Engineering bright sub-10-nm upconverting nanocrystals for single-molecule imaging

Abstract

Imaging at the single-molecule level reveals heterogeneities that are lost in ensemble imaging experiments, but an ongoing challenge is the development of luminescent probes with the photostability, brightness and continuous emission necessary for single-molecule microscopy. Lanthanide-doped upconverting nanoparticles overcome problems of photostability and continuous emission and their upconverted emission can be excited with near-infrared light at powers orders of magnitude lower than those required for conventional multiphoton probes. However, the brightness of upconverting nanoparticles has been limited by open questions about energy transfer and relaxation within individual nanocrystals and unavoidable tradeoffs between brightness and size. Here, we develop upconverting nanoparticles under 10 nm in diameter that are over an order of magnitude brighter under single-particle imaging conditions than existing compositions, allowing us to visualize single upconverting nanoparticles as small (d = 4.8 nm) as fluorescent proteins. We use advanced single-particle characterization and theoretical modelling to find that surface effects become critical at diameters under 20 nm and that the fluences used in single-molecule imaging change the dominant determinants of nanocrystal brightness. These results demonstrate that factors known to increase brightness in bulk experiments lose importance at higher excitation powers and that, paradoxically, the brightest probes under single-molecule excitation are barely luminescent at the ensemble level.

Lanthanide upconversion luminescence at the nanoscale: fundamentals and optical properties

“…For example, (a) optimizing the types and concentrations of Ln 3+ ions can result in brighter particles, but equally important is (b) the composition and geometry of the host matrix: intuitively a high Ln 3+ concentration would yield a higher UCL brightness, however, it was shown early on that high concentrations result in quenching of UCL 10 . This can be overcome by (c) separating the ions in core-shell structures 51 , (d) clustering of sensitizers 52 , or (e ) using high excitation power density schemes 23,25 . Finally, (f) external materials can be used to enhance the upconversion brightness, for example using metal substrates to induce surface plasmon resonance 53 .…”

“…The rate equation approach centres on population behaviour and, although simplified, it enables quantitative predictions, as long as the required multiple parameters are known with sufficient accuracy. In the rate equations approach 23,25,46,71,100 , individual energy transfers and other processes are represented by their population averages. This is a fairly coarse approximation for upconversion which generally requires a more nuanced and detailed understanding of random walks of excitation energy through the entire sensitizer-activator system.…”

“…The process of UCL is mediated by real electronic states, while in multiphoton fluorescence and second (third, etc) harmonic generation, such real intermediate electronic states do not take part. Due to this difference, the two-photon fluorescence and second harmonic generation based on simultaneous interaction of two or more photons require 5-10 orders of magnitude higher excitation powers but yield more than 5 orders of magnitude lower quantum efficiency in comparison to UCL 20,[23][24][25] .…”

Glass and Process Development for the Next Generation of Optical Fibers: A Review

“…The ever-advancing synthetic techniques of NCs using a wide range of techniques such as wet chemistry [72][73][74], laser ablation [75], ball milling [58,59] and detonation [60] have shown exceptional control over emitting center concentration, crystallite phase, size, shape, composition and nanostructure. The technique, which consists of the synthesis of the NCs and then of their addition in the glass, gives a clear advantage as compared to the glass-ceramic technique for the synthesis of hybrid material, as with this novel fabrication technique, one can control the composition and nanostructure of the as-prepared NCs and glasses, overcoming the limitation of the glass ceramics method.…”

Deposited BSA/IgG Conjugated Up-Conversion Nanoparticles by the Matrix-Assisted Pulsed Laser Evaporation (MAPLE)

“…For past fifty years, the researchers made enormous progress on developing the application of up-conversion nanoparticles, especially in the bioapplication field. The no-linear property of up-conversion nanoparticles could be applied in vitro cell and tissue imaging [8], fluorescence diffuse optical tomography [9] and photo-thermal agents for cancer cell treatment [10].…”