Optical painting and fluorescence activated sorting of single adherent cells labelled with photoswitchable Pdots

Abstract

The efficient selection and isolation of individual cells of interest from a mixed population is desired in many biomedical and clinical applications. Here we show the concept of using photoswitchable semiconducting polymer dots (Pdots) as an optical 'painting' tool, which enables the selection of certain adherent cells based on their fluorescence, and their spatial and morphological features, under a microscope. We first develop a Pdot that can switch between the bright (ON) and dark (OFF) states reversibly with a 150-fold contrast ratio on irradiation with ultraviolet or red light. With a focused 633-nm laser beam that acts as a 'paintbrush' and the photoswitchable Pdots as the 'paint', we select and 'paint' individual Pdot-labelled adherent cells by turning on their fluorescence, then proceed to sort and recover the optically marked cells (with 90% recovery and near 100% purity), followed by genetic analysis.

Photostable Ratiometric Pdot Probe for in Vitro and in Vivo Imaging of Hypochlorous Acid

“…46,52 Briefly, to form the fluorescent Pdots, the semiconducting polymer PFOBT 36 SeTBT 3,5,7 (see Scheme 1 for the chemical formula) was co-condensed with a small amount of the amphiphilic polymer carboxyl-functionalized polystyrene- g -poly(ethylene oxide) (PS-PEG-COOH). During nanoparticle formation, the hydrophobic polystyrene backbone of PS-PEG-COOH were most likely embedded inside the Pdots, while the hydrophilic PEG chains and functional carboxyl groups extended out into the aqueous environment.…”

“…37–39 Significant advancements in biological applications of Pdots have been made recently in biological/chemical sensors, fluorescence imaging, and gene and drug delivery. 38,40–46 To date, however, the application of Pdots for ROS sensing remains largely unexplored. Compared with other fluorescent probes, a probe based on Pdots could offer increased imaging contrast accuracy and high stability for long-term observations.…”

Recent Advances in the Development of Highly Luminescent Semiconducting Polymer Dots and Nanoparticles for Biological Imaging and Medicine

“…As an example, we recently described an approach to optically sort cells that used photoswitchable Pdots as cell paint. 41 …”

Recent Advances of Activatable Molecular Probes Based on Semiconducting Polymer Nanoparticles in Sensing and Imaging

“…Facile PEGylation (where PEG is polyethylene glycol) generally endows SPNs with good biodistribution, allowing them to act as whole‐body imaging agents to detect the target of interest in living animals after systemic administration 27. With all these advantages, SPNs have been widely used in cells imaging,[[qv: 27a,d]] activated cell sorting,28 sensing of chemical mediators,[[qv: 17b,c]] tumor imaging[[qv: 27e,29]] hemodynamic imaging,[[qv: 27g]] and optogenetics 30…”