Nanoscale Localization Sampling Based on Nanoantenna Arrays for Super‐resolution Imaging of Fluorescent Monomers on Sliding Microtubules

Abstract

Sub-diffraction-limited imaging of fluorescent monomers on sliding microtubules in vitro by nanoscale localization sampling (NLS) is reported. NLS is based on periodic nanohole antenna arrays that create locally amplified electromagnetic hot spots through surface plasmon localization. The localized near-field hot spot temporally samples microtubular movement for enhanced spatial resolution. A fourfold improvement in spatial resolution compared to conventional wide-field microscopy is demonstrated. The resolution enhancement is achieved by imaging rhodamine-labeled microtubules that are sampled by the hot spots to provide sub-diffraction-limited images at 76 nm resolution in the direction of movement and 135 nm orthogonally. The intensity distribution produced by the NLS is measured to be broader than that of conventional imaging, which is consistent with the improvement of imaging resolution. Correlation studies between neighboring nanoantennas are also performed. This confirms the possibility of measuring microtubular transport dynamics. NLS can be useful for moving objects that have a high labeling density or for performing fluctuation spectroscopy in small volumes, and may allow "super-resolution on demand" by customizing nanoantenna structures for specific resolution needs.

Electromagnetic Near-Field Nanoantennas for Subdiffraction-Limited Surface Plasmon-Enhanced Light Microscopy

“…The ellipticity in this case was 2.1 (=110/53). The smallest spot size obtained with circular nanoantennas was 60 × 251 nm 2 (ellipticity = 4.2), which is roughly consistent with recent experimental results [36]. With rhombic nanoantennas, it was 52 × 129 nm 2 (ellipticity = 2.5).…”

“…For example, random nanoantenna patterns were used to create hot spots for cell imaging [33], [34]. Periodic arrays of nanopatterns were preferred for complete reconstruction of super-resolved images [35] and used to imaging microtubules that slide on motor proteins [36].…”

High frame rate multi-resonance imaging refractometry with distributed feedback dye laser sensor

“…30,33 So far, the spectrometer was used with a grating with 150 g mm 21 resulting in a spectral bandwidth of 0.17 nm. By using a grating with 1800 g mm 21 , the spectral bandwidth can be increased such that one spectrometer pixel in wavelength direction corresponds to 12 pm. However, this implies that only 11 laser peaks fit into the whole spectral range corresponding to the length of the spectrometer CCD.…”

“…Here, the spectrometer was used with a grating with 150 g mm 21 resulting in a spectral bandwidth of 0.17 nm corresponding to the length of one of the 1340 pixels in the wavelength direction of the 13403100 pixel CCD array. Accordingly, there are 100 pixels in the y-direction.…”

“…This imaging refractometry has e.g., been applied to detect nanoparticles, 4,5 proteins, [6][7][8] viruses [9][10][11] and cells [12][13][14][15][16] and has enabled imaging as well as multiplexed sensing. 10,17 In this context, label-free signal transduction is based on surface plasmon resonance, 9,12,13,17,18 plasmonic hole arrays, [19][20][21][22][23] photonic crystals 5,[14][15][16] and interferometry. 8,10,11 Most of these methods rely on detecting a resonance shift in the optical spectrum.…”