Microscopic imaging and flow cytometry of fluorophore dark state transitions for fundamental biophysical studies and cellular diagnostics

Exploiting the high environmental sensitivity of fluorescence blinking we will develop new readouts of local metabolic conditions and molecular interactions in cells.

Using sample imaging with systematic excitation modulation, our development will focus on two cellular phenomena, having features not within reach by state-of-the-art fluorescence methods:Altered cellular metabolism and redox control is a central hallmark of cancer.

We will optimize imaging of fluorescence blinking, strongly affected by local oxygen concentrations and redox conditions, to identify individual cancer cells with such alterations.Protein-lipid interactions in plasma membranes modulate central cellular functions, but are often too intermittent to be readily observable.

We will optimize our readout to detect such interactions, by long-lived, dark triplet states of fluorophores being influenced by spin-labels in the membranes.The optimized readouts will next be combined with flow cytometry, for the first time demonstrating high-throughput identification of individual cells based on these metabolic and interaction features, then combined with super-resolution microscopy to demonstrate imaging of membrane microenvironments with tens of nanometer resolution.This project, engaging several PhD students in our lab, will establish new sensitive cellular readouts, analyses with high-throughput and high resolution, and will be of large relevance for fundamental biophysical research and clinical diagnostics.