Feel the force

Back in the 1960s, mankind took on the big challenges of space exploration and putting a man on the moon. Today’s challenges are not to look towards such distant horizons, but to explore the smallest details of matter. Here, we learn of the advances in applying nanotechnology to the life sciences

AS WE explore this route down to molecular and atomic levels, we find ourselves on the edge of new discoveries, primarily in the medical and life sciences areas.

Enabling nanoscale discoveries has required new tools. This has been critical in the life sciences where researchers are not working in vacuum as in areas of physics but require environments where liquid and gas are controlled to mimic real life parameters. Since light microscopy and spectroscopy techniques are often vital to life science research, the design of appropriate tools must also take this into account.

Ten years ago in Berlin, a group of young research scientists founded an instrument company to provide nanoanalytical solutions for research in life sciences and soft matter.
The last decade has seen the development of a research-grade platform atomic force microscope (AFM) which enables simultaneous scanning probe and light microscopy coupled to various spectrometers. It has seen basic applications taken to levels of automation and quantification while introducing tools to manipulate molecules and measure forces at levels undreamed of in the previous decades.

When JPK Instruments was launched, the company benefited from public start-up subsidies which strongly contributed to their success. Conscious of this important part of the company’s history, JPK has joined the sponsors of the German Museum of Masterpieces of Technology and Science in Munich by donating one of their leading NanoWizard atomic force microscope systems to support nano research at the Glass Laboratory at the museum. This will not be a stereotypical museum piece; visitors will be encouraged to observe the genuine research being carried out in the Laboratory. And so JPK adds a commitment to education to its mainstay of developing ever more capable and flexible nanotechnology tools for life sciences.

The NanoWizardII BioAFM provides the most stable platform for highest resolution in imaging and force measurements fitting all standard inverted research microscopes enabling integration with advanced optical imaging techniques such as DIC, CLSM, TIRF and FRET. Direct Overlay software provides the ability to combine AFM and optical images free from distortion with in-situ imaging in biological/chemical fluids or in air.

Applications cover a broad range of fields including live cell imaging and investigations of the cell membrane to single molecule and binding studies.

The importance of cell membranes has long been recognised since they serve a wide variety of functions such as physically separating the intracellular components from the extracellular environment, cross membrane substance transport and cellular adhesion. Cell membranes predominantly contain proteins and lipids. Membrane proteins are responsible for very specific tasks that include surface recognition, signalling and ion conductance. The image (image1) shows an AFM topography image of a 2D crystal of Outer membrane protein F (OmpF) adsorbed onto HOPG. OmpF is a trans-membrane protein obtained from Escherichia coli. This image was obtained in low force contact mode AFM with x and y closed-loop switched on in buffer conditions. The image shows the extracellular surface clearly. Here, OmpF trimers are organised in an orthogonal crystal structure where they are packed two-by-two.

In the next example single molecule fluorescence detection is combined with high resolution AFM imaging of Rad51 DNA. Rad51 is a recombinase protein involved in so-called homologous recombination, an intricate biological process that is responsible for both crossing over of genes during meiosis (reductional cell division in which the number of chromosomes per cell is cut in half) and the reliable repair of double-stranded breaks. Rad51 assembles into filaments along double-stranded DNA that lead to an increased stiffness and thickness of the DNA. Here, Rad51 proteins chemically labelled with Alexa-555 were used to allow for optical detection of Rad51-bound DNA, while AFM imaging was exploited to zoom in to the structural changes to the DNA. These proteins were kindly provided by Dr Mauro Modesti, CNRS Marseille.

Quantitative AFM experiments have been commonly used for many years to measure forces. With a basic AFM system, such measurements have been time-consuming singular events requiring intense operator interactions. Making this into a routine operation was the goal for the design of the ForceRobot 300 system which automates routine procedures and provides software support for experimental design, data acquisition and evaluation. It gives cutting-edge force spectroscopy and force mapping in combination with single molecule fluorescence with automated laser and detector alignment enabling cantilever drift correction. 

The force associated with bond rupture or protein unfolding is known to be a dynamic property that depends on the force loading rate applied. From the study of the unfolding forces recorded at different loading rates, thermodynamic information on the stability of the complex can be deduced. Both the length scale of the unfolding barrier during the transition (usually in the range of a few Ångströms) and the unfolding rate at zero force can be derived from a set of experimental data.

In this study, an I27 octamer was unfolded by applying force loading rates ranging from 40 to 4000nm/s. The I27 domain is located in the distal immunoglobulin-like region of the giant muscle protein titin, and its secondary structure consists of antiparallel β-strands. Its mechanical properties suggest that certain domains of the titin serve as a molecular spring. Using an artificial homopolymer of eight domains of I27, the force spectroscopy studies result in typical sawtooth pattern traces appearing when one domain after the other unfolds due to thermal fluctuations in the stretched molecule. As soon as the first domain has unfolded, the force drops, shown as a negative slope in the force-extension profile. A typical force-extension profile is shown below. Fitting the WLC model into the force-extension traces reveals a change in contour length when a domain unfolds of 28nm in average.

Optical tweezers systems enable the trapping and manipulation of nanoparticles, microparticles and biological species in fluid media. JPK's unique NanoTracker system extends this technology to enable measurement of interaction forces with sub picoNewton sensitivity. In addition, particles are simultaneously tracked in 3D to quantify dynamics, viscosity, diffusion and host of other processes. For the first time, dual beam force-sensing optical tweezers seamlessly integrate on inverted optical microscopes combining advanced optical and confocal techniques including single molecule fluorescence in a small footprint, easy to use system.

This unique tweezers technology (also known as Photonic Force Microscopy) enables the quantification of molecular, cellular and micro-rheological processes. Applications include molecular motor mechanics, binding/elasticity of DNA and proteins, cell membrane dynamics and particle uptake.

The NanoTracker characterised the binding and motion of the kinesin motor protein as it steps across microtubule filaments. Sub pN force resolution allows the reduction in motor protein speed to be characterised with increasing counteracting trapping force and measurement of the motion stalling force.

BioAFM and advanced life sciences applications to measure forces through probe-sample interactions have come a long way in the past decade as illustrated by the examples shown here. What is the future? What will be the next major breakthrough? One of JPK’s founders, Torsten Jähnke, thinks that it will become increasingly important to introduce new technology into biology as a turnkey solution. Sample handling is another critical factor to consider. Experimental workflow can be completely different to other fields and biologists often take the same sample, mostly in liquid, from one tool to the next to build up a complete profile of the structure.