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Holo-Tomographic Flow Cytometry is a new technology for single-cell analysis that combine Phase-Contrast Tomography and Flow Cytometry opening to a new approach in biomedical field by high-throughput, tri-dimensional imaging of unstained cell populations. Tomographic Phase Microscopy is a label-free phase contrast imaging method able to supply quantitative and volumetric refractive index distribution at single cell level in adherent or fixed populations. Here, we demonstrate that phase-contrast tomography can be achieved also for cells into a microfluidic environment obtaining accurate 3D tomographic imaging of thousands of flowing and rotating cells thanks to a robust and reliable computational strategy. Recording setups are based on Digital Holography in microscopy configurations integrated with microfluidic apparatus to record interference fringes (hologram) of rotating cells. Computational pipeline includes 3D cell tracking into the microfluidic channel, quantitative 2D phase-contrast maps retrieval for each acquired hologram, robust angle recovery code, tomographic processing to measure the inner refractive index distribution. Holo-Tomographic Flow Cytometry surpasses the limits of conventional Imaging flow cytometers because make available the recording of hundreds of informative images for each flowing cells avoiding the employment of fluorescent tags. Holo-Tomographic Flow Cytometry allows to retrieve the unique all-optical 3D fingerprint for each cell flowing into the field-of-view opening to a wide range of applications such as: (i) identification of inner subcellular compartments; (ii) recognition of nanoparticle uptake and (iii) phenotyping of different subclasses in heterogeneous populations. Future perspectives are presented in the fields of liquid biopsy, drug resistance and genetic disfunctions.
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