Jörg Bewersdorf

Multifocal Multiphoton Microscopy.

We present a real-time, direct-view multiphoton excitation fluorescence microscope that provides three-dimensional imaging at high resolution. Using a rotating microlens disk, we split the near-infrared light of a mode-locked titanium-sapphire laser into an array of beams that are transformed into an array of high-aperture foci at the object. We typically scan at 225 frames per second and image the fluorescence with a camera that reads out the images at video rate. For 1.4 aperture oil and 1.2 water immersion lenses at 780-nm excitation we obtained axial resolutions of 0.84 and 1.4mum , respectively, which are similar to that of a single-beam two-photon microscope. Compared with the latter setup, our system represents a 40-100-fold increase in efficiency, or imaging speed. Moreover, it permits the observation with the eye of high-resolution two-photon images of (live) samples.

Fis1p and Caf4p, but not Mdv1p, determine the polar localization of Dnm1p clusters on the mitochondrial surface.

The mitochondrial division machinery consists of the large dynamin-related protein Dnm1p (Drp1/Dlp1 in humans), and Fis1p, Mdv1p and Caf4p. Proper assembly of Dnm1p complexes on the mitochondrial surface is crucial for balanced fission and fusion events. Using quantitative confocal microscopy, we show that Caf4p is important for the recruitment of Dnm1p to the mitochondria. The mitochondrial Dnm1p assemblies can be divided into at least two morphologically distinguishable fractions. A small subset of these assemblies appear to be present as Dnm1p-spirals (or rings) that encircle tubule constrictions, with seldom more than seven turns. A larger fraction of the Dnm1p assemblies is primarily present at one side of the mitochondrial tubules. We show that a majority of these mitochondria-associated Dnm1p clusters point towards the cell cortex. This polarized orientation is abolished in fis1Δ and caf4Δ yeast cells, but is maintained in mdv1Δ cells and after disruption of the actin cytoskeleton. This study suggests that Caf4p plays a key role in determining the polarized localization of those Dnm1p clusters that are not immediately involved in the mitochondrial fission process.

Comparison of I5M and 4Pi-microscopy

The axial (z‐) resolution of ∼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.

Single sharp spot in fluorescence microscopy of two opposing lenses

We demonstrate theoretically, experimentally, and in an imaging application the possibility to generate a single predominant sharp diffraction maximum in the effective point-spread function of a fluorescence microscope that coherently uses two opposing lenses. This is achieved through binary pupil filters that preclude the origination of the unfavorable strong interference side maxima that are otherwise present in these systems. Mathematical postprocessing, which has so far been a prerequisite to gain artifact-free images, is now optional or obsolete.

Multifocal Multi-Photon Microscopy

Multi-photon processes relying on the cooperative action of two or more photons can broadly be divided into two families that are distinguished by the fact that the photons are either absorbed or scattered (Shen, 1984). Whereas the scattering events relevant to microscopy are second and third harmonic generation (SHG, THG), as well as coherent anti-Stokes Raman scattering (CARS), the useful multi-photon absorption events are two- and threephoton excitation (2PE, 3PE).

Determination of the unknown phase difference in 4Pi-confocal microscopy through the image intensity

We investigate the effect of the phase difference of the interfering wavefronts on the image intensity in 4Pi-confocal microscopy and thereby introduce a method to diminish the problem of the unknown phase difference in this microscope. An efficient implementation of our method utilizes binary amplitude filters reshaping the point-spread-function such that a significant disparity arises between the maximum fluorescence intensity in the constructive and destructive interference case. In the presence of planar and point-like features in the sample this property enables the system to unambiguously determine the mode of interference.

Phase determination in interference-based superresolving microscopes through critical frequency analysis

Utilizing the interference of wave fronts of two opposing lenses, 4Pi-confocal and I(5)M microscopy improve the axial resolution of far-field fluorescence microscopy as much as threefold to sevenfold. However, establishing the phase difference of the wave fronts in the sample is a problem yet to be solved. Here we show that the phase difference is encoded in the microscopes transfer of the spatial frequencies that match the distance of the interference peaks. As a result the phase difference is readily extracted through a Fourier transform of the image. Our method is relevant to all microscopes that exploit the interference of counterpropagating waves to improve the axial and the lateral resolution.