Nanobiotechnology Scanning Probe Microscopies Jan Přibyl CEITEC MU Kamenice 5/A35, CZ-62500 Brno pribyl@nanobio.cz Atomic Force Microscopy AFM AFM microscope basic scheme AFM microscope block scheme Tip and cantilever Cantilever and tip tl. 1 mm cantilever hrot Reflex layer (metal) 6 mm 2mm Cantilever holder hrot 100-200 um 50-100 um 5 um6 mm 5 um • Cantilever holder is quite universal • Cantilever and tip – a variety of various types Cantilevers Material properties – Stiffness Force Constant [N/m] Force const.[N/m] 10-130 1-10 0.1-1.0 0.005-0.1 Material cryst. silicon pol. silicon glass Si3N4 Res. f. [kHz] 200-500 100-200 15-100 1-20 Special applications – conductive, colloid, magnetic, tip less, … Cantilever field choose the one you like/need Cantilever characterization you may find on box AFM probes (micro)fabrication is quite complex Tip properties Shape – Curvature Radius R [nm] Supersharp Standard Special app. Tip less R R 1 nm 10 nm 100 nm NA Supersharp Standard Special app. Tip less Cantilever fabrication FIB (Focus Ion Beam) post-fabrication of AFM probes (tip) Plateau Tip Idealized force-distance curve describing a single approach-retract cycle of the AFM tip, which is continuously repeated during surface scanning. Victor Shahin et al. J Cell Sci 2005;118:2881-2889 ©2005 by The Company of Biologists Ltd (DFL deflection) Torsion forces (LF latheral forces) Cantilever bending – how to detect Contact with surface Change of cantilever properties (DFL/LF) is detected by laser beam DFL LF Curvature radius (R) effect SuperSharp tip = real image Standard tip = R ~ 5-10nm Blunt tip = affecting real shape and size Laser, photodiode a cantilever Laser + photodiode Detection of cantilever bending Upper side of cantilever - reflective Laser beam reflects to detector Detector = photodiode Laser beam movement – bending detection ovrch vzorku PZT skener (PiezoElectric Tube) PZT skener (PiezoElectric Tube) Laser beam movement – bending detection DFLAiming – 1st step of microscope setting LF Change of laser beam position – during scanning over the sample Automatic transformation to the 2D image (software) Aiming: initial setting of AFM microscope 1. Laser position close to the top-side + highest signal possible (relative intensity, check guide) Aim of aiming (2 steps): 1. Highest possible reflection of laser beam from cantilever 2. Center beam position on the detector Not in the center Screws to adjust (labeled as LASER) Aiming Aim of aiming (2 steps): 1. Highest possible reflection of laser beam from cantilever 2. Center beam position on the detector DFL = deflection LF = lateral force Position on detector Screws to adjust (labeled as PHOTODIODE) Automatic adjustment available JPK Force Robot head Bruker Icon/FastScan NTMDT Solver Next AFM modes of operation Contact mode • Measured parameter - cantilever bending (= deflection, DFL) • Deflection ~ tip sample force interaction • Hook`s law: F = - k * ΔhF = - k * Δh F – force k – force constant (stiffness) Δh – change of height (=deflection) Cantilever deflection (DFL ) SetPoint = basic value of tip-sample interaction DFLint DFL(contact) DFL0DFL0 no DFL = no contact SetPoint Semicontact mode (tapping mode, AC mode, oscillation mode, …) • Measured parameter amplitude of oscillation (= magnitude, MAG, …) • Measured as:• Measured as: - relative parameter, e.g. as MAG [nA] - absolute parameter – A [nm] A0 free amplitude Aint damped amplitude Relative to absolute amplitude calibration Amplitude is proportional: • Voltage of an oscillator • SetPoint MAG – height spectroscopy slope AMPreal = (dX/dY)*SetPoint [nm] Semicontact mode: Amplitude of oscillation ~ size of object SetPoint = damping of free oscillation amplitude (relative/absolute) A0 free amplitude Aint damped amplitude PZT Piezoelectric tubes Piezoelectric tubes PZT Piezoelectrodes • Hollow ceramic tubes • Metal covered in selected parts • Voltage application change of size Notes + cautionsNotes + cautions • Fragile • High voltage applied PZT – construction approaches of AFM Scanning by probe construction Scanning by sample construction •x,y,z axes movement byconstruction •x,y,z axes movement by head •Oscillator in head •Range x,y 100-150 um •Range z 10-15 um •x,y,z axes movement by sample •Oscillator in head •Range x,y 1-10 um •Range z 1-3 um •Low noise Piezo-tubes PZT in software 1. Approaching to surface - first approach with step motors - after wards Z-piezos Z-piezo position Jump to surface by Z-piezos Z-piezo position 2. FBloop (FeedBack Loop) - feed back driving of cantilever deflection (=constant) over the surface - ON/OFF of Fbloop leads to tip-sample interaction ON/OFF: Piezo-tubes PZT in software OFF OFF PZT ON ZAPNUTO ON ZAP vzorek OFF ZAP 3. SCANNING OF SAMPLE: parameters driven by PZT Velocity of scanning (typically 0.35 – 0.7 Hz) Resolution (pixels): typically 128x128; 256x256,Resolution (pixels): typically 128x128; 256x256, 512x512 a 1024x1024 pix. Size of area (10 nm up 150 um) Step of scanning PZT electrodes Detailed view PZT: voltage-extension dependency non-linear Native (raw) AFM data are shifted. Removed e.g. by polynomal regression of data. Oscillator Oscillator(PV,Oscillator(PV, PiezoVibrations) always located in the head Oscillator setting Amplitude = voltage [V] Gain = Lock-In amplifier DFL x = numerical amplifier Other components ClosedLoop (X, Y - axis) Capacitance sensors ClosedLoop • Temperature drift correction • PZT non-linearity correction • Increasing noise Step motors •Help to drive sample in the appropriate area of PZT action •Driven automatically / manually•Driven automatically / manually