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Increased Reliability and Accuracy in Optical Metrology
The Olympus LEXT OLS4000 is being used for the development of good practice guidelines and new calibration standards in optical metrology. These reference standards are being developed by a team led by Professor Richard Leach, Principal Research Scientist at the National Physical Laboratory (NPL). Preliminary data, presented by Professor Leach at the 10th International Symposium on Measurement Technology and Intelligent Instruments (ISMTII) demonstrated that these calibration standards will help users to eliminate background noise and data uncertainties, thus increasing reliability and accuracy. The good practice guide on the calibration of confocal microscopes will also provide input to the upcoming ISO/WD 25178-607 specification standard, which is currently in development.
As an important component of these calibration standards, Professor Leach’s team is defining good practice guidelines using typical examples of: stylus instruments; white light interferometers; focus variation instruments; and confocal microscopes. This project has established robust and easy-to-use calibration steps and reference standards for all the major areal surface measurement instrumentation technologies. As part of this, NPL is developing a box of artefacts (optical flat, step heights, lateral grids, star patterns, sphere on plane, deterministic) to allow calibration in compliance with the latest draft ISO standard. Once finalised, users can implement these to gain a level of confidence in the accuracy and trueness of their resulting data.
Further demonstrating the reliability and accuracy of the LEXT OLS4000, Mr Hitoshi Suzuki, Olympus Corporation, Japan presented his findings on ‘A study of surface measuring limitation on a laser scanning microscope by the examination of response properties of surface texture and slope detection on a ruby sphere sample’, at the EUSPEN conference in May. Since identifying and rejecting doubtful measurements is key to maintaining accuracy, instruments need to be able to produce repeatable results. Accuracy when using a CLSM depends on the numerical aperture (NA) of the objective lens; steep slope measurements up to 80º can be accurately obtained with the LEXT OLS4000 when using a 0.95 NA lens.
Circle no. 271
Research into Expanding the use of AFM for Cell Biological Applications
Dr Clemens Franz leads a group of researchers at the DFG-Centre for Functional Nanostructures at Karlsruhe Institute of Technology where he works on expanding the use of AFM for cell biological applications. Given that AFM cantilevers are ultrasoft springs, they can be used to measure inter- and even intramolecular bonds. To study cell adhesion, Dr Franz and his team often employ AFM-based single-cell force spectroscopy (SCFS). Here, a living cell is attached to the AFM cantilever and brought into contact with a substrate under defined contact conditions. With a force sensitivity spanning over four orders of magnitude, SCFS provides a unique opportunity to measure cellular adhesion forces from the single-molecule level to overall adhesion in the same experimental setup. AFM-based SCFS has provided important insight into molecular mechanisms involved in adhesion force generation, such as the transition from single-receptor mediated to cooperative receptor binding during the initial phase of cellular contact with an extracellular substrate. Furthermore, the adhesive properties of different surfaces can be accurately characterised.
Their latest challenge is to combine AFM with advanced light microscopy techniques, such as laser scanning confocal or total internal reflection (TIRF) microscopy. In this way, the clustering of receptors can be followed in living cells by optical time-lapse-microscopy and directly correlated to the adhesion force information. The group now has built a functional TIRF/AFM setup.
The motivation for this work is to better understand fundamental mechanisms of cell adhesion, particularly initial adhesion events when cells first encounter components of the extracellular matrix (ECM). Through the use of micro- or nano-patterned artificial cell adhesion substrates, the goal is to emulate the natural cell environment and to manipulate cell adhesion. Dr Franz describes why he likes to use the JPK AFM systems: "JPK's products are particularly suitable for biological research because of their relative ease of use; their good hardware/software integration; the availability of heated sample chambers and the possibility to image in fluid to keep biological sample functional. Furthermore, the JPK offers excellent integration of AFM and optical microscopy techniques (such as phase contrast, confocal or TIRF microscopy) while the unique 100 µm z-piezo pulling range (CellHesion®
modules) are essential for complete cell-substrate separation in single-cell force spectroscopy." Circle no. 272
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