The challenge of segmenting neurospheres (NSPs) from brightfield images includes uneven background illumination (vignetting), low contrast and shadow-casting appearance near the well wall. We propose a pipeline for neurosphere segmentation in brightfield images, focusing on shadow-casting removal. Firstly, we remove vignetting by creating a synthetic blank field image from a set of brightfield images of the whole well. Then, radial line integration is proposed to remove the shadow-casting and therefore facilitate automatic segmentation. Furthermore, a weighted bi-directional decay function is introduced to prevent undesired gradient effect of line integration on NSPs without shadow-casting. Afterward, multiscale Laplacian of Gaussian (LoG) and localized region-based level set are used to detect the NSP boundaries. Experimental results show that our proposed radial line integration method (RLI) achieves higher detection accuracy over existing methods in terms of precision, recall and F-score with less computational time.
IRSp53 is a Cdc42 effector and a member of the Inverse-Bin-Amphiphysins-Rvs (I-BAR) domain family which can induce negative membrane curvature. IRSp53 generates filopodia by coupling membrane protrusion (I-BAR domain) with actin dynamics through its SH3 domain binding partners. Dynamin 1 (Dyn1), a large GTPase associated with endocytosis, is a novel interacting partner of IRSp53 that localises to filopodia. Using rapid time-lapse TIRF microscopy we have shown that Dyn1 localized to a subcellular region just behind Mena at the leading edge, or in filopodial tip complexes when co-expressed with IRSp53. Dyn1-GFP was strongly localized in the filopodial shaft during the early phase of elongation, after which it moved rearward, suggestive of a role in early filopodia assembly. Mena and Eps8, accumulate at the tip complex in sequence and are involved in filopodial extension and retraction, respectively (Chou at al, 2014 submitted). Here we describe the use of dSTORM to investigate the molecular architecture of filopodia and in particular the size of the F-actin bundle in these structures. The data suggest that <i>direct Stochastic Optical Reconstruction Microscopy</i> (dSTORM) in combination with other techniques will allow the molecular architecture of
Recently, a class of single-molecule based localization techniques such as the Photo-activated Localization Mi-
croscopy (PALM) or the Stochastic Optical Reconstruction Microscopy (STORM) has ingeniously brought light-
microscopy beyond the diraction limit. However, as the single-molecule images contain point source objects
(which have no clear edges, alignment and usually superimposed to the background), traditional restoration
techniques used for industrial vision images do not give satisfactory result on the PALM/STORM dataset. In
this work, we apply the multi-scale product of sub-band images resulting from the wavelet transformation, a
technique originally used for astronomical image restoration, for the noise ltering and single-molecule detection
in the Super-resolution images. This is an extension of the work by J.C Olivo-Marin1 on spot detection in bio-
logical images. Experimental results on real and synthetic datasets with ground-truth show that our approach
achieves very good detection rates as compared to the QuickPALM or the rapidSTORM software.
We report the detection of interactions between a photosensitizer, hypericin (HY), and its solvent system prepared with a formulation additive, polyvinylpyrrolidone (PVP), a commonly used pharmaceutical excipient. Fluorescence correlation spectroscopy (FCS) and fluorescence lifetime imaging microscopy (FLIM) were used to study aggregation and binding of HY in the presence of PVP. Digitized fluorescence endoscopic imaging (DFEI) was used to study the effect of the pharmaceutical formulation in the in vivo tumor implanted chick chorioallantoic membrane (CAM) model. The results presented reveal the coordination of HY-PVP binding, HY disaggregation in the presence of PVP, and strengthened HY tumor uptake selectivity. PVP is thus suggested as a potential adjuvant to previously investigated N-methyl pyrrolidone (NMP) in the HY delivery system as well as a replacement for the conventionally used albumin in the HY bladder instillation fluids preparation for clinical use.