Researchers in the life science community are starting to successfully apply nanotechnology in their work. Understanding processes at the molecular level is now regarded as essential in order to produce better products in a more reliable and cost effective manner. This article looks at how the scanning probe microscope has opened up the field of nanoscale research and looks at novel tools that are also shaping our understanding of particle and cellular interactions.
by Torsten Jaehnke
The last twenty five years have seen the rapid growth of new ways to study materials at the nanometre scale. Key to this has been the development of the scanning probe microscope (SPM). Early commercial systems were designed to meet the needs of the semiconductor industry and it has only been since the turn of the century that the basic experimental needs of the life scientist have been considered in the design of applicable instruments. This has been principally due to a development around one specific form of SPM known as the Atomic Force Microscope (AFM).
In this article, the basics of applying SPM to study “soft matter” in optimal experimental conditions will be described and the applications of new and exciting tools such as optical tweezers will be reviewed together with the many research possibilities these developments are opening up.
Developing a new AFM system
The beginning of a new generation of BioAFM systems designed specifically to address the needs of life science and soft matter researchers has been heralded by the introduction of the NanoWizard II instruments from JPK instruments of Berlin, Germany. The new system combines high-resolution imaging in air and fluid, force measurements, nanomanipulation and lithography, with advanced optical imaging through the interface with an inverted light microscope.
Most AFMs were developed for engineering, materials science or physics applications and were not optimised for life science applications, where samples are mainly studied in fluids. Furthermore, measurements on single molecules demand the ultimate resolution in imaging and highest sensitivity force spectroscopy modes. Long-term experiments for time-lapse imaging require a closed loop scanner to be integrated into the SPM and optical microscope platform with the utmost stability and zero-drift.
To develop an instrument that delivers what can only be described as imaging wizardry, JPK’s interdisciplinary development team comprising engineers, physicists, software developers, chemists and biologists, worked in close cooperation with many leading AFM experts from around the world.
Tip scanning operation
The NanoWizardII employs tip-scanning imaging. The sample remains stationary, thus eliminating the blurring of optical images that are observed whilst imaging with sample scanning systems. Another important advantage of tip-scanning is that the sample does not get shaken during operation, so ensuring that species such as weakly bound live cells remain in place and feel no shear stress from the scanning motion.
The short working distance design of the system enables numerous fluorescence sources, as well as both standard and specialist condensers, to be used to acquire images using optical contrast enhancement methods such as phase contrast and differential interference contrast (DIC).
Fluorescence techniques such as TIRF (total internal reflection fluorescence), FRET (Förster resonance energy transfer) and laser scanning microscopy (LSM) also require that the sample does not move but remains in focus at all times throughout the experiment. The NanoWizard II is already being used widely with these techniques.
Liquid imaging
The system has been specifically designed for imaging samples in liquids and is compatible with all commonly used biological substrates including Petri dishes, cover slips and microscope slides. All parts that come into contact with the liquid have been made to be easy to exchange, replace and clean rigorously.
In addition, a whole range of fluid cells and temperature control options have been developed to ensure the instrument excels in the widest possible range of applications that require high performance. JPK’s patent-pending BioCell embodies this as the only AFM fluid cell capable of temperature control from 15-60°C, perfusion and gas-flow with standard cover slip substrates.
State-of-the-art accessories enable new applications. Accessories such as the CellHesion module automate cell mechanics and adhesion measurements. The Tip Assisted Optics module (TAO) for advanced AFM-optical experiments adds a three degrees of freedom nanopositioning system, and thus additional unique capabilities. For example, users can integrate tip enhanced Raman spectroscopy (TERS) or perform single molecule FRET and nanomanipulation within a confocal laser spot.
Software and analysis
Of course, no matter how powerful an instrument is, high data throughput requires user-friendly software. The SPMControl II software enables easy and intuitive imaging combined with specialist modules for force measurement with advanced cantilever calibration, force mapping, nanomanipulation and other lithographic functions.
