(Invited) The Evolution of Inorganic Nanocrystals for Bioimaging

The first applications of luminescent nanocrystals to bioimaging were semiconductor quantum dots with optoelectronic properties that largely mirror 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,1,2 anti-Stokes emission a billion-fold more efficient than 2-photon excitation,3 and most recently, photon avalanches hosted within nanostructures.4 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. We recently reported the realization of PA at room temperature in sub-30 nm Tm3+-doped NaYF4 upconverting nanoparticles (UCNPs) and demonstrated their use in high-resolution imaging at wavelengths that fall within NIR spectral windows of maximal biological transparency. Avalanching nanoparticles (ANPs) can be pumped by either continuous-wave or pulsed lasers and exhibit all of the defining features of PA: clear excitation power thresholds, exceptionally long rise time at threshold, and a dominant excited-state absorption that is >10,000 times larger than ground-state absorption. Beyond the avalanching threshold, ANP emission scales with up to the 31st power of pump intensity, an extreme nonlinearity caused by the induced positive optical feedback within each nanocrystal. This enables sub-70 nm spatial resolution using only simple scanning confocal microscopy and before any computational data analysis. NaYF4 ANPs with 8-20% Tm3+content can be excited at either 1064 or 1450 nm, with avalanching emission at 800 nm. Pairing the steep nonlinearity of ANPs with existing superresolution techniques and computational methods allows for imaging with higher resolution and at ca. 100-fold lower excitation intensities than is possible with other probes. 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. Wu, S. et al. Non-blinking and photostable upconverted luminescence from single lanthanide-doped nanocrystals. Proc. Natl. Acad. Sci. 106, 10917–10921 (2009). Fernandez-Bravo, A. et al. Continuous-wave upconverting nanoparticle microlasers. Nat. Nanotechnol. 13, 572–577 (2018). Tian, B. et al. Low irradiance multiphoton imaging with alloyed lanthanide nanocrystals. Nat. Commun. 9, 3082 (2018). Lee, C. et al. Giant nonlinear optical responses from photon-avalanching nanoparticles. Nature 589, 230–235 (2021). Pedroso, C. C. S. et al. Immunotargeting of nanocrystals by SpyCatcher conjugation of engineered antibodies. ACS Nano 15, 18374–18384 (2021). Figure 1