Imaging technique implemented in CellTracks system.

BACKGROUND: We developed the CellTracks cell analysis system that, similar to flow cytometry, yields multiparameter information by which the cells can be differentiated. We describe the implementation of a laser scanning imaging method in the system. Image analysis of the cells improves the specificity of cell classification, especially in cases where the particular cells are found relatively infrequently and one has to discriminate between artifacts and real events. METHODS: Fluorescent images of immunomagnetically labeled and aligned cells are obtained by passing the cells through a laser focus. The laser focus is smaller than the objects and subsequent frames captured by a regular surveillance CCD camera with a frame grabber board represent different parts of the cells. Complete images of the cells are constructed by shifting each image with respect to each other and adding individual pixel values. RESULTS: The power of combining a fluorescent image with multiparametric data is demonstrated by imaging fluorescent and magnetically labeled beads and cells. The image gives additional information about the dye distribution across the objects. Changes in dye distribution as a function of time were observed in leukocytes labeled with the red fluorescent label, Oxazine750, which are imaged at different time intervals. CONCLUSIONS: An imaging technique implemented in the CellTracks system provides high-resolution fluorescent images of events previously identified by the system. The images of the fluorescent cells enhance the ability to classify rare events.

Magnetic field design for selecting and aligning immunomagnetic labeled cells.

BACKGROUND: Recently we introduced the CellTracks cell analysis system, in which samples are prepared based on a combination of immunomagnetic selection, separation, and alignment of cells along ferromagnetic lines. Here we describe the underlying magnetic principles and considerations made in the magnetic field design to achieve the best possible cell selection and alignment of magnetically labeled cells. Materials and Methods Computer simulations, in combination with experimental data, were used to optimize the design of the magnets and Ni lines to obtain the optimal magnetic configuration. RESULTS: A homogeneous cell distribution on the upper surface of the sample chamber was obtained with a magnet where the pole faces were tilted towards each other. The spatial distribution of magnetically aligned objects in between the Ni lines was dependent on the ratio of the diameter of the aligned object and the line spacing, which was tested with magnetically and fluorescently labeled 6 microm polystyrene beads. The best result was obtained when the line spacing was equal to or smaller than the diameter of the aligned object. CONCLUSIONS: The magnetic gradient of the designed permanent magnet extracts magnetically labeled cells from any cell suspension to a desired plane, providing a homogeneous cell distribution. In addition, it magnetizes ferro-magnetic Ni lines in this plane whose additional local gradient adds to the gradient of the permanent magnet. The resultant gradient aligns the magnetically labeled cells first brought to this plane. This combination makes it possible, in a single step, to extract and align cells on a surface from any cell suspension.

Cell analysis system based on compact disk technology.

BACKGROUND: A cell analysis system was developed to enumerate and differentiate magnetically aligned cells selected from whole blood. The cellular information extracted is similar to the readout of musical information from a compact disk (CD). Here we describe the optical design and data processing of the system. The performance of the system is demonstrated using fluorescent-labeled cells and beads. Materials and Methods System performance was demonstrated with 6-microm polystyrene beads labeled with magnetic nanoparticles and allophycocyanin (APC) and immunomagnetically aligned leukocytes, fluorescently labeled with Oxazine750 and CD4-APC, CD8-Cy5.5, and CD14-APC/Cy7 in whole blood. RESULTS: The sensitivity of the system was demonstrated using APC-labeled beads. With this system, beads containing 333 APC molecules could easily be resolved from the background. This level of sensitivity was not achievable with a commercial flow cytometer. A maximum of 20,000 immunomagnetically labeled cells could be aligned and analyzed in between 0.6 m of Ni lines, distributed over a surface area of 18 mm(2) and extracted from a blood volume that depended on the height of the chamber. The utility of the system was demonstrated by performing a three-color CD4-CD8-CD14 assay. CONCLUSIONS: We built a cell analysis system based on immunomagnetic cell selection and alignment and analysis of fluorescent signals employing CD-technology that is as good or better than current commercial analyzers. The cell analysis can be performed in whole blood or any other type of cell suspension without extensive sample preparation.