3D sub-diffraction imaging in a conventional confocal configuration by exploiting super-linear emitters

Abstract

Sub-diffraction microscopy enables bio-imaging with unprecedented clarity. However, most super-resolution methods require complex, costly purpose-built systems, involve image post-processing and struggle with sub-diffraction imaging in 3D. Here, we realize a conceptually different super-resolution approach which circumvents these limitations and enables 3D sub-diffraction imaging on conventional confocal microscopes. We refer to it as super-linear excitation-emission (SEE) microscopy, as it relies on markers with super-linear dependence of the emission on the excitation power. Super-linear markers proposed here are upconversion nanoparticles of NaYF 4 , doped with 20% Yb and unconventionally high 8% Tm, which are conveniently excited in the near-infrared biological window. We develop a computational framework calculating the 3D resolution for any viable scanning beam shape and excitation-emission probe profile. Imaging of colominic acid-coated upconversion nanoparticles endocytosed by neuronal cells, at resolutions twice better than the diffraction limit both in lateral and axial directions, illustrates the applicability of SEE microscopy for sub-cellular biology.

Controlling the non-linear emission of upconversion nanoparticles to enhance super-resolution imaging performance

“…The ability to resolve single UCNP probes in biological samples is important since this allows binding of individual nanoparticles to target structures to be visualised and quantified. 14,17 Despite the unique advantages of UCNPs, their relatively moderate luminescence brightness represents a major challenge. Applications in STED nanoscopy in particular require a critical balance between high excitation laser intensities, which are potentially harmful for biological samples, and excessive acquisition or processing times.…”

Lanthanide-Doped Upconversion Nanoparticles for Super-Resolution Microscopy

“…(D) Non-linear photoresponses of NaYF 4 :Yb,Tm UCNPs at 455 nm under 980-nm excitation (top) and comparative imaging of two adjacent UCNPs under 980-nm excitation with different power densities (bottom). Reproduced with permission from Denkova et al ( 2019 ). Copyright 2019 Springer Nature Publishing Group.…”

“…The four-photon 455-nm emission of Tm 3+ is a good paradigm for achieving high-resolution imaging. Denkova (Denkova et al, 2019 ) and Zvyagin (Kostyuk et al, 2019 ) et al realized a ca . 200-nm resolution in heavily doped NaYF 4 :Yb,Tm UCNPs under 980-nm excitation ( Figure 2D ).…”

High resolution imaging with anomalous saturated excitation

“…Meanwhile, the nonlinear optical response of the fluorescence is also explored to improve the spatial resolution of the microscopy [15][16][17][18][19][20]. The key is to increase the high spatial frequency component of the image, and suppress the low spatial frequency component.…”

“…The nonlinearity of the fluorescence determines the resolution and signal-to-noise ratio of the images. Therefore, emitters with a high nonlinearity of the fluorescence emission are required for the high resolution imaging [16,24,25]. Combined with STED microscopy, the nonlinear fluorescence emission has also been utilized for the super-resolution imaging with upconversion nanoparticles [26][27][28].…”