Yael Paran

Laser autofocusing system for high-resolution cell biological imaging

Automated acquisition of high resolution, light microscope images of cells is becoming a common requirement in modern proteomic and cellomic research. A prerequisite for such microscopy is fine focus tuning, commonly optimized by multiple exposures, followed by image sharpness analysis. We describe here an extremely fast and accurate laser autofocusing system with distinct advantages for large‐scale cell‐based screening.

Evaluation of the anticancer action of a permanently charged tamoxifen derivative, tamoxifen methiodide: an MRI study

Abstract The anticancer action of a permanently charged tamoxifen derivative, tamoxifen methiodide (TMI), was assessed in a model of breast cancer. In addition, analysis of MCF-7 cells in cultures was performed to consider the anticancer mechanism of TMI. Nude mice were implanted with MCF-7 human cancer cells in the ventral fat pad at the level of the milkline (breast). The effect of TMI (administered as a slow-release pellet) and placebo were evaluated using magnetic resonance imaging (MRI) as well as by histological evaluation. The action of TMI and tamoxifen on cell growth of MCF-7 cells in cultures was also assessed. TMI induced tumor regression, while placebo-treated animals manifested tumor masses that grew monotonically throughout the experimental period (25 days). MRI revealed and histopathology confirmed that TMI treatment resulted in tumor necrosis which (1) had a faster onset; (2) was more extensive; and (3) was more intense than that observed by partial estrogen ablation (placebo-treated animals). Studies with MCF-7 cells in culture suggested that tamoxifen and TMI are equipotent in vitro. Given various reports that TMI is more potent than tamoxifen in vivo and that the anticancer efficacy between TMI and tamoxifen cannot be explained by differences in estrogen receptor interaction or effects on MCF-7S cell in culture, other mechanism may differentially contribute to the anticancer action of TMI.

Parametric MRI of Water Diffusion in Breast Cancer

Diffusion MRI studies revealed specific morphological and physiological properties of MCF7 tumors implanted in the mammary gland of immunodeficient mice. These tumors mimic the histological and pathophysiological properties of human breast cancer in patients. The experiments were conducted by (1) applying varying diffusion gradient strengths, G d , from 0 to 20 G/cm and a short diffusion time (t d = 16 ms) in order to minimize the effect of restriction and exchange of water between the intra- and extracellular compartments, and (2) applying a strong constant gradient and diffusion times up to 96 ms, revealing water restriction and exchange. The normalized signal intensity was plotted against the diffusion weighting factor b, taking into account interaction with the imaging gradients. The curves were analyzed by applying a bi-exponential decay function assuming two exchanging water compartments, with fast and slow diffusion coefficients. The amplitudes and decay constants of the two exponents, a fast and a slow one, were related to the fraction and apparent diffusion coefficients of the extra- and intracellular water, respectively, considering contributions of restriction and exchange. During tumor progression the distribution of the diffusion parameters for the same experimental protocol varied and became less homogeneous. This was predominantly due to variations in the cellularity and increased necrosis. Upon treatment of the tumors with a new anti-estrogenic drug, tamoxifen methiodide, the changes in the diffusion parameters indicated increased cell swelling. Hence, this cytostatic response to treatment was detected before actual cell death was apparent. The potential capacity of diffusion MRI is of high clinical relevance and may help improve the noninvasive diagnosis and followup of treatment of this devastating disease.

Cell-based screening for function

Biological image analysis software packages offer tools to analyze microscope images of cells. Some of these tools allow quantitative analysis through interactive processing. High-throughput applications employing microscopy for cell-based assays require analysis of large number of images. We describe here acquisition and analysis of cell images in high throughput automated mode aiming to screen for effects in structure and molecular organization of cellular components recorded by high resolution cell images and in cell motility.

Image acquisition and understanding in high-throughput high-resolution cell-based screening applications

Quantitative interpretation of microscope images is more challenging the higher the resolution of the images is. The reward is rich multi-parametric characterization of subcellular structures and detailed description of cell responses to perturbations. This information is the basis of high- throughput cell-based screening, searching to discover new drugs and understand molecular mechanisms at the cell level. We have developed a fast screening microscope acquiring high-resolution images from cells cultured in plastic-bottom multi-well plates, [1-4] and are writing an automated pipeline for the analysis of Tera Bytes of images from high throughput screens. The platform includes database for storage and retrieval of images, visualization with easy linkage of the analyzed results to the original multi-color images, segmentation of objects in images (including cells, nuclei, cytoskeletal fibers and sub-cellular organelles), multi-parametric quantification of morphological and multicolor fluorescence intensities, and statistical comparisons to control wells displayed in color coded scores on the plate graphics. This system was successfully employed for screening of the effect of drugs, gene over-expression and siRNA of diverse cellular properties, including cell adhesion, migration, survival and cytoskeletal organization.