Live Cell Imaging Applications

Fluorescent labeling and detection of target biological molecules in live cells is an essential approach for studying complex and dynamic cellular processes. We designed an amine-containing poly(phenylene ethynylene) (PPE) and fabricated it into conjugated polymer nanoparticles (CPNs) for live cell imaging.

Confocal microscopy studies suggest that CPNs are taken up by various cells without inhibiting cell viability and accumulate in vesicular structures such as early or late endosomes. CPNs also exhibit high resistance to photobleaching compared with commercially available dyes.

We are interested in developing highly fluorescent CPNs through molecular design and aggregation control. To provide target specificity, we apply bioconjugation techniques to introduce targeting ligands onto CPNs.

Multiphoton Probes

Fluorophores are typically excited by absorbing photons of a particular energy (e.g., 400 nm) and release energy as fluorescence emission. With extremely high photon density, molecules can also be excited by the simultaneous absorption of two lower-energy photons (two-photon, 2P; e.g., 800 nm).

Because 2P excitation occurs primarily at the focal point, it enables applications such as deep-tissue microscopic imaging. Our initial experiments indicate that CPNs exhibit extremely large 2P action cross sections, photostability comparable to quantum dots, and low toxicity.

Through a collaboration with Prof. Peter So (MIT), we have investigated CPNs as promising 2P probes for in vivo tissue imaging.

Biochemical Kinase Assays

Many diseases (including cancers, diabetes, and inflammation) are associated with dysregulated protein kinases that phosphorylate substrates in signaling pathways. These pathways are prime targets for drug discovery. Fluorescence-based homogeneous assays are widely used because they sensitively detect phosphorylation events and avoid radioisotopic labels.

We exploit efficient energy transfer in CPNs for sensitive detection of rhodamine-labeled peptides used as kinase substrates. Energy transfer from CPNs to rhodamine enables quantitative monitoring of phosphorylation via both fluorescence quenching (FQ) and FRET.

We demonstrated a multiwell-format biochemical kinase assay for inhibitor screening. Ratiometric analysis using CPN fluorescence quenching and FRET to rhodamine supports sensitive and reliable detection of substrate phosphorylation, aided by the large spectral separation between excitation and emission wavelengths.