One of the most exciting aspects of the software is the picture-in-picture display that automatically overlays new and old images to allow easy comparison. Uniquely, the software can also directly import calibrated optical images into the AFM software allowing users to directly set force spectroscopy points in special features without the need to prescan the image surface, thus preserving delicate, chemically modified probe tips.
Overlaying AFM and optical images traditionally involved time-consuming post-processing, cutting and stretching because the images were frequently offset and taken at different magnifications. However, the DirectOverlay function enables data from transmission optics, fluorescence and AFM to be combined with the touch of a button.
Combining AFM and laser confocal microscopy
Atomic force microscopy and fluorescence microscopy provide complementary information about biological samples. When laser scanning confocal microscopy is used, the out-of-focus light is suppressed, so the information from the fluorescence signal can be much better localised. The cell can be optically sectioned, for instance to specifically image labelled proteins at the surface of the cell, for co-localisation with surface structures seen in the AFM images.
Calibration of the confocal image with the absolute dimensions obtained by AFM, allows precise matching of surface structures, such as cavaeoli and clathrin-coated pits [Figure 2], acquired by AFM to specifically labelled proteins or bio-molecules imaged by confocal microscopy. Additionally, confocal imaging in combination with manipulation with the AFM allows imaging of biological processes such as signal transduction.
The next step – optical tweezers
The properties of biological systems are, to a large extent, determined by the behaviour and interactions of individual molecules. Molecules bind together to form aggregates, compartments, cells, tissues, organs and organisms. The basic mechanism of such binding events involves rapid molecular recognition at the nm scale, where Brownian motion is the driver of the binding processes. The concept of using nanoparticles as local sensors or probes enables important developments in medicine, molecular and cellular biology. For example, the dream of many life science researchers is to be able to observe the entrance process of a particle into a cell in real time, in 3D and without perturbation of the biological specimen. Existing technologies such as epifluorescence, TIRF, LSM and video particle tracking have several drawbacks. Labelling is time consuming and causes perturbation in single molecule experiments.
The best resolution in confocal microscopy (CLSM) is ~300nm in the X & Y and ~500nm in the Z axis. Fluorescence is not label-free and has poor performance for processes occurring on cell membranes. TIRF measures only the first 200nm from the surface of the cell and gives no access to the whole cell volume. Particle tracking by video microscopy is only a 2D technique with best resolution of 15nm.
Given these drawbacks of the classical techniques, the question is: can the dream of observing the entrance process of a particle into a cell in real time, in 3D and without perturbation of the biological specimen be fulfilled, and if so how?
The answer is a live cell imaging technique with high temporal and spatial resolution applied to non-labelled nanoparticles. JPK Instruments’ new NanoTracker optical tweezers and 3D particle tracking system delivers live cell imaging at a new level. With the NanoTracker, the user can trap and track particles from several µm down to 30nm and control, manipulate and observe vesicles, endosomes, gene and drug spheres, viruses and bacteria, nanoprobes or carriers, biomarkers or even
whole cells in real time with nanomtre precision.
This opens up new applications in many different disciplines including biophysics, biochemistry, cellular and medical research in microbiology, developmental and system biology, infection research and immune response, toxicity of nanoparticles
and many more.
NanoTracker technology provides precisely quantifiable and reproducible measurements of particle / cell interactions. The system delivers precise information about single molecule mechanics and may also be used to determine mechanical characteristics such as adhesion, elasticity or stiffness.
Managing future developments
The JPK company strongly believes that success comes only by listening and reacting to the detailed needs of customers to develop an understanding of the challenges they face so that a reliable and innovative solution can be found. To achieve this the development team at JPK works in collaboration with an international network of leading scientists and industry experts to develop premium class instruments. As a qualified and reliable partner, JPK is firmly committed to help users apply these exciting technologies to their research.
The author
Torsten Jaehnke,
Chief Technical Officer,
JPK Instruments AG
Bouchéstrasse 12
12435 Berlin,
Germany
Tel. +49 30 5331 12070
www.jpk.com
info@jpk.com
