Ernst H. K. Stelzer

Dynamic organization of the actin cytoskeleton during meiosis and spore formation in budding yeast.

During sporulation in Saccharomyces cerevisiae, the four daughter cells (spores) are formed inside the boundaries of the mother cell. Here, we investigated the dynamics of spore assembly and the actin cytoskeleton during this process, as well as the requirements for filamentous actin during the different steps of spore formation. We found no evidence for a polarized actin cytoskeleton during sporulation. Instead, a highly dynamic network of non‐polarized actin cables is present underneath the plasma membrane of the mother cell. We found that a fraction of prospore membrane (PSM) precursors are transported along the actin cables. The velocity of PSM precursors is diminished if Myo2p or Tpm1/2p function is impaired. Filamentous actin is not essential for meiotic progression, for shaping of the PSMs or for post‐meiotic cytokinesis. However, actin is essential for spore wall formation. This requires the function of the Arp2/3p complex and involves large carbohydrate‐rich compartments, which may be chitosome analogous structures.

Optical transfer functions for confocal theta fluorescence microscopy

Optical transfer functions are presented for the confocal theta fluorescence microscope, which uses an orthogonally placed objective lens to detect the fluorescence emission. Therefore the transfer functions do not exhibit cylindrical symmetry about the illumination axis. We show that in an ideal noise-free system, confocal theta microscopy increases the cutoff spatial frequency along the illumination axis by a factor of 3.5 to 3.7 for a numerical aperture of 0.75. In a system with 10% noise the improvement by confocal theta microscopy is even greater, between a factor of 4.1 and 4.4. This explains the improved three-dimensional imaging properties of theta microscopy.

Confocal theta microscopy and 4Pi-confocal theta microscopy

Confocal theta microscopy is a novel method by which the axial resolution in confocal fluorescence microscopy can be substantially improved. The basic idea is to observe the sample with two or more objective lenses and to detect the emission light at an angle theta to the illumination axis. The observation volume is considerably decreased when the detection axis is rotated by 90 degree(s) relative to the illumination axis. This leads to an almost spherical observation volume, which is three times smaller than in a comparable confocal fluorescence microscope. Confocal theta microscopy can be combined with 4Pi-confocal microscopy and is the first viable method proposed for fully exploiting the resolution increase achievable with 4Pi-techniques. The axial side lobes of the 4Pi-point spread function are suppressed, and the observation volume is reduced by a factor of 4. The resolution properties of confocal theta fluorescence microscopies are investigated for single- and two-photon absorption. Evaluations of confocal theta point spread functions are presented and the resolution improvement achieved by theta observation is discussed.

Developments and applications of confocal theta microscopy

Confocal theta microscopy improves the resolution of confocal laser scanning microscopes by solving the problem of the inferior axial resolution instrumentally. The specimens are observed at an angle theta relative to the illumination axis (ideally 90 degrees). The resulting observation volume, defined by the product of illumination and detection point spread functions (PSFs), is reduced by 2.5 and has an isotropic shape. Single-Lens Theta Microscopy (SLTM) is a technical variation of confocal theta microscopy. It is designed to be easily adapted to any common confocal laser scanning microscope. It is based on the use of a mirror unit between the microscope objective lens and its focal plane. This mirror unit deflects the incoming and outcoming light in such a way, that the detection axis is perpendicular to the illumination axis. With SLTM different kinds of other microscopical techniques (such as 4Pi microscopy) are possible. The quantitative evaluation of physical test systems underline the feasibility of SLTM and prove the excellent resolution. The extensions of the experimentally determined point spread functions fit well with the predicted theoretical values. The technique was applied to the investigation of GFP labelled organelles in HeLa cells as well as for the analysis of embryos.