulses were directly compared with respect to this cumulative photodamage. The variation of the pulse length at a constant two-photon excitation rate indicated that a two-photon excitation mechanism is mainly responsible for the cumulative photodamage within the investigated window of 75 fs to 3.2 ps. As a direct consequence, at low excitation rates, the same image quality is achieved irrespective of whether two-photon Ca(2+)-imaging is carried out with femto- or picosecond laser pulses.

Absorption↗
PubMed1998

4Pi-confocal microscopy provides three-dimensional images of the microtubule network with 100- to 150-nm resolution.

We show the applicability of 4Pi-confocal microscopy to three-dimensional imaging of the microtubule network in a fixed mouse fibroblast cell. Comparison with two-photon confocal resolution reveals a fourfold better axial resolution in the 4Pi-confocal case. By combining 4Pi-confocal microscopy with Richardson-Lucy image restoration a further resolution increase is achieved. Featuring a three-dimensional resolution in the range 100-150 nm, the 4Pi-confocal (restored) images are intrinsically more detailed than their confocal counterparts. Our images constitute what to our knowledge are the best-resolved three-dimensional images of entangled cellular microtubules obtained with light to date.

3T3 Cells↗
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↗