Measurement and autocorrelation analysis of two‐dimensional light‐scattering patterns from living cells for label‐free classification (original) (raw)

We incorporate optics and an ICCD to record the two-dimensional angular optical scattering (TAOS) patterns retrieved from single aerosolized cells. We analyze these patterns by performing autocorrelations and demonstrate that we are able to retrieve cell size from the locations of the secondary maxima. Additional morphological information is contained in the autocorrelation functions and decay rate of the heights of the autocorrelation peaks. We demonstrate these techniques with C6 and Y79 cells, which are readily distinguishable. One key advantage of this methodology is that there is no requirement for antibody and fluorescent labeling molecules. Published 2011 Wiley-Liss, Inc. y Key terms elastic scattering pattern; autocorrelation; label-free; cell CELL-BASED therapies are being used increasingly in modern clinical practices, from bone-marrow transplantation to stem-cell replacement. The demands on sensitivity for cell classification and identification are continually increasing. Flow cytometry has developed into one of the key techniques for counting, analyzing, and sorting microscopic particles, such as cells and chromosomes. It is used widely in molecular biology, pathology, immunology, and other fields, such as diagnosing disordered blood cancers in pathology. Flow cytometry is used to classify particles in the subto super-micron size range by examining the intensities of fluorescence and elastic scattering when particles move through a fluidic system. Information is captured from one or more fluorescence bands at different wavelength regions and from an elastic scattering channel located at one or several scattering angles, usually at intermediate and near-forward scattering angles. The cells may also be illuminated by one or more lasers, depending on the application. Fluorescence emissions typically result from fluorescent molecular markers that attach to specific antibodies for specific cell labeling, but can also result from intrinsic fluorescence (often called autofluorescence) of the examined cell itself. Different kinds of cells can be well discriminated, identified, and sorted using such techniques (1-5). One recent advance is the use of polychromatic approaches that have entered the mainstream in flow cytometry (6,7). Instruments for measuring 17 bands of fluorescence and two parameters of elastic scattering using multiple laser excitations have been reported (7). Other advances include introducing image-intensified charge coupled devices (ICCDs) to record the dispersed fluorescence spectrum (6), and using photo-diodes, avalanche diodes (APD), or photomultiplier tube (PMT) (8-10) linear arrays as spectral detectors. Using the dispersed fluorescence spectrum can improve the accuracy and precision of ratiometric measurements and increase the probability of recording more discrete fluorescence bands.