Space non-invariant point-spread function and its estimation in fluorescence microscopy

In this research report, we recall briefly how the diffraction-limited nature of an optical microscope's objective, and the intrinsic noise can affect the observed images' resolution. A blind deconvolution algorithm can restore the lost frequencies beyond the diffraction limit. However, under other imaging conditions, the approximation of aberration-free imaging, is not applicable, and the phase aberrations of the emerging wavefront from a specimen immersion medium cannot be ignored any more. We show that an object's location and its original intensity distribution can be recovered by retrieving the refracted wavefront's phase from the observed intensity images. We demonstrate this by retrieving the point-spread function from an imaged microsphere. The noise and the influence of the microsphere size can be mitigated and sometimes completely removed from the observed images by using a maximum a posteriori estimate. However, due to the incoherent nature of the acquisition system, phase retrieval from the observed intensities will be possible only if the phase is constrained. We have used geometrical optics to model the phase of the refracted wavefront, and tested the algorithm on some simulated images

Point-spread function retrieval for fluorescence microscopy

International audienceIn this paper we propose a method for retrieving the Point-Spread Function (PSF) of an imaging system given the observed images of fluoroscentmicrospheres. Theoretically calculated PSFs often lack the experimental or microscope specific signatureswhile empirically obtained data are either over sized or (and) too noisy. The effect of noise and the influence of the microsphere size can be mitigated from the experimental data by using a Maximum Likelihood Expectation Maximization (MLEM) algorithm. The true experimental parameters can then be estimated by fitting the result to amodel based on the scalar diffraction theory. The algorithm was tested on some simulated data and the results obtained validate the usefulness of the approach for retrieving the PSF from measured data

Focal Contacts as Mechanosensors: Externally Applied Local Mechanical Force Induces Growth of Focal Contacts by an Mdia1-Dependent and Rock-Independent Mechanism

The transition of cell–matrix adhesions from the initial punctate focal complexes into the mature elongated form, known as focal contacts, requires GTPase Rho activity. In particular, activation of myosin II–driven contractility by a Rho target known as Rho-associated kinase (ROCK) was shown to be essential for focal contact formation. To dissect the mechanism of Rho-dependent induction of focal contacts and to elucidate the role of cell contractility, we applied mechanical force to vinculin-containing dot-like adhesions at the cell edge using a micropipette. Local centripetal pulling led to local assembly and elongation of these structures and to their development into streak-like focal contacts, as revealed by the dynamics of green fluorescent protein–tagged vinculin or paxillin and interference reflection microscopy. Inhibition of Rho activity by C3 transferase suppressed this force-induced focal contact formation. However, constitutively active mutants of another Rho target, the formin homology protein mDia1 (Watanabe, N., T. Kato, A. Fujita, T. Ishizaki, and S. Narumiya. 1999. Nat. Cell Biol. 1:136–143), were sufficient to restore force-induced focal contact formation in C3 transferase-treated cells. Force-induced formation of the focal contacts still occurred in cells subjected to myosin II and ROCK inhibition. Thus, as long as mDia1 is active, external tension force bypasses the requirement for ROCK-mediated myosin II contractility in the induction of focal contacts. Our experiments show that integrin-containing focal complexes behave as individual mechanosensors exhibiting directional assembly in response to local force

POINT-SPREAD FUNCTION MODEL FOR FLUORESCENCE MACROSCOPY IMAGING

International audienceIn this paper, we model the point-spread function (PSF) of a fluo- rescence MACROscope with a field aberration. The MACROscope is an imaging arrangement that is designed to directly study small and large specimen preparations without physically sectioning them. However, due to the different optical components of the MACRO- scope, it cannot achieve the condition of lateral spatial invariance for all magnifications. For example, under low zoom settings, this field aberration becomes prominent, the PSF varies in the lateral field, and is proportional to the distance from the center of the field. On the other hand, for larger zooms, these aberrations become gradually ab- sent. A computational approach to correct this aberration often relies on an accurate knowledge of the PSF. The PSF can be defined either theoretically using a scalar diffraction model or empirically by ac- quiring a three-dimensional image of a fluorescent bead that approx- imates a point source. The experimental PSF is difficult to obtain and can change with slight deviations from the physical conditions. In this paper, we model the PSF using the scalar diffraction approach, and the pupil function is modeled by chopping it. By comparing our modeled PSF with an experimentally obtained PSF, we validate our hypothesis that the spatial variance is caused by two limiting optical apertures brought together on different conjugate planes

Quantitative Multicolor Compositional Imaging Resolves Molecular Domains in Cell-Matrix Adhesions

Background: Cellular processes occur within dynamic and multi-molecular compartments whose characterization requires analysis at high spatio-temporal resolution. Notable examples for such complexes are cell-matrix adhesion sites, consisting of numerous cytoskeletal and signaling proteins. These adhesions are highly variable in their morphology, dynamics, and apparent function, yet their molecular diversity is poorly defined. Methodology/Principal Findings: We present here a compositional imaging approach for the analysis and display of multicomponent compositions. This methodology is based on microscopy-acquired multicolor data, multi-dimensional clustering of pixels according to their composition similarity and display of the cellular distribution of these composition clusters. We apply this approach for resolving the molecular complexes associated with focal-adhesions, and the time-dependent effects of Rho-kinase inhibition. We show here compositional variations between adhesion sites, as well as ordered variations along the axis of individual focal-adhesions. The multicolor clustering approach also reveals distinct sensitivities of different focaladhesion-associated complexes to Rho-kinase inhibition. Conclusions/Significance: Multicolor compositional imaging resolves ‘‘molecular signatures’ ’ characteristic to focaladhesions and related structures, as well as sub-domains within these adhesion sites. This analysis enhances the spatial information with additional ‘‘contents-resolved’ ’ dimensions. We propose that compositional imaging can serve as

Identification of Novel Pro-Migratory, Cancer-Associated Genes Using Quantitative, Microscopy-Based Screening

Background: Cell migration is a highly complex process, regulated by multiple genes, signaling pathways and external stimuli. To discover genes or pharmacological agents that can modulate the migratory activity of cells, screening strategies that enable the monitoring of diverse migratory parameters in a large number of samples are necessary. Methodology: In the present study, we describe the development of a quantitative, high-throughput cell migration assay, based on a modified phagokinetic tracks (PKT) procedure, and apply it for identifying novel pro-migratory genes in a cancer-related gene library. In brief, cells are seeded on fibronectin-coated 96-well plates, covered with a monolayer of carboxylated latex beads. Motile cells clear the beads, located along their migratory paths, forming tracks that are visualized using an automated, transmitted-light screening microscope. The tracks are then segmented and characterized by multi-parametric, morphometric analysis, resolving a variety of morphological and kinetic features. Conclusions: In this screen we identified 4 novel genes derived from breast carcinoma related cDNA library, whose over-expression induces major alteration in the migration of the stationary MCF7 cells. This approach can serve for high throughput screening for novel ways to modulate cellular migration in pathological states such as tumor metastasis and invasion