STED nanoscopy reveals molecular details of cholesterol- and cytoskeleton-modulated lipid interactions in living cells.

Details about molecular membrane dynamics in living cells, such as lipid-protein interactions, are often hidden from the observer because of the limited spatial resolution of conventional far-field optical microscopy. The superior spatial resolution of stimulated emission depletion (STED) nanoscopy can provide new insights into this process. The application of fluorescence correlation spectroscopy (FCS) in focal spots continuously tuned down to 30 nm in diameter distinguishes between free and anomalous molecular diffusion due to, for example, transient binding of lipids to other membrane constituents, such as lipids and proteins. We compared STED-FCS data recorded on various fluorescent lipid analogs in the plasma membrane of living mammalian cells. Our results demonstrate details about the observed transient formation of molecular complexes. The diffusion characteristics of phosphoglycerolipids without hydroxyl-containing headgroups revealed weak interactions. The strongest interactions were observed with sphingolipid analogs, which showed cholesterol-assisted and cytoskeleton-dependent binding. The hydroxyl-containing headgroup of gangliosides, galactosylceramide, and phosphoinositol assisted binding, but in a much less cholesterol- and cytoskeleton-dependent manner. The observed anomalous diffusion indicates lipid-specific transient hydrogen bonding to other membrane molecules, such as proteins, and points to a distinct connectivity of the various lipids to other membrane constituents. This strong interaction is different from that responsible for forming cholesterol-dependent, liquid-ordered domains in model membranes.

Biological dose estimation of UVA laser microirradiation utilizing charged particle-induced protein foci.

The induction of localized DNA damage within a discrete nuclear volume is an important tool in DNA repair studies. Both charged particle irradiation and laser microirradiation (LMI) systems allow for such a localized damage induction, but the results obtained are difficult to compare, as the delivered laser dose cannot be measured directly. Therefore, we revisited the idea of a biological dosimetry based on the microscopic evaluation of irradiation-induced Replication Protein A (RPA) foci numbers. Considering that local dose deposition is characteristic for both LMI and charged particles, we took advantage of the defined dosimetry of particle irradiation to estimate the locally applied laser dose equivalent. Within the irradiated nuclear sub-volumes, the doses were in the range of several hundreds of Gray. However, local dose estimation is limited by the saturation of the RPA foci numbers with increasing particle doses. Even high-resolution 4Pi microscopy did not abrogate saturation as it was not able to resolve single lesions within individual RPA foci. Nevertheless, 4Pi microscopy revealed multiple and distinct 53BP1- and gamma H2AX-stained substructures within the lesion flanking chromatin domains. Monitoring the local recruitment of the telomere repeat-binding factors TRF1 and TRF2 showed that both proteins accumulated at damage sites after UVA-LMI but not after densely ionizing charged particle irradiation. Hence, our results indicate that the local dose delivered by UVA-LMI is extremely high and cannot be accurately translated into an equivalent ionizing radiation dose, despite the sophisticated techniques used in this study.

A compact STED microscope providing 3D nanoscale resolution.

The advent of supercontinuum laser sources has enabled the implementation of compact and tunable stimulated emission depletion fluorescence microscopes for imaging far below the diffraction barrier. Here we report on an enhanced version of this approach displaying an all-physics based resolution down to (19 +/- 3) nm in the focal plane. Alternatively, this single objective lens system can be configured for 3D imaging with resolution down to 45 x 45 x 108 nm in a cell. The obtained results can be further improved by mathematical restoration algorithms. The far-field optical nanoscale resolution is attained in a variety of biological samples featuring strong variations in the local density of features.

Reversible photoswitching enables single-molecule fluorescence fluctuation spectroscopy at high molecular concentration.

We demonstrate that photoswitchable markers enable fluorescence fluctuation spectroscopy at high molecular concentration. Reversible photoswitching allows precise control of the density of fluorescing entities, because the equilibrium between the fluorescent ON- and the dark OFF-state can be shifted through optical irradiation at a specific wavelength. Depending on the irradiation intensity, the concentration of the ON-state markers can be up to 1,000 times lower than the actual concentration of the labeled molecular entity. Photoswitching expands the range of single-molecule detection based experiments such as fluorescence fluctuation spectroscopy to large entity concentrations in the micromolar range.

Nanoscale organization of nicotinic acetylcholine receptors revealed by stimulated emission depletion microscopy.

Acetylcholine receptor (AChR) supramolecular aggregates that have hitherto only been accessible to examination by electron microscopy were imaged with stimulated emission depletion (STED) fluorescence microscopy, providing resolution beyond limits of diffraction of classical wide-field or confocal microscopes. We examined a Chinese hamster ovary cell liner CHO-K1/A5, that stably expresses adult murine AChR. Whereas confocal microscopy displays AChR clusters as diffraction-limited dots of approximately 200 nm diameter, STED microscopy yields nanoclusters with a peak size distribution of approximately 55 nm. Utilizing this resolution, we show that cholesterol depletion by acute (30 min, 37 degrees C) exposure to methyl-beta-cyclodextrin alters the short and long range organization of AChR nanoclusters on the cell surface. In the short range, AChRs form larger nanoclusters, possibly related to the alteration of cholesterol-dependent protein-protein associations. Ripley's K-test on STED images reveals changes in nanocluster distribution on larger scales (0.5-3.5 microm), which possibly are related to the abolition of cytoskeletal physical barriers preventing the lateral diffusion of AChR nanoclusters.

Comparison of I5M and 4Pi-microscopy.

The axial (z-) resolution of approximately 100 nm provided by 4Pi and I5M fluorescence microscopy relies on the coherent addition of spherical wavefronts of two opposing high aperture angle lenses. Both microscopes feature a point-spread function (PSF) with a sharp central spot that is accompanied by axially shifted sidelobes which leads to replication artefacts in the raw image data. In a 4Pi-microscope the sidelobes are less pronounced than in I5M and without relevant lateral (x,y) substructure, making their posterior removal in the image reliable and fast. On the other hand, high speeds of raw data acquisition are more easily gained by I5M. Moreover, I5M features a stronger signal as compared to the commonly employed two-photon excitation (2PE) 4Pi-imaging mode. We investigate here the capability of both techniques to image (aqueous) specimens without artefacts. To this end, we consider the optical transfer function (OTF) of the two microscopes in conjunction with the signal-to-noise-ratio (SNR) of the object to be imaged. The imaging of E. coli bacteria with an interconvertable setup enabled a direct comparison of the two imaging modes. As both systems rely on high aperture angles, water-immersion lenses of the largest numerical aperture available (NA = 1.2) were employed. The experimental results are corroborated by simulations assuming the signal strength encountered in the experiment. The comparison of the theoretical with the experimental PSFs/OTFs showed that our setup operated close to theory in both imaging modes. Although I5M provided about 10 times brighter raw image data as compared to (2PE) 4Pi-microscopy, the I5M data could not be entirely cleared of artefacts. In conclusion, with the current aperture angles and fluorescence signal strengths, it is not advisable to trade in the suppression of the sidelobes for a larger image signal.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Time-multiplexed multifocal multiphoton microscope.

We resolve the classical conflict between parallelization and axial resolution in three-dimensional fluorescence microscopy through time-multiplexed multifocal multiphoton excitation. A rotating array of microlenses on a disk splits ultrafast laser pulses in such a way that an array of high-aperture foci are created in the sample. Two rigidly mounted corotating glass disks with suitable arrays of holes ensure that adjacent foci illuminate the sample at different time points. Recordings of biological specimens demonstrate elimination of out-of-focus haze for densely packed foci and concomitant substantial improvement of contrast and resolution.

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.

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.