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

Denitza Denkova,Lindsay M Parker,Martin Ploschner,Minakshi Das,James A Piper,Xianlin Zheng,Nicolle H Packer,Antony Orth,Yiqing Lu

doi:10.1038/s41467-019-11603-0

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 NaYF4, 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.

Highlights

The resolution of conventional lens-based microscopes is limited by diffraction to about λ/2 in lateral and λ in the axial direction[1]
Each technique has its own set of tradeoffs, but typically suffers from one or more of the following drawbacks: (i) requires purpose-built optical system and software, usually with high cost and complexity (STED, PALM, STORM, SIM); (ii) necessitates acquisition of many images and their subsequent post-processing, which is time consuming and prone to imaging artifacts (PALM, STORM, SIM); (iii) needs high laser fluence, often at visible wavelengths, which is potentially damaging to biological samples, results in rapid bleaching of the fluorophores and obstructs long-time imaging and tracking[13]; (iv) lacks biomarkers working in the less photo-toxic, biologically convenient near-infrared window[14,15]
We achieve sub-diffraction imaging via super-linear excitation-emission (SEE) microscopy by scanning a super-linear upconversion nanoparticles (UCNPs) in a conventional confocal configuration (Fig. 1a)

Summary

Introduction

The resolution of conventional lens-based microscopes is limited by diffraction to about λ/2 in lateral (in-plane) and λ in the axial (out-of-plane) direction[1]. The growing demand from the biological sciences to optically image nanoscale features has led to the development of numerous techniques, which push the resolving power of optical microscopes beyond this diffraction barrier These super-resolution methods have helped answer biological questions by visualization of nano-sized structures and interactions which were otherwise not experimentally resolvable[2,3,4]. From the library of luminescent nanoparticles, nanodiamonds work only in the visible spectrum, and require complex conditions to enter the superlinear regime of operation, involving several different lasers and specific illumination sequence[24] This has so far prevented the use of super-linear nanodiamonds in a biological setting. The studied quantum dots work only in the visible range and require a tri-exciton process, which is excited at 2 orders of magnitude higher power than the standard diffraction-limited mono-exciton emission

Methods

Results

Conclusion