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Keywords:

  • cell analysis;
  • image cytometry;
  • TDI;
  • rare event detection;
  • CTC

Abstract

Characterization of rare cells usually requires high sensitivity quantification of multiple parameters. Detection of morphological features of these cells is highly desired when routinely identifying circulating tumor cells (CTC) in blood of patients. We have designed an image cytometer intended for fast and sensitive routine analysis of CTC. After an initial scan, prospective events can be revisited for more detailed analysis. The image cytometer features: 375, 491, and 639 nm laser lines, a 40×/0.6NA objective, a CCD camera operating in TDI mode, servo stages to move the sample in two dimensions and a piëzo microscope objective positioner to move the objective in the third dimension. ImageJ is used for dedicated image analysis. A homogeneous illumination area, measuring 180 × 180 μm2, was created by the use of a rotating diffuser in combination with two micro-lens arrays. For feed-forward automatic focusing of the sample during a scan, a 3D spline was fitted through 30 predetermined focus positions before scanning the sample. Continuous signal acquisition is made possible by using a CCD operating in TDI mode synchronized to the movement of two servo scan stages. The limit of fluorescence sensitivity is 120 PE molecules on a bead with a diameter of 6.8 μm, at a scanning speed of 1.0 mm s−1. The resolution of the imaging system is 0.76 μm in the TDI scan direction at a wavelength of 580 nm. Identification of cells is facilitated by scatter plots of the fluorescent parameters in which each individual event can be viewed for its morphological features by fluorescence as well as bright field. The image cytometer measures quantitative fluorescence and morphological features at a high sensitivity, high resolution, and with minimal overhead time. It has the ability torelocate events of interest for further detailed analysis. The system can be used for routine identification and characterization of rare cells. © 2011 International Society for Advancement of Cytometry.