Search for: All records

Abstract The photoluminescence (PL) saturation of CdSe/ZnS core/shell inorganic semiconductor quantum dots (QDs) and its utility as a probe for saturated excitation (SAX) microscopy are reported. Under saturating excitation power densities, the PL signal was demodulated and recorded at harmonics of the fundamental frequency. For commercially available Qdot® 655 ITK™ QDs, the power density required to achieve saturation was dependent upon the local environment of the QDs. For QDs deposited and dried on a glass substrate, the excitation power density required for PL saturation was less than 1 kW/cm². Compared to this, saturation of PL for QDs dispersed in water required an excitation power density greater than 200 kW/cm². This observation is manifested as a limitation in the imaging of hydrated samples, as demonstrated for HeLa cells labelled with biotinylated‐phalloidin followed by labelling with streptavidin‐coated QDs. As saturation affects the obtained spatial resolution in several imaging formats, including confocal imaging, the provided data will aid in obtaining the optimal spatial resolution when using QD probes to image biological samples. 

Abstract The synthesis and application of a photoactivatable boron‐alkylated BODIPY probe for localization‐based super‐resolution microscopy is reported. Photoactivation and excitation of the probe is achieved by a previously unknown boron‐photodealkylation reaction with a single low‐power visible laser and without requiring the addition of reducing agents or oxygen scavengers in the imaging buffer. These features lead to a versatile probe for localization‐based microscopy of biological systems. The probe can be easily linked to nucleophile‐containing molecules to target specific cellular organelles. By attaching paclitaxel to the photoactivatable BODIPY, in vitro and in vivo super‐resolution imaging of microtubules is demonstrated. This is the first example of single‐molecule localization‐based super‐resolution microscopy using a visible‐light‐activated BODIPY compound as a fluorescent probe. 

Abstract Developing improved fluorescent probes for imaging the endoplasmic reticulum (ER) is necessary for structure‐activity studies of this dynamic organelle. Two coumarin‐based compounds with sulfonamide side groups were synthesized and characterized asER‐targeting probes. Their selectivity to target theERin HeLa andGM07373 mammalian cells was shown with co‐localization experiments using commercially available probes that localize in theER, mitochondria, or lysozymes. The hydrophobicity of the coumarin‐based probes was comparable to known probes that partition into theERmembrane. Their cytotoxicity in mammalian cells was low withIC50 values that range from 205 to 252 μm. The fluorescent quantum yields of the coumarin‐based probes when excited with 400 nm light were 0.60, and they have a much narrower emission spectrum (from 435 to 525 nm in methanol) than that of the only commercially availableERprobe that is exited with 400 nm light (ER‐Tracker™ Blue‐WhiteDPX). Thus, the coumarin‐based probes are more useful for multicolor imaging with yellow and red emitting fluorophores. In addition to the above benefits,ERlabeling was achieved with the coumarin‐based probes in both live cells and fixed cells, revealing their versatility for a wide range of cellular imaging applications.