bright field microscopy as an alternative to whole cell fluorescence in automated analysis of macrophage images亮视场显微镜替代整个细胞荧光巨噬细胞图像的自动分析.pdf

Bright Field Microscopy as an Alternative to Whole Cell Fluorescence in Automated Analysis of Macrophage Images 1,2 1,2 1 1 1 Jyrki Selinummi , Pekka Ruusuvuori , Irina Podolsky , Adrian Ozinsky , Elizabeth Gold , Olli Yli- 2 1 1,2,3,4 Harja , Alan Aderem , Ilya Shmulevich * 1 Institute for Systems Biology, Seattle, Washington, United States of America, 2 Department of Signal Processing, Tampere University of Technology, Tampere, Finland, 3 Department of Bioengineering, University of Washington, Seattle, Washington, United States of America, 4 Department of Electrical Engineering, University of Washington, Seattle, Washington, United States of America Abstract Background: Fluorescence microscopy is the standard tool for detection and analysis of cellular phenomena. This technique, however, has a number of drawbacks such as the limited number of available fluorescent channels in microscopes, overlapping excitation and emission spectra of the stains, and phototoxicity. Methodology: We here present and validate a method to automatically detect cell population outlines directly from bright field images. By imaging samples with several focus levels forming a bright field z -stack, and by measuring the intensity variations of this stack over the z -dimension, we construct a new two dimensional projection image of increased contrast. With additional information for locations of each cell, such as stained nuclei, this bright field projection image can be used instead of whole cell fluorescence to locate borders of individual cells, separating touching cells, and enabling