44 Summary and Outlook


The transfer of an immunoassay onto a biosensor chip device can be greatly facilitated by the use of these types of antibody binding monolayers. The surfaces are stable and amenable to long-term storage.


The self-assembly from aqueous buffer using a simple ‘apply and wash’ method is highly manufacturable and protein production is also easily scaled up. The technology is amenable to microcontact, or dip-pen lithography for the generation of antibody-binding arrays.


Figure 6. Injection of 30 µg mL-1 rabbit polyclonal antibody and regeneration with 100


mM HCl over 30 cycles. The first cycle is shown in purple and the last in blue. Antibody injection was from 60-360 seconds with a 15 min wash with TBS-T buffer prior to regeneration. The flow rate was 5 µL min-1


throughout. Acknowlegements


Many thanks are due to Dr Alison Young for the analysis of IgG binding on Biacore 2000, Dr Andrei Soliakov for the purification of influenza A NP and Dr Anton Le Brun and Prof Jeremy Lakey of Newcastle University for their amazing intellectual input and neutron reflection data.


Thanks also to Dr Dale Athey for critical reading of the manuscript. The Influenza A NP work was part funded by the Technology Strategy Board under the Virasens grant.


References


[1] Terretaz et al, Protein Science 11: 1917-1925 (2002) [2] Shah et al., Biochem Soc Trans 35: 522-526 (2008) [3] Sjödahl, Eur. J. Biochem. 73: 343-351 (1977) [4] Sjöbring et al, J. Biol. Chem. 266: 399-405 (1991) [5] Nilson et al, J. Biol. Chem. 267: 2234-2239 (1992) [6] Le Brun et al, Eur. Biophys. J. 37: 639-645 (2008) [7] Stenberg et al. J Colloid Interface Science 143: 513-519 (1991)


Beyond the examples presented above, the technology has been applied to the design and manufacture of highly specialised surfaces for cell culture. It is possible to create fusions with a huge variety of proteins and peptides e.g. the fusion of single chain Fv domains of antibodies to create highly specific capture molecules with self-assembling properties.


DirectOverlay Display and Analysis Software


JPK Instruments provides a unique software package to make the display of light microscopy images together with atomic force microscopy images a seamless exercise. This package is called DirectOverlay™. Atomic force microscopy (AFM) is a powerful tool to investigate a huge variety of different samples with nanometre scale resolution under physiological conditions. As well as providing topographic measurements, information about interaction forces and mechanical properties like adhesion and elasticity can also be obtained. Perfect integration of AFM with an optical setup can increase the range of applications and opens up many possibilities for correlating structural information with optical information such as functionalised labelling of certain components. To achieve the perfect combination of optics and AFM at the molecular scale, distortions must be prevented. This will result in two images, such as optical and AFM images, that do not perfectly overlay. Reasons for distortions include aberrations arising from the lenses and mirrors of the optics system. This nonlinear stretching, rotating and offsetting of optical images are present in nearly all types of optical setups.


To generate a seamless overlay of both techniques, JPK developed a cutting-edge calibration method, called DirectOverlay, which uses the accuracy of the AFM closed-loop scanning system to enable a true display of absolute angles and length coordinates. The calibration procedure is done automatically and uses the known positions and offsets of the cantilever to calibrate the optical image into the AFM coordinates. To generate a perfect match of the optical and AFM image, 25 or more points are used in the calibration algorithm. At each point, an optical image is acquired and the position of the cantilever tip is automatically detected in each optical image without needing input on cantilever angle, shape or magnification. The algorithm then performs a nonlinear conversion and, as a result, the optical image is corrected for any lens imperfections and converted into the linearised AFM length coordinates. This provides a perfect integration of optical and AFM data with sub-diffraction limit precision. Finally, the calibrated optical image is transferred into the JPK SPM software, so that AFM scan regions can be selected within the optical image. Direct ‘in optical image’ selection of AFM measurements (imaging, mapping and force spectroscopy) leads to more efficient experiments and reduces dramatically overview image scanning in AFM.


