Shack-Hartmann wave front measurements in cortical tissue for deconvolution of large three-dimensional mosaic transmitted light brightfield micrographs

We present a novel approach for deconvolution of 3D image stacks of cortical tissue taken by mosaic/optical-sectioning technology, using a transmitted light brightfield microscope. Mosaic/optical-sectioning offers the possibility of imaging large volumes (e.g. from cortical sections) on a millimetre scale at sub-micrometre resolution. However, a blurred contribution from out-of-focus light results in an image quality that usually prohibits 3D quantitative analysis. Such quantitative analysis is only possible after deblurring by deconvolution. The resulting image quality is strongly dependent on how accurate the point spread function used for deconvolution resembles the properties of the imaging system. Since direct measurement of the true point spread function is laborious and modelled point spread functions usually deviate from measured ones, we present a method of optimizing the microscope until it meets almost ideal imaging conditions. These conditions are validated by measuring the aberration function of the microscope and tissue using a Shack-Hartmann sensor. The analysis shows that cortical tissue from rat brains embedded in Mowiol and imaged by an oil-immersion objective can be regarded as having a homogeneous index of refraction. In addition, the amount of spherical aberration that is caused by the optics or the specimen is relatively low. Consequently the image formation is simplified to refraction between the embedding and immersion medium and to 3D diffraction at the finite entrance pupil of the objective. The resulting model point spread function is applied to the image stacks by linear or iterative deconvolution algorithms. For the presented dataset of large 3D images the linear approach proves to be superior. The linear deconvolution yields a significant improvement in signal-to-noise ratio and resolution. This novel approach allows a quantitative analysis of the cortical image stacks such as the reconstruction of biocytin-stained neuronal dendrites and axons

Focused and defocused retinal images with Bessel and axicon pupil functions.

Retinal image light distributions in a standard optical model of a diffraction-limited eye with round pupils are presented for several patterns of amplitude and phase modulation of the light admitted into the eye. Of special interest are circularly symmetrical configurations of truncated Bessel amplitude transmission functions, and of light subjected to axicon deviation. It is shown by several examples that this kind of beam shaping allows generation of retinal imagery, which can be more robust to defocus while maintaining minimal image degradation, and it points to situations of two separate zones simultaneously in sharp focus, several diopters apart

Image Reconstruction with Analytical Point Spread Functions

The image degradation produced by atmospheric turbulence and optical aberrations is usually alleviated using post-facto image reconstruction techniques, even when observing with adaptive optics systems. These techniques rely on the development of the wavefront using Zernike functions and the non-linear optimization of a certain metric. The resulting optimization procedure is computationally heavy. Our aim is to alleviate this computationally burden. To this aim, we generalize the recently developed extended Zernike-Nijboer theory to carry out the analytical integration of the Fresnel integral and present a natural basis set for the development of the point spread function in case the wavefront is described using Zernike functions. We present a linear expansion of the point spread function in terms of analytic functions which, additionally, takes defocusing into account in a natural way. This expansion is used to develop a very fast phase-diversity reconstruction technique which is demonstrated through some applications. This suggest that the linear expansion of the point spread function can be applied to accelerate other reconstruction techniques in use presently and based on blind deconvolution.Comment: 10 pages, 4 figures, accepted for publication in Astronomy & Astrophysic

Smearing effect due to the spread of a probe-particle on the Brownian motion near a perfectly reflecting boundary

Quantum fluctuations of electromagnetic vacuum are investigated in a half-space bounded by a perfectly reflecting plate by introducing a probe described by a charged wave-packet distribution in time-direction. The wave-packet distribution of the probe enables one to investigate the smearing effect upon the measured vacuum fluctuations caused by the quantum nature of the probe particle. It is shown that the wave-packet spread of the probe particle significantly influences the measured velocity dispersion of the probe. In particular, the asymptotic late-time behavior of its $z$-component, $$, for the wave-packet case is quite different from the test point-particle case ($z$ is the coordinate normal to the plate). The result for the wave-packet is \sim 1/\t^2 in the late time (\t is the measuring time), in stead of the reported late-time behavior $\sim 1/z^2$ for a point-particle probe. This result can be quite significant for further investigations on the measurement of vacuum fluctuations.Comment: 8 page

The optical system of the H.E.S.S. imaging atmospheric Cherenkov telescopes, Part II: mirror alignment and point spread function

Mirror facets of the H.E.S.S. imaging atmospheric Cherenkov telescopes are aligned using stars imaged onto the closed lid of the PMT camera, viewed by a CCD camera. The alignment procedure works reliably and includes the automatic analysis of CCD images and control of the facet alignment actuators. On-axis, 80% of the reflected light is contained in a circle of less than 1 mrad diameter. The spot widens with increasing angle to the telescope axis. In accordance with simulations, the spot size has roughly doubled at an angle of 1.4 degr. from the axis. The expected variation of spot size with elevation due to deformations of the support structure is visible, but is completely non-critical over the usual working range. Overall, the optical quality of the telescope exceeds the specifications.Comment: 23 pages, 13 figure

Actin, α-actinin, and tropomyosin interaction in the structural organization of actin filaments in nonmuscle cells

During the spreading of a population of rat embryo cells, approximately 40% of the cells develop a strikingly regular network which precedes the formation of the straight actin filament bundles seen in the fully spread out cells. Immunofluorescence studies with antibodies specific for the skeletal muscle structural proteins actin, α-actinin, and tropomyosin indicate that this network is composed of foci containing actin and α-actinin, connected by tropomyosin-associated actin filaments. Actin filaments, having both tropomyosin and α-actinin associated with them, are also seen to extend from the vertices of this network to the edges of the cell. These results demonstrate a specific interaction of α-actinin and tropomyosin with actin filaments during the assembly and organization of the actin filament bundles of tissue culture cells. The three-dimensional network they form may be regarded as the structural precursor and the vertices of this network as the organization centers of the ultimately formed actin filament bundles of the fully spread out cells

Principal Component Analysis of the Time- and Position-Dependent Point Spread Function of the Advanced Camera for Surveys

We describe the time- and position-dependent point spread function (PSF) variation of the Wide Field Channel (WFC) of the Advanced Camera for Surveys (ACS) with the principal component analysis (PCA) technique. The time-dependent change is caused by the temporal variation of the $HST$ focus whereas the position-dependent PSF variation in ACS/WFC at a given focus is mainly the result of changes in aberrations and charge diffusion across the detector, which appear as position-dependent changes in elongation of the astigmatic core and blurring of the PSF, respectively. Using >400 archival images of star cluster fields, we construct a ACS PSF library covering diverse environments of the $HST$ observations (e.g., focus values). We find that interpolation of a small number ($\sim20$) of principal components or ``eigen-PSFs'' per exposure can robustly reproduce the observed variation of the ellipticity and size of the PSF. Our primary interest in this investigation is the application of this PSF library to precision weak-lensing analyses, where accurate knowledge of the instrument's PSF is crucial. However, the high-fidelity of the model judged from the nice agreement with observed PSFs suggests that the model is potentially also useful in other applications such as crowded field stellar photometry, galaxy profile fitting, AGN studies, etc., which similarly demand a fair knowledge of the PSFs at objects' locations. Our PSF models, applicable to any WFC image rectified with the Lanczos3 kernel, are publicly available.Comment: Accepted to PASP. To appear in December issue. Figures are degraded to meet the size limit. High-resolution version can be downloaded at http://acs.pha.jhu.edu/~mkjee/acs_psf/acspsf.pd