Single-molecule imaging flow cytometry

Flow cytometry (FC) enables the analysis of large cell populations, with multiple cellular parameters measurable in a single experiment. These parameters allow categorisation of cells and identification of heterogeneous populations. FC is routinely used for high-throughput quantification of membrane protein expression and phenotype isolation across large populations. However, conventional FC systems lack the sensitivity to detect individual molecules on the cell surface, with the lower detection limit typically around 100 molecules per cell.

In contrast, single-molecule microscopy techniques are capable of visualising individual molecules on cells, in some cases achieving a resolution down to sub-10 nm. Yet, these methods are inherently low throughput and often restricted to fixed-cell imaging.

Bridging this gap by pushing the sensitivity of FC towards single-molecule detection would enable the identification of low-abundance proteins, including targetable antigens implicated in essential signalling pathways and disease processes.

This thesis presents the development of a single-molecule sensitive imaging flow cytometry (smIFC) platform. The approach integrates a state-of-the-art imaging modality with a microfluidic system for flow-based cell loading, combined with optimised immunolabelling protocols and the use of bright, photostable fluorophores. Proof-of-concept experiments demonstrate the ability to detect and count individual molecules on the surface of cells, highlighting the potential of smIFC to extend the capabilities of conventional flow cytometry.