Flexible non-diffractive vortex microscope for three-dimensional depth-enhanced super-localization of dielectric, metal and fluorescent nanoparticles

• Published in: Journal of optics (2010) 2017-09, Vol.19 (10), p.105606
• Main Authors: Bouchal, Petr, Bouchal, Zden k
• Format: Article
• Language: English
• Subjects: non-diffractive beams; optical vortices; phase modulation; self-imaging effects
• ISSN: 2040-8978; 2040-8986
• DOI: 10.1088/2040-8986/aa87fb

In the past decade, probe-based super-resolution using temporally resolved localization of emitters became a groundbreaking imaging strategy in fluorescence microscopy. Here we demonstrate a non-diffractive vortex microscope (NVM), enabling three-dimensional super-resolution fluorescence imaging and localization and tracking of metal and dielectric nanoparticles. The NVM benefits from vortex non-diffractive beams (NBs) creating a double-helix point spread function that rotates under defocusing while maintaining its size and shape unchanged. Using intrinsic properties of the NBs, the dark-field localization of weakly scattering objects is achieved in a large axial range exceeding the depth of field of the microscope objective up to 23 times. The NVM was developed using an upright microscope Nikon Eclipse E600 operating with a spiral lithographic mask optimized using Fisher information and built into an add-on imaging module or microscope objective. In evaluation of the axial localization accuracy the root mean square error below 18 nm and 280 nm was verified over depth ranges of 3.5 m and 13.6 m, respectively. Subwavelength gold and polystyrene beads were localized with isotropic precision below 10 nm in the axial range of 3.5 m and the axial precision reduced to 30 nm in the extended range of 13.6 m. In the fluorescence imaging, the localization with isotropic precision below 15 nm was demonstrated in the range of 2.5 m, whereas in the range of 8.3 m, the precision of 15 nm laterally and 30-50 nm axially was achieved. The tracking of nanoparticles undergoing Brownian motion was demonstrated in the volume of 14 × 10 × 16 m3. Applicability of the NVM was tested by fluorescence imaging of LW13K2 cells and localization of cellular proteins.