Amir Rahmani

Flow cytometry (FC) is a powerful tool for high-throughput characterisation of cell populations, yet its ensemble fluorescence detection and the autofluorescence of cells impose a sensitivity limit of ∼100-1,000 fluorophores per cell. This precludes detection of low-abundance proteins and weak signalling events with biological importance and therapeutic potential. We introduce single-molecule Flow Cytometry (smFC), integrating high-numerical aperture oblique plane microscopy (OPM) with microfluidics to achieve optical sectioning and photon collection efficiencies suitable for single-molecule detection on flowing cells. Using super-bright, large Stokes shift fluorophores, smFC can detect labelled membrane proteins on cells with digital precision down to ∼2 molecules per cell, compared to an unlabelled control. This represents an improvement in the detection limit of 10- to 80-fold depending on the probe. We apply smFC to quantify the distribution of the membrane receptor c-kit in triple-negative breast cancer cells with unprecedented sensitivity. This revealed a previously unidentified distribution of 60% c-kit negative cells and 40% with low-abundance but heterogenous (1-200 mol/cell) surface distribution that is undetectable by conventional FC. These results establish smFC as a robust platform for high-throughput, quantitative single-molecule analysis in cell populations, opening new avenues for detecting rare biomarkers and quantifying the presence of low-abundance membrane proteins and targetable surface molecules with digital precision.

Focus stabilisation is vital for long-term fluorescence imaging, particularly in the case of high-resolution imaging techniques. Current stabilisation solutions either rely on fiducial markers that can be perturbative, or on beam reflection monitoring that is limited to high-numerical aperture objective lenses, making multimodal and large-scale imaging challenging. We introduce a beam-based method that relies on astigmatism, which offers advantages in terms of precision and the range over which focus stabilisation is effective. This approach is shown to be compatible with a wide range of objective lenses (10x-100x), typically achieving <10 nm precision with >10 μm operating range. Notably, our technique is largely unaffected by pointing stability errors, which in combination with implementation through a standalone Raspberry Pi architecture, offers a versatile focus stabilisation unit that can be added onto most existing microscope setups.