A brief introduction to the major techniques that our group uses to conduct research.

Atomic force microscopy (AFM)

Atomic force microscopy is a type of scanning probe microscopy (SPM), with very high resolution (on the order of fractions of a nanometer). The basic principle of AFM is the detection of inter-atomic interactions through a sharp probe (ideally a single atom) that is mounted on a cantilever.

Credit: https://upload.wikimedia.org/wikipedia/commons/e/e0/Atomic_Force_Microscope.ogv

AFM has three basic modes: topographic imaging, force measurement, and manipulation. These can be combined with advanced modes to characterize samples in ambient conditions or in buffers that mimic the natural chemical environment of the samples. This way material properties, including friction, electrical forces, capacitance, magnetic forces, conductivity, viscoelasticity, surface potential, and resistance, can be quantified using AFM.

A Bruker NanoScope V system equipped with MultiMode 8 AFM is available in the lab for high-resolution AFM imaging and quantitative nanomechanical property mapping. This system is named Hestia, for its steadfast and perpetual performance.

Fast-scanning AFM with photothermal excitation

Conventional AFM techniques have limited temporal resolution, and generally operate at scan rates between 1-10 lines/second (Hz). Meaning that a typical image of 512×512 pixels will take ~8.5 minutes to acquire at 1 Hz. Advancement in material science, piezo technology, and AFM probe manufacturing have made it possible to exceed the conventional limits and scan at faster rates. These commercial and academic advancements have been described and promoted using different terms, however fast scan or fast-scanning is the popular term used to collectively describe AFM imaging at scan rates up-to 400 Hz; which translates to an image of 512×512 pixels taking ~1.3 seconds to acquire. The significant speed increase allowed researchers to elucidate the dynamics of biological and non-biological systems that had been out of reach for other techniques.

A system built around a Bruker MultiMode 8 platform with custom controller and AFM head (with help from LBNI @ EPFL) is available for high resolution imaging at speeds up-to 100 Hz. This system is named ZagZag, after the late Karen Zagorski (1990-2023) whose ebullient character and unbridled enthusiasm for science is mirrored in this system.

High-speed AFM (video rate)

High-speed AFM, or video rate AFM, is typically AFM imaging at scan rates beyond 400 Hz and reaching upward 50 frames per second (fps) for specific cases. The temporal resolution has allowed researchers to directly visualize dynamic phenomena for biological processes (for cells, proteins, DNA, and others), synthetic polymer chains, detergents, reactions at solid-liquid interfaces (e.g. crystallization), electrochemical reactions, and others.

A Bruker JPK NanoWizard UltraSpeed 3 system capable of 1400 Hz scan rate is available in the lab for high-resolution and high-speed scanning under temperature-controlled environment. The system is sitting on top of an inverted microscope capable of super-resolution structured illumination microscopy (SIM). This system is named Huma, after the auspicious mythical bird that spends its entire life flying.

Single-molecule force spectroscopy (SMFS)

Mechanical interactions/forces govern fundamental aspects of recognition, response, and signaling mechanisms. Manipulation and quantification of the strength of individual molecular bonds and characterization of the force-dependent behavior of complexes provide insights into the mechanical properties, folding pathways, and kinetics of molecules being studied.

Single-molecule force spectroscopy is an experimental method that allows the study and manipulation of the mechanical properties of individual molecules. Several instruments are capable of performing single-molecule force measurements, the main difference is the force range they are operating in.

Credit: DOI: 10.1101/2021.01.04.425265

AFM-based SMFS

AFM can be used to study the intramolecular forces between molecules. The molecules of interest are immobilized on the AFM tip and the surface (e.g. mica), typically either through non-specific physisorption or by selective chemistry. A linker or spacer molecule (e.g. PEG or artificial multi-domain polyprotein) between the molecule of interest and the tip, sample, or both can help reduce unwanted interactions and act as an internal control/gauge to identify specific interactions. Bringing the AFM tip close to the surface, and withdrawing it thereafter allows the probing of molecular interactions, and the characterization of such based on the recorded force distance curves.

In addition to all AFM systems available in the lab, a dedicated Bruker JPK ForceRobot 300 system capable of advanced automated single molecule force spectroscopy is available for high-throughput, quantitative single-molecule experiments. The system is named HK-47 (this is the droid you are looking for).

Magnetic tweezers (MT)

Magnetic tweezers are straightforward instruments, the most basic consisting of a pair of permanent magnets placed above an inverted microscope outfitted with an imaging device. A magnetic particle is then used to tether the molecule of interest to the surface and apply a force (proportional to the gradient of the square of the magnetic field). An advantage of magnetic tweezers is the alignment of the magnetic particle within the externally applied magnetic field, thus enabling application of torque by rotating the magnet – ideal for the study of nucleic acid-enzyme interactions.

A magnetic tweezer instrument, combined with single-molecule total internal reflection fluorescence microscope, is currently under construction. This system will be called Krølle.

Credit: DOI: 10.1016/j.ymeth.2016.03.025

Single-molecule fluorescence microscopy

Optical microscopy has historically been used to generate magnified images of small objects. More modern variants are capable of visualizing molecules labelled with specific fluorophores. However, these experiments yield ensemble properties, which may not be sufficient to fully describe a specific molecule, and therefore observation of a few or single molecules are necessary.

Single molecule fluorescence microscopy (SMFM) allows the imaging, localization, and tracking of single molecules. This makes possible the study of the kinetics and dynamics of molecules, and can elucidate their behavior such as if the molecule exists in several states. Förster resonance energy transfer (FRET) and total internal reflection fluorescence (TIRF) are popular SMFM techniques.

Total internal reflection fluorescence microscopy (TIRFm)

A stand-alone TIRFm is available in the lab capable of simultaneous two-color experiments. This system is called MulMul (Babylonian name for the Pleiades).

Super-resolution structured illumination microscopy (SIM)

A four-color SIM microscope available in the lab and is combined with the high-speed AFM system.