αplan apochromat 100

Unless stated otherwise, all commercial reagents were used as received. Thin-layer chromatography (TLC) was performed on silica gel 60F₂₅₄ (Merck). ¹H NMR spectra were obtained on a Varian Mercury 400 spectrometer with residual non-deuterated solvents (CD₃OD: 3.31 ppm for ¹H) as the internal reference. ESI mass spectra were recorded using an Exactive (Thermo Scientific). Reversed-phase HPLC (RP-HPLC) was carried out on a Hitachi Chromaster system equipped with a diode array and YMC-Pack Triart C18 or ODS-A columns. All runs used linear gradients of acetonitrile (ACN) containing 0.1% trifluoroacetic acid (TFA) and 0.1% aqueous TFA. The images of confocal laser scanning microscopy (CLSM) were acquired by a FV1000 (Olympus) and a LSM800 (Carl Zeiss Microscopy). UPlanSApo 100× (1.40 numerical aperture, oil immersion, Olympus), UPlanSApo 4× (0.16 numerical aperture, Olympus), Plan-Apochromat 20× (0.8 numerical aperture, Carl Zeiss), and αPlan-Apochromat 100× (1.46 numerical aperture, Carl Zeiss) were used. The fluorescent intensity of the CLSM images was calculated by Fiji^{49 (link)}.

A continuous-wave laser centered at 445 nm (iBeam smart, Toptica) is collimated and focused onto the back focal plane of an oil-immersion microscope objective (α Plan-Apochromat ×100, NA 1.46, Zeiss). A coverglass is positioned at the focus of the microscope objective using a piezo positioner (Nano-LPQ, Mad City Labs). The iSCAT field is imaged using a scientific CMOS camera (MV1-D1024E-160-CL, Photonfocus).
TIRF illumination was done with a laser beam at 631 nm, which was directed into the iSCAT pathway via a dichroic mirror (D1, Chroma ZT647rdc-UF3) mounted on a translation stage and a second dichroic mirror (D2, Chroma T480spxxr-UF3). The fluorescence signal was collected via the same microscope objective that was used for the iSCAT measurements. D2 separated the fluorescence from the iSCAT path and transmitted it through D1 onto a CCD (charge-coupled device) camera (Hamamatsu Orca Flash). Here, we also used a band pass filter (ET700/75) in front of the camera (S1).