In the fields of bioimaging, photonics, and quantum science, it is equally crucial to combine high brightness with a nanoscale size in short-wave infrared (SWIR) emitters. However, such nanoemitters are currently lacking. Here, we report that when functionalized with luminescent color centers, ultrashort carbon nanotubes with a length much shorter than 100 nm are surprisingly bright in the near-infrared second-biological window (NIR-II) of the SWIR domain. We discuss the origin of this exceptional brightness based on the uncontrollable presence of quenching defects in dispersed carbon nanotubes. We further investigate the nonlinear photoluminescence behavior of color center-functionalized carbon nanotubes in response to varying excitation conditions, spanning from ensemble measurements to single-nanotube experiments. We discuss how this behavior influences the determination of their photoluminescence quantum yields, which can reach values as high as 20% for ultrashort ones detected at the single-nanotube level. Notably, the corresponding NIR-II brightness exceeds that of well-known visible emitters, including quantum dots. After rendering them biocompatible, we demonstrate point-spread function engineering and high-resolution, 3-dimensional single-particle tracking using these bright ultrashort carbon nanotubes allowing nanoscale imaging in the NIR-II window within thick brain tissue.

Single-molecule localization microscopy has proved very promising to unravel the dynamics and molecular architecture of thin biological samples down to the nanoscale. However, achieving meaningful results in complex, thick biological tissues requires shifting the observation wavelengths to the shortwave-infrared (SWIR) region, where biological tissues are most transparent. In consequence, nanomaterials with optical activity in the SWIR exhibiting brightness and photostability suitable for detection at the single-molecule level are needed. Currently mainly single-walled carbon nanotubes (SWCNTs) satisfy this, but are inherently 1D objects. Here we present 0D ultra-small gold nanoclusters (AuNCs, <3nm) and ~25 nm AuNC-loaded-polymeric particles that can be detected at the single-particle level in the SWIR. Thanks to their high brightness and excellent photostability, we show that the dynamics of the spherical polymeric particles can be followed at the single-particle level in solution at video rates for minutes. Analysis of the mean square displacement confirms the diameter of the particles in aqueous media, and enables us to compare their brightness with that of biocompatible SWCNTs. This extends the library of SWIR emitting nanomaterials to 0D nano-objects of variable size for single-molecule localization microscopy in the second biological window, opening unprecedented possibilities for mapping structure and dynamics of complex biological systems.

Important applications of single-particle tracking (SPT) aim at deciphering the diffusion properties of single fluorescent nanoparticles immersed in heterogeneous environments, such as multi-cellular biological tissues. To maximize the particle localization precision in such complex environments, high numerical aperture objectives are often required, which intrinsically restrict depth-of-focus (DOF) to less than a micrometer and impedes recording long trajectories when particles escape the plane of focus. In this work, we show that a simple binary phase mask can work with the spherical aberration inevitably induced by thick sample inhomogeneities, to extend the DOF of a single-molecule fluorescence microscope over more than 4 μm. The effect of point-spread-function (PSF) engineering over spherical aberration regularizes inhomogeneities of the PSF along the optical axis by restricting it to a narrow distribution. This allows the use of a single fitting function (i.e. Gaussian function) to localize single emitters over the whole extended DOF. Application of this simple approach on diffusing nanoparticles demonstrate that SPT trajectories can be recorded on significantly longer times.

Measuring carbon nanotube diffusion is complex in 3D liquid environments. Single molecule fluorescence microscopy commonly provides nanotube trajectories in the 2D imaging plane with nanometer precisions but assessing the third dimension is more challenging task. To this aim, We will present two strategies based on point-spread function (PSF) engineering [1] or self-interfering PSF [2]. Because nanotubes are not spherical objects, 3D angular diffusion of the nanotubes shall also be considered. We will show that using a high-frame rate imaging (kHz) of nanotube movements, the autocorrelation time of nanotube fluorescence intensity can be computed in order to measure the rotational diffusion coefficient of the nanotubes. This further allows to estimate the length of the nanotubes either from the rotational diffusion coefficients alone, or by combining translational and rotational diffusion coefficients which has the advantage to avoid the requirement of knowing the solution viscosity or the SWCNT hydrodynamic diameter [3]. References [1] Gresil, Lee, et al. In preparation [2] Caceido, Lee, et al. In preparation [3] Lee & Cognet J. Appl. Phys. 128 (2020) 224301