Standard optical microscopes are limited by diffraction, which limits the ability to differentiate features at the nanoscale level. Over the last 20 years, microscope technology has advanced to provide super-resolution capabilities. However, these techniques are expensive and require highly complex instrumentation.

The researchers set out to find a method for 3D sub-diffraction imaging on a standard confocal microscope without the need for setup modifications or image processing. The methodology developed in the paper is termed as superlinear excitation–emission (SEE) microscopy. If the imaging is realized with upconversion nanoparticles, we will refer to it as upconversion super-linear excitation–emission (uSEE) microscopy.

The lead authors found fluorescent markers called upconversion nanoparticles, or super-linear emitters, which when excited light emitted grows abruptly in a super-linear fashion. The researchers describe the effect as “whenever a laser beam scans over such a marker, it is only the most intense, central part of the beam that yields significant emission. As this occurs in a region smaller than the size of the beam itself, the imaging resolution is effectively improved. The stronger the super-linearity, the smaller the region of significant emission, and therefore the better the resolution.” The unconventional class of luminescent markers used by the researchers included nanoparticles of sodium yttrium fluoride (NaYF4), doped with 20% ytterbioum (Yb) and unconventionally high 8% thulium (Tm), which are conveniently excited in the near-infrared biological window.

In SEE methodology, the luminescent markers allow higher order multiphoton processes to be excited at the same wavelength. This increase of resolution is due to super-linearity allows sub-diffraction imaging. Moreover, the scientists demonstrated that highly doped particles reach superior ultimate resolution. They achieved resolution twice better than the diffraction limit both in axial and lateral directions. This capability can be easily utilized in laboratories already using super-linear emitters, by simply tuning the imaging conditions (excitation power) or by adjusting the super-linear emitter composition (emitter doping).

Also included in the paper is the computational framework calculating the 3D resolution for any viable scanning beam shape and excitation-emission probe profile. The will guide other researchers to adapt the methodology to their unique needs.

While the concept behind this method has been around for quite some time, it combines the latest research in the fields of biology, material science, optical engineering and physics. Lead author, Dr. Denitza Denkova, says that "Best of all, super-resolution can be achieved without setup modifications and image processing. Thus, this method has the potential to enter any biological lab, at practically no extra cost."

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