Atomic Force Microscopy (AFM) Facility

Bruker Atomic Force Microscope Nanowizard 5 (NW5)

The NW5 instrument performs tip-scanning imaging and single-point probing of objects in liquids and air with atomic lattice resolution (closed loop). NW5 measures height, topography, roughness, and mechanical properties (elasticity, adhesion, deformation, and adhesion work) of live cells, cell clusters, spheroids, organoids, tissue slices, and other materials.

In addition, the NW5 measures adhesion forces between cells, cell and tissues or biomaterials. It can inject and collect fL of liquids from cells and tissue simultaneously performing AFM imaging, or non-chemically transfer cells with FluidFM. NW5 can image macromolecules such as proteins, nucleic acids, and their complexes with a high resolution and follow their dynamics.

Mounted on a dedicated inverted fluorescence microscope (Nikon Ti Eclipse) allowing direct visual observation and control of the imaging and collecting light and fluorescence images using a broad range of microscopic techniques (TIRS, FRET, etc). The laser and detector alignment are fully motorized with the head sealed against elements which additionally allows for unattended long-term experiments.

Equipped with automated cantilever calibration. The NW5 Z axis is mechanically decoupled from an XY scanner. Its Z-noise is lower than 30 pm RMS. Vortis controller adds several high speed channels for advanced fluorescence microscopy signals capture and super speed, super resolution data acquisition.

The Top View optics allow for observation of semi and non-transparent samples. It includes a Petri Dish Heater and CO2 controller, 3-port small volume SmallCell™ ultra-small volume samples with perfusion, a probe side viewer for cell-cell adhesion visual control, AFM remote control hardware, and software.

The system is placed in a dedicated acoustic enclosure and mounted on an active vibration isolation table. Instrumental control, data acquisition, and analysis are performed using dedicated modules of SPM software. Additional software modules allow for overlay of AFM and optical images, JPK DirectTiling software module V7 – automated mapping of large sample areas, SmartMapping – scanning of large areas with automatic z adjustment for HybridStage, exploration of mechanical properties of soft and viscous materials and testing rheological properties with an extended range of modulation frequencies.

Technical capabilities of Nanowizard 5:

• Image topography of proteins, protein complexes, nucleic acids and their complexes with proteins with nm spatial resolution using the same setup applied for imaging cells (smooth transition between imaging biomolecules and tissues).
• Image spatiotemporal dynamics of macromolecules.
• Image topography, roughness, and mechanical properties of tissue sections, organoids, and spheroids.
• Measure strength of cell to cell and cell to tissue interactions (adhesion) and determine mechanisms of their separation even with very elastic or sticky materials.
• Probe side view capabilities for direct control of tip-object interactions
  Spatial positioning of single cells to study biology of their interactions with environment.
• Testing content of a single cell by aspiration of its cytoplasm for downstream applications (sequencing, proteomics, metabolomics) provided by FluidFM (Fluidic Force Microscopy) from Cytosurge®.
• Controlled injection of liquid content to a single cell (CRISPR, drugs, stains).
• Conduct all the above measurements under strictly controlled environmental conditions.

It automatically optimizes critical imaging parameters including setpoint, gains, scan rate and Z limit for imaging in liquid and air. Directly controlling the tip-sample imaging force down to at least 50 pN automatically compensates for a drift of the deflection signal. Switching between sample scanning and tip scanning (sample moving or probe moving during scanning) is performed seamlessly in the software without physically disturbing the sample.

Automation and optical overlay software allow for the selection of any custom combination of multiple AFM images, modulus maps, and user-defined force spectroscopy ramps of areas anywhere within the full sample travel range to be acquired in a batch fashion, fully automatically.  The 1MHz data transfer bandwidth allows capturing all force curves without compromising the imaging speed.

Available Instruments

1. Nanoscope IIIa (1997) departmental
2. Bioscope Catalyst (2013) BASiC
3. NanoWizard V (2023) BASiC –
   NIH grant S10OD034419
   Hosted in BASiC – STRF Rm. 253

Features

Standard Scanner:

• Scan Range 100x100x15µm
• Sample size 14cm dia x 1.8cm tall
• Speed 400 lines/sec

Hybrid Stage:

• Scan range 200x200x200µm
• Sample size 14 x 14 cm x no height limits

Image Modes

1. PeakForce-QI mode
2. Quantitative Imaging QI™
3. PeakForce™ Tapping
4. ScanAsyst
5. Nested scanner capabilities
6. Contact mode with lateral force microscopy (LFM)
7. Tapping™ mode with PhaseImaging
8. Static and dynamic force spectroscopy