Burton Kevin
Department of Radiation Oncology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, USA.
Cytometry A. 2003 Aug;54(2):125-31. doi: 10.1002/cyto.a.10062.
Rapid measurements of large numbers of cells in 3D are often required for measurements of cell migration. A method is presented for quantifying the position of cells in three-dimensional gels using brightfield microscopy.
Images were recorded using transmitted light with a closed condenser aperture diaphragm positioned off axis to produce oblique illumination. Two or more images, each at the same focal plane, were obtained with the aperture shifted equally to either side of the optical axis. All in-focus objects were at the same position in the two images, but out-of-focus objects were displaced parallel to the aperture movement.
The method was tested using gels containing 12-microm-diameter glass beads or cells that had migrated several hundred micrometers into the gel. The position in the image plane varied linearly with axial position, being reversed for objects above and below the focal plane. Beads and cells could be visualized up to a depth of >1 mm in gels.
The method facilitates measurements of positions of cells in 3D matrices by eliminating the need to perform optical sectioning and can be used with any brightfield microscope. Supplementary material for this article can be found at http://www.interscience.wiley.com/jpages/0196-4763/suppmat/54_2/v54.125.html
细胞迁移测量通常需要快速测量三维空间中的大量细胞。本文介绍了一种利用明场显微镜定量三维凝胶中细胞位置的方法。
使用透射光记录图像，关闭聚光器孔径光阑并使其偏离光轴以产生斜射照明。在同一焦平面上获得两张或更多张图像，孔径向光轴两侧等距移动。所有聚焦的物体在两张图像中的位置相同，但失焦物体平行于孔径移动方向发生位移。
该方法通过含有直径为12微米的玻璃珠或已迁移至凝胶中数百微米的细胞的凝胶进行测试。图像平面中的位置随轴向位置呈线性变化，焦平面上方和下方的物体情况相反。在凝胶中，珠子和细胞在深度大于1毫米处仍可被观察到。
该方法无需进行光学切片即可方便地测量三维基质中细胞的位置，并且可与任何明场显微镜配合使用。本文的补充材料可在http://www.interscience.wiley.com/jpages/0196-4763/suppmat/54_2/v54.125.html获取
