Recently, we realized a simple technique, which spontaneously bypasses the diffraction limit in a conventional confocal microscope by exploiting super-linear effects in nanoparticle bio-markers: super-linear excitation-emission (SEE) microscopy. Here, we present a theoretical framework and its practical implementation for optimizing and expanding this technique. We accurately predict the expected 3D super-resolution by accounting for all crucial parameters affecting the resolution: the empirically measured/modelled excitation-emission curve, the filling factor of the microscope objective back pupil, the polarization and the pinhole setting. The presented theoretical framework is a practical tool, which enables end-users to augment their own confocal microscopes with super-resolution capabilities.
We achieve spontaneous 3D super-resolution on a standard confocal microscope by exploiting bio-friendly fluorescent markers with super-linear excitation-emission dependence (upconversion nanoparticles of NaYF4: Yb, Tm). We refer to this approach as upconversion super-linear excitation-emission (uSEE) microscopy. To demonstrate the applicability of the method for biological applications, we image sugar-coated upconversion nanoparticles in neuronal cells and we achieve resolution twice better than the diffraction limit both in lateral and axial directions. We envision that due to the application simplicity of the developed methodological toolbox, uSEE microscopy can be widely incorporated as an everyday super-resolution method in biological laboratories.
Lanthanide-doped upconversion nanoparticles (UCNPs) are capable of converting near-infrared (NIR) excitation to visible and ultraviolet emission via stepwise multiphoton processes. They offer unique advantages (e.g. sharp emission bands, superb photostability and long lifetimes1) compared to conventional dyes and quantum dots, enabling advanced detection and imaging for biologically important molecules. However, their application to live cells and small animals has been practically limited due to the potential damage under high-power laser illumination, as well as concerns for life scientists who do not necessary possess sufficient knowledge and experience in laser safety. Therefore, we have been exploring new strategies to develop UCNPs that are capable of excitation by incoherent sources such as light-emitting diodes (LEDs), in particular via increased number of sensitizer ions and coating of inert shells.
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