Development and Characterization of a Quantum Dot (QD)-based Photoresponsive Fluorescence Probe for Multicolor Super-Resolution Imaging

Abstract

Probe-based super-resolution imaging technique is becoming a rising tool for visualizing unknown sub-cellular life phenomenon, because of their overcoming Abbe diffraction limit. A number of researchers have jumped into the new super-resolution bio-imaging field carrying on their unique photoactivable fluorescent probes, however, there are some problems being pointed out such as low brightness and lack of photocontrollability of organic-based fluorescent probes. Here, I used fluorescent semiconductor nanocrystals, quantum dots (QDs), surface-conjugated with photolabile molecules, crystal violets (CVs). Non-radiative recombination of excitons in QD was accelerated by the CV molecules near the surface of QD, as a result, the photoluminescence (PL) of QD was quenched. The QD-CV conjugates were dispersed in phosphate buffered saline (PBS, pH 7.4), which is commonly referred to as a simulated human body fluid. The solution of QD-CV conjugates responds to visible light, showing their luminescent modulation from quenched state to photoactivated state and subsequent photobleached state (PLon -> PLoff -> PLon). In the process of the photoactivation by irradiation of visible light, the PL of QD-CV conjugates recovers significantly; the irradiated QD-CV conjugates shows the PL enhancement with > 1000 % compared with that of non-irradiated QD-CV conjugates. At this point, I analyzed the illuminated QD-CV conjugates using 1H NMR spectroscopy and fast atom bombardment (FAB)-mass spectrometer, and confirmed the presence of 4,4’-bis(dimethylamino)benzophenone (BDBP) as a byproduct of photodegradation of CV. This indicates CV molecules degrade to benzophenone derivative via the electron acceptance from photoexited QDs, and it is the very the reason that QD-CV conjugates turn on their PL in the irradiation condition. Meanwhile, PL decrease again down to the PL level of non-irradiated QD-CV conjugates after certain irradiation time. Photoexcited QD-CV conjugates were disclosed to accelerate the production of hydroxyl radicals which are one of the reactive oxygen species, and furthermore the hydroxyl radical bleach out the luminescent property of QDs. Time-resolved measurements of the fluorescence decay reveal the conjugation of CV accelerates non-radiative recombination of carriers in exited QDs, and multi-exponential kinetics and an average lifetime that lengthens during the illumination period and shortens when QDs are returned to darkness. A shoulder in the fluorescence correlation spectroscopy (FCS) curve of QD-CV conjugates appears at microsecond-correlation times, which means that blinking dynamics changes from the power law to increased population of transitory on/off states with microsecond-dwell time. Therefore, changes in FCS curves as a function of CV amount could reflect changes in blinking parameters, as a result of carrier-trapping mechanism. These results inform us that when an electron transfers to LUMO of CV and a hole remains in the valence band of the QD, and the dwell time of the charge separation is in microsecond time scale. In addition, FCS measurements show the quenching process of QD-CV conjugates can be believed as the turning the bright QDs into the “completely” dark QDs by QD-nearing CV molecules. I demonstrate functional patterning of optically active QD-CV conjugate films, which combines some advantages of high-throughput as well as and simplicity of photolithography. Importantly, the same technique was used to produce not only monochrome but also multicolor images. It also was confirmed that QD-CV conjugates remain their unique photoresposive luminescent modulation in a biomodel. QD-CV conjugates incubated in HeLa cells exhibited their luminescent recovery and subsequent bleaching in the irradiation of visible light, where PL dynamic range, defined as an intensity ratio of bright state to dark state, reaches to ~10. Furthermore, under single particle fluorescence measurement based on total internal reflection (TIRF) microscope, I observed that PL of small aggregates composed of two QD-CV conjugates showed stochastic photoactivation and photobleaching. The two QD-CV conjugates were localized via the two-dimensional Gaussian function fitting process on the individual CCD frames, and the resultant distance between the QD-CV conjugates is 46.6 nm. The same approach could be extended to reconstruct super-resolution image using simultaneously two color imaging system. As a result, the two green- and two red-emitting QD-CV conjugates in diffraction-limited area were resolved as a 147 nm distance through the reconstruction process.