Materials Discovery One Standard Deviation from The Mean: Finding Avalanches in Nanoparticles

The first applications of luminescent nanocrystals to bioimaging were semiconductor quantum dots with optoelectronic properties that largely mirrored those of organics and proteins, but with substantially increased stability and brightness that have enabled single molecule and other challenging imaging applications. Building on this success, newer nanocrystals have been engineered with optical properties unlike anything found in traditional probes, including perfect photostability,¹ anti-Stokes emission a billion-fold more efficient than 2-photon excitation,² and most recently, photon avalanches hosted within nanostructures.³ Avalanches are steeply nonlinear events in which outsized responses arise from a series of minute inputs. With light, photon avalanching (PA) had been observed only in bulk materials and aggregates, often at cryogenic temperatures, preventing its application to bioimaging. In two recent studies,^3,4 we describe the engineering and imaging of avalanching nanoparticles (ANPs), which are ~25-nm Tm³⁺-doped NaYF₄upconverting nanoparticles that efficiently convert near infrared excitation to higher energy emission. Avalanches are steeply nonlinear events in which outsized responses arise from a series of minute inputs and, with light, photon avalanching had been observed only in bulk materials, often at cryogenic temperatures. The extreme nonlinearity of ANP emission enables sub-70 nm spatial resolution using only simple scanning confocal microscopy and before any computational analysis. Two-way NIR photoswitching of ANPs enables full optical control of photodarkening and photobrightening, and we find indefinite photoswitching of individual nanoparticles in ambient or aqueous conditions without measurable photodegradation. This enables unlimited photon collection for calculation of sub-Ångstrom localization accuracies, and we can distinguish individual ANPs within tightly packed clusters. For application of ANPs to live-cell imaging, we have developed synthetic chemistry-free methods for conjugating engineered antibodies to NP-surface SpyCatcher proteins,⁵ which bind and spontaneously form covalent isopeptide bonds with cognate SpyTag peptides. This enables controlled and irreversible attachment of antibodies to nanoparticle surfaces, for specific targeting of cell-surface receptors in quantitative live-cell study of their distribution, trafficking, and physiology.<br/>1. Wu, S. <i>et al.</i> Non-blinking and photostable upconverted luminescence from single lanthanide-doped nanocrystals. <i>Proc. Natl. Acad. Sci. U. S. A.</i> <b>106</b>, 10917–10921 (2009).<br/>2. Tian, B. <i>et al.</i> Low irradiance multiphoton imaging with alloyed lanthanide nanocrystals. <i>Nat. Commun.</i> <b>9</b>, 3082 (2018).<br/>3. Lee, C. <i>et al.</i> Giant nonlinear optical responses from photon-avalanching nanoparticles. <i>Nature</i> <b>589</b>, 230–235 (2021).<br/>4. Lee, C. <i>et al.</i> Indefinite and bidirectional near-infrared nanocrystal photoswitching. <i>Nature</i> <b>618</b>, 951–958 (2023).<br/>5. Pedroso, C. C. S. <i>et al.</i> Immunotargeting of nanocrystals by SpyCatcher conjugation of engineered antibodies. <i>ACS Nano</i> <b>15</b>, 18374–18384 (2021).