Gold nanospheres

Imaging was performed using a custom-built upright 2P-STED microscope as previously described (see [10 (link),19 (link),23 (link)] for a complete description). In all the following, 2D-STED and z-STED refer to images acquired using pure donut or pure bottle beams, respectively. Gold bead samples were used to assess the donut or bottle beam quality, while optical resolution was assessed by imaging fluorescent beads immobilized on a glass coverslip. Samples were prepared with either gold beads (150 nm Gold nanospheres, Sigma-Aldrich) or fluorescent beads (yellow-green fluorescent beads, 170 nm in diameter, Invitrogen) placed on polylysine-coated #1 coverslips. Samples were left to dry overnight and then transferred to a microscope glass slide, where they were sealed with nail polish. In order to evaluate the effect of the coverslip tilt angle, samples were placed on a tiltable mechanical stage (TTR001/M, Thorlabs), enabling us to vary the tilt angle relative to the microscope objective axis in a precise and systematic way. STED PSFs were originally checked and optimized at a tilt angle of 0 ± 0.03°.

Samples were prepared
by spin coating 40 × 120 nm² gold nanorods (Nanoseedz,
Hong Kong) at 2000 rpm for 2 min onto glass coverslips. Before spin-coating
the glass coverslips were sonicated in methanol and UV/ozone-cleaned
for 90 min. After spin coating the samples were rinsed with methanol,
PBS and water and blown dry under N₂-flow.
To induce
gold nanospheres binding to the gold nanorods we used cysteine-cysteine
coupling.^{22 (link)} Cysteine binds covalently to
the particles via a gold–thiol interaction. Coupling of spheres
to rods was achieved at a pH of 2.3 at which cysteine is zwitterionic
and allows for nanosphere-nanorod coupling by electrostatic interactions.^{22 (link)} The immobilized gold nanorods were first incubated
in 100 μM l-cysteine (Sigma-Aldrich) in pH 2.3 Milli-Q
water. A solution containing 1.2 nM of 20 nm gold nanospheres (Sigma-Aldrich)
was then flown in and incubated for 30 min while a time-trace was
recorded. Afterward the sample was flushed with MQ water adjusted
to pH 2.3 to wash away unbound gold nanospheres and cysteine. On the
basis of the dimensions of two bound cysteine molecules an interparticle
spacing of ∼1 nm is assumed.^{22 (link)}