Quanta 3d feg dual beam fib sem

A Pro-4 four point system (Lucas Labs, Gilroy, CA, USA) was used in resistivity measurements. The system was made up of a Keithley 2400, a Pro4, and a notebook with the Pro4 V1.2.4 software installed. The system uses the dual configuration test method of ASTM standard F84-99 to compensate for errors in probe spacing and errors caused by proximity to the edge of the conducting layer. A V/I measurement was taken and recorded and the subsequent resistivity was computed.
Scanning electron microscope (SEM) micrographs were obtained using a Quanta 3D FEG Dual Beam FIB/SEM and its xT Microscope Control Software (FEI, Hillsboro, OR, USA). Fluorescence studies were performed on samples using a DM RXA2 fluorescence microscope (Leica, Wetzlar, Germany) fitted with a 100× objective lens with a 1.4 N.A. Slidebook version 4.1 was used for image acquisition from a SensiCam QE camera (Cooke, Auburn Hills, MI, USA) attached to the microscope.
Optical images were obtained from a 40 CFL inverted microscope (Axiovert, Thornwood, NY, USA) fitted with a 20× objective with a 1.0 N.A. AxioVision 4.6.1.0 was used for image acquisition from an AxioCam MRc camera attached to the microscope. Percentage area covered by the NMBs on the glass slides were determined using the ImageJ 1.46r software (Wayne Rasband, National Institutes of Health, USA).

Grids imaged by light microscopy were mounted on a transfer shuttle designed for a cryo-loading system (Rigort et al, 2010 (link)) and loaded into a Quanta 3D FEG dual-beam FIB/SEM (FEI). Grids were sputtered with platinum (10 mA, 30 s) in a PP3000T loading system (Quorum) to reduce charging effects during electron imaging. Grids were loaded into the FIB chamber and coated with organometallic platinum as a protective layer for ion beam milling. Grids were imaged with the scanning electron beam operated at 5 kV/12 pA and ROI were identified via cryo-LM/SEM three-point correlation using MAPS 2.1 software. ROIs were thinned down at tilt angles of 18°–20° with the focused ion beam operated at 30 kV. The beam currents where set to 1 nA at ∼1 μm distance from the ROI, 500 pA at 750 nm, 300 pA at 400 nm, 100 pA at 250 nm, 50 pA at 100 nm, and 30 pA at 75 nm for polishing. Grids were sputtered once more with platinum (10 mA, 5 s) after milling to increase conductivity of the lamellae for VPP imaging.