Microscopy & Microtechniques 87 Innovative Stage used for the Imaging of Mammalian Cells with Cryo-CLXM Microscopy


Linkam Scientifi c Instruments report on the use of their innovative CMS196 cryo stage for the study of mammalian cells at the London Research Institute, Cancer Research UK. In a recent publication in the journal, Ultramicroscopy (Duke et al., 2013), Dr Lucy Collinson (LRI Electron Microscopy Unit), in collaboration with Dr Sharon Tooze (LRI Secretory Pathways Lab), imaged forming autophagosomes in whole mammalian cells. The structures are particularly diffi cult to capture in cells prepared for electron microscopy, so they are now using a powerful new technique called cryo-soft X-ray tomography, cryo-SXT, working with Dr Liz Duke at the Diamond Light Source synchrotron. The cells are grown on tiny gold grids and plunged into liquid ethane to preserve the cells in the frozen state.


In order to fi nd the autophagosomes within the cells, they are labelled with green fl uorescent protein (GFP). The fl uorescent autophagosomes are then located using a technique called correlative cryo-fl uorescence and cryo-soft x-ray microscopy (cryo-CLXM). Cryo-fl uorescence microscopy is performed using the Linkam CMS196 stage prior to the cells being transported in cryo-containers to synchrotrons in Oxfordshire, Berlin and Barcelona for imaging. One of the major advantages of this new correlative approach is that the CMS196 stage allows the cells to be screened for quality and protein localisation in the research laboratory before actually travelling to the synchrotron. The combination of cryo-fl uorescence microscopy and cryo-SXT allows scientists to link the functionality of proteins to their near native-state structure.


The Linkam CMS196 stage was designed specifi cally to solve the problem of how to get vitrifi ed EM grids from the fl uorescence microscope into the cryo-TEM without devitrifi cation and contamination through condensation. The stage has been optimised optically to enable the use of high NA lenses. Up to 3 grids can be loaded into a specially designed cassette for transportation from the plunge freezer to the upright fl uorescent microscope. The cassette is then easily loaded onto the viewing bridge using special manipulation tools. The sample viewing chamber is perfectly dry and below -180ºC while the sample bridge itself is at -196°C. The grids can be quickly and effi ciently scanned using a 100X 0.75NA lens and manipulated using high precision micrometers. The cassette is then simply manipulated back into the transportation device and is then transported to the cryo-TEM under liquid nitrogen.


27512pr@reply-direct.com Report on the Characterisation of Nanostructures and Nanosized Systems using AFM


JPK Instruments reports on how the NanoWizard AFM is being applied in the study of nanostructures and nano-sized systems at the École Polytechnique Federale de Lausanne.


Dr Giovanni Longo is a Research Scientist in the LPMV under the supervision of Professor Giovanni Dietler. He works on a number of projects which require the accuracy and reproducible performance at high resolution of an atomic force microscope, AFM, and has chosen the JPK NanoWizard®


3 for his work. For example,


he uses the AFM cantilevers for the rapid detection of bacterial resistance to antibiotics. Living, moving specimens are attached to an AFM cantilever and their movements are transduced by the lever to the AFM electronics and recorded. Describing this work, Dr Longo said “We recently moved this activity to the JPK NanoWizard microscope since it demonstrated an excellent sensitivity and since the optical microscope is invaluable to couple the macroscopic movements of the biological systems with the microscopic movements we detect using our nanomotion sensor.” Dr Longo describes a second project. “We also study osteoblasts for bone-integration and to characterise their response to microgravity conditions. In this fi eld of research we have coupled the fl uorescence microscopy capabilities of the Axiovert inverted microscope with the high-resolution AFM images to obtain stunning images of cells.”


There are three main features of the NanoWizard which makes it the ideal tool for AFM studies. First, it operates with remarkable stability and the images it delivers are of high-quality and with a very high rate of repeatability. This is particularly impressive since the X/Y/Z ranges are not small and yet the images are of excellent quality both at very large and at very small ranges.


The JPK Quantitative Imaging mode (QI™) is excellent allowing fast imaging of the specimens while, at the same time, collecting their mechanical properties. This is invaluable especially when characterising many samples or when a large number of cells must be probed. For example, Dr Longo used it to determine the stiffness of hundreds of bacteria in different environmental conditions in just a few weeks.


Lastly, when the microscope is coupled with an excellent optical inverted microscope, it is capable of fl uorescence imaging. 27681pr@reply-direct.com


The CRANN Institute of Trinity College Dublin Applies Kleindiek Manipulators for Electrical Probing Experiments


In partnership with German manufacturers, Kleindiek Nanotechnik, Agar Scientifi c has supplied manipulation systems for electrical probing to a variety of customers in the UK and Ireland. One such group is the Advanced Microscopy Laboratory at the CRANN Institute at Trinity College Dublin.


CRANN (the Centre for Research on Adaptive Nanostructures and Nanodevices) is a Science Foundation Ireland (SFI) funded Centre for Science, Engineering and Technology (CSET). The institute works at the frontiers of nanoscience developing new knowledge of nanoscale materials, with a particular focus on new device and sensor technologies for the ICT, biotechnology and medical technology sectors, with a growing interest in energy related research.


Dermot Daly works on the FEI Strata 235 and Zeiss Auriga Focused Ion Beam systems. The tools are ideally suited to failure analysis and cross sectioning, and TEM sample preparation of a wide range of academic or industrial samples and material sets. He also uses a Zeiss Ultra Plus Scanning Electron Microscope featuring STEM capability and a number of specialised detectors with sophisticated contrast and detection mechanisms.


To advance CRANN’s work, they have chosen to use Kleindiek Nanotechnik manipulators for various electrical probing projects and sample prep. These are also used for TEM lift-out while also fi nding applications for particle, fl ake and wire manipulation. Describing his work, Mr Daly says “As well as using the micro needles for TEM prep, and probing, we are now working with the Kleindiek microgrippers to help lift


and move small objects under SEM and FIB. I have always worked with Kleindiek as I had a Zeiss 1540XB system with 4 MM3As in a previous role doing semiconductor failure analysis in Xilinx. They are very precise and easy to use and a lot of users are modifying and updating them to do new things. Kleindiek love collaborations and taking on new ideas for their products that people come up with.”


Agar Scientