Multi-luminescent up-converting nanohybrids with controlled blinking for super-resolution imaging and single particle tracking – BLINK

An innovative approach to the synthesis of ultrasmall UCNP (US-UCNP) is currently developed involving the use of dielectric heating tanks to a microwave oven. The photochromic dye chosen (spiropyran SP) belongs to the negative thermal photochromic dye family, being colored (shutter ON) in its dark state and colorless (shutter OFF). While the ON-to-OFF process requires light, the reverse reaction is spontaneous, restoring the OFF state. The polymer chosen is able to bind electrostatically to the UCNP’s surface. The understanding of the energy flows inside the UCNP and toward the dye is an important task involving time-resolved measurements. Finally the observation of isolated nanohybrids requires a dedicated microscope with a NIR illumination source that has to be constructed. An image analysis software with a high density of weakly fluctuating luminescent sources had to be developed as well.

At the present stage, the microwave based protocol to prepare US-UCNP is validated, as well as the choice of the photochromic dye. In particular, our approach allowed us to establish the UCNP minimal size to observe the upconversion phenomenon. Observation set-ups are also operating, and the data analysis software is available.

Next steps are now to observe the luminescent nanoprobes in a biological context. Thus conditions optimized in our preliminary abiotic experiments will be transposed to cultured cells. Once fixed, wide field observation of these cells will be undertaken under a NIR excitation. In the field of UCNP preparation, our industrial partner will implement the DLS and Microwave oven combination. DLS data recorded throughout all synthesis phases are already available and will be used to calibrate the instrument.

Several conferences in International Symposia have been already given ; 3 publications on the image analysis methods are available.

The interest in new non invasive, far-field super-resolution microscopies breaking the diffraction limit is now a hot topic, crowned by the 2014 Nobel Prize. Expected fallout spans from fundamental biology to medicine. Among the super-resolution microscopies, the two main classes of single molecule localization microscopy (PALM, STORM, GSDIM...) and stochastic optical fluctuation microscopy (SOFI, pcSOFI, 3B analysis) use the blinking of a fluorescent probe to break the diffraction limit. This blinking can be either intrinsic (as in the case of quantum dots) or results from the coupling of the fluorescent probe with a molecular switch (photochrom).
Lanthanides-doped inorganic nanoparticles present the unique feature of emitting a discrete spectrum up to the blue region of the visible spectrum when excited in the near infrared (NIR). Because biological samples have a limited absorption in the NIR, these so-called Up-Converting NanoParticles (UCNPs) are attractive candidates for biological applications. In particular, since the higher emission energies can operate the photochrom switch, the UCNPs which are naturally not blinking, can blink when the UCNPs are coupled to a molecular switch. Thus the non blinking emission of UCNPs (in the NIR range) can be used in a conventional way for nanoparticle tracking, whereas the blinking emission (in the visible range) will be used for super-resolution. The specific challenge of the present BLINK project is the control of the blinking and lifetime of the high energy emission of UCNPs via grafted molecular photoswitch. These hybrids nanoparticles will be used simultaneously for nanoparticle tracking and super-resolution fluorescence microscopy.
Two situations will be explored:
a: the blinking is obtained by activating the photochrom switch via dye specific irradiations.
b: the switch of the photochrom is directly sensitized by the nanoparticle itself, via the high energy emission. Spontaneous reverse switch occurs via thermal relaxation.
The blinking nanohybrids consist in two parts: the UCNPs and the polymer supported photochrom. The polymer supported photochrom will be synthesized following a well established protocol, by grafting negative T photochrom (spiropyran) on a polymer able to coat and stabilize the UCNPs. Concerning the UCNPs, we will first used commercially available, then to get a better control on fluorescence lifetime and thus on the blinking dynamics, we will synthesized tailored UCNPs. To this aim, we will use a micro-wave synthesis, and develop a technique of monitoring by dynamic light scattering, in partnership with the company Anton Paar France. The optimization of fluorescence and lifetime will guide the selection of the UCNPs. The behavior of the individual UCNPs will be assayed on single particle using confocal and broad field fluorescence microscopies in order to extract the photophysical properties (lifetime, brightness) and the blinking dynamics. Eventually, the nanohybrids will be tested for super-resolution fluorescence microscopy on HeLa cells.
The main expected outcomes of this project, beyond the understanding of the UCNP-photochrom interaction, are (i) the design of new biocompatible “blinking” UCNPs nanoparticles for super-resolution fluorescence microscopy coupled to tracking; and (ii) and the development of a new bench-top instrument for the synthesis of nanoparticles, a combination of a monomode microwave oven and remote DLS probe.