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

Abstract

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 μm and 13.6 μm, respectively. Subwavelength gold and polystyrene beads were localized with isotropic precision below 10 nm in the axial range of 3.5 μm and the axial precision reduced to 30 nm in the extended range of 13.6 μm. In the fluorescence imaging, the localization with isotropic precision below 15 nm was demonstrated in the range of 2.5 μm, whereas in the range of 8.3 μm, 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 μm3. Applicability of the NVM was tested by fluorescence imaging of LW13K2 cells and localization of cellular proteins.

Roadmap on Recent Progress in FINCH Technology

“…Here, we present concepts combining the imaging potential of coherent optical vortices with the advantages of FINCH experiments conducted using incoherent light. Our efforts resulted in optical and digital vortex FINCH modalities, which we demonstrate in their use for isotropic and anisotropic edge-contrast enhancement of 3D holographic images [38] and for super-localization of point-like emitters [89].…”

“…By determining the lateral position of the DH PSF and the angular rotation of its lobes, the point object is accurately localized in 3D space. A flexible non-diffractive vortex microscope was designed for 3D depth-enhanced super-localization of dielectric, metal, and fluorescent nanoparticles by implementing the DH PSF into FINCH imaging [89]. Subwavelength gold and polystyrene beads were localized with isotropic precision below 10 nm in the axial range of 3.5 µm and the axial precision reduced to 30 nm in the extended range of 13.6 µm (Figure 18).…”

Optical topography of rough surfaces using vortex localization of fluorescent markers

“…To maximize the dynamic range of the topographic measurement, we deployed the DH PSF created by non-diffracting vortex beams. This DH PSF previously succeeded in the structured illumination topography of smooth surfaces [18] and the localization of dielectric, metal, and fluorescent nanoparticles in the axial range significantly exceeding the depth of focus [19].…”

“…Higher rotation sensitivity can also be achieved by increasing the magnification of the imaging path. This results in a shortening of the axial measuring range and a reduced field of view due to the limited size of the CCD [18,19]. Hence, the system is designed to find a trade-off between accuracy, axial measuring range, and field of view.…”

“…The parameters of our setup were chosen adopting the theoretical model in Refs. [18,19]. The system allows the measurement with a rotation rate 12 deg/µm, an axial range z = 16 µm, and a field of view 380 × 380 µm 2 (limited by CCD area) when evaluated in the object space.…”