Circle no. 434


Temperature Controlled Cryo Stage to Study Biological Specimens


Linkam Scientific Instruments announce the use of their THMS600 temperature stage at the Leiden University Medical Centre to aid the cryo-study of biological specimens. Professor A.J. Koster (Leiden University Medical Centre) is one of the founders of NeCEN - the Netherlands Centre of Electron Nanoscopy. Professor Koster's own group focuses on applications in cell biology. His goal is to localise molecular structures in cells using fluorescence microscopy and then transfer the sample to a cryo-electron microscopy (Cryo-EM) set up to image the corresponding macromolecular structures in 3D with nm-scale resolution. Two areas are of particular interest: the study of viral infections and viral replication where fluorescence may be used to pinpoint areas worthy of enhanced investigation. The other is in the field of vascular biology to study the process of regulated exocytosis of Weibel-Palade bodies (WPBs) that is a pivotal mechanism via which vascular endothelial cells initiate repair in response to injury and inflammation. The goal of the studies was to develop a setup and workflow for cryo-CLEM (correlative light and electron microscopy) that permits visualisation of structures in cryo-FM (fluorescence microscopy) at sub-cellular resolution, and that retrieves those structures accurately in cryo-EM for the purpose of cryo electron tomography. The group wanted a cryo-FM setup that was easy to implement. To this end, Professor Koster and his colleagues opted for a commercially available heating and freezing stage (the Linkam THMS 600), which was modified in order to accommodate EM support grids. As Professor Koster said: "Before we found the Linkam system in the literature and the ability


to correlate microscopies, combining modalities was next to impossible. We have now been using this cryo-CLEM method for more than three years. It has certainly enabled us to produce results quickly and hence get to publication more rapidly too," (European Journal of Cell Biology 88 (2009) 669-684). More than 3,000 THMS600 stages are in use in laboratories worldwide. It is used in many applications where high heating/freezing rates and 0.1°C accuracy and stability are needed. Samples can be quickly characterised by heating to within a few degrees of the required temperature at a rate of up to 150°C/min with no overshoot, then slowed down to a few tenths of a degrees per minute to closely examine sample changes.


Circle no. 435


Digital Cameras Ideal for All Contrast Methods in Light Microscopy Warner Instruments is pleased to introduce its new line of ProgRes®


in the development and production of high-end solutions for digital imaging. The highly versatile and cost effective ProgRes®


Digital CMOS and CCD Microscope Cameras. These cameras are the result of more than two decades experience CMOS Camera range allows for quick and precise setting


of both specimen and microscope. The fast live images coupled with the outstanding CMOS technology and high resolutions make these cameras the first choice in education institutes and training labs. The ProgRes®


CCD Research range of cameras feature excellent colour reproduction and high-resolution. With up to 7 megapixel resolution, these cameras are ideal


tools for high quality image documentation and elementary image analysis. To facilitate precise focusing and positioning of specimens, a high resolution, fast, live image frame rate (up to 50 fps) is available. Particularly well-suited for acquiring brilliant images under low-light conditions, the ProgRes®


CCD Research range of cameras has been optimised for applications


requiring exacting capabilities. Smooth operation is facilitated by sensitive CCD sensors, optionally available with cooling, that offer high frame rates and abroad dynamic range. Sophisticated Microscanning Technology allows for capturing images of up to 12.5 megapixels, even in true colour. All ProgRes® image capture software, an intuitive professional software suite that operates with Windows®


microscope cameras come with ProgRes® or Mac® OSX Circle no. 436 CapturePro


Page 1  |  Page 2  |  Page 3  |  Page 4  |  Page 5  |  Page 6  |  Page 7  |  Page 8  |  Page 9  |  Page 10  |  Page 11  |  Page 12  |  Page 13  |  Page 14  |  Page 15  |  Page 16  |  Page 17  |  Page 18  |  Page 19  |  Page 20  |  Page 21  |  Page 22  |  Page 23  |  Page 24  |  Page 25  |  Page 26  |  Page 27  |  Page 28  |  Page 29  |  Page 30  |  Page 31  |  Page 32  |  Page 33  |  Page 34  |  Page 35  |  Page 36  |  Page 37  |  Page 38  |  Page 39  |  Page 40  |  Page 41  |  Page 42  |  Page 43  |  Page 44  |  Page 45  |  Page 46  |  Page 47  |  Page 48  |  Page 49  |  Page 50  |  Page 51  |  Page 52