In this work, we developed a new method for high throughput and high content spectral imaging flow cytometry based on structured linear spot array excitation. This method leverages equally spaced laser spots for illumination, scanning of a single cell with cell movement, and the cell image is reconstructed by splitting and assembling PMT signals. To demonstrate this method, we first built an imaging flow cytometer with dual-laser and five imaging channels (Bright-field, FITC, PE, PI, APC). More specially, due to the excellent scalability of this method, for the first time, we demonstrated a high-throughput hyperspectral imaging flow cytometer by integrating a high speed 32-channel spectrometer. This system obtains 32 spectral images of 1 μm resolution at the cell flowing speed of 5 m/s with the maximum throughput up to 5000 eps.
In this work, we developed a new method for high throughput imaging flow cytometry, using diffractive optics elements to generate linear laser spot array for illumination, and single-pixel detectors for detection. The illumination spots are arranged in a line at equal intervals and form a small angle with the direction of the cell movement. When the cell passes through the illumination area, the two-dimensional information of the cell's fluorescence and scattered intensity profile is encoded into signals detected by the PMTs. Fluorescence and scattering imaging were experimentally demonstrated for beads and cells traveling at a velocity of 4.7 m/s in a microfluidic chip, with a resolution of 1 μm and a maximum throughput of 5000cell/s.
Flow cytometer is a powerful tool for the quantitative analysis of a large population of cells. A key factor determining the measurement accuracy and system stability is the illumination beam. The conventional beam shaper is composed of multiple geometrical optical elements, which should be precisely calibrated to produce elliptical Gaussian beams. It is difficult to control the sizes and positions of the focal spots formed by different wavelengths. A new beam shaping method based on diffractive optical element (DOE) is developed for rectangular flattop spots with multi-wavelength illumination. Its benefits include high simplicity, design flexibility, uniform illumination, and multifunctionality.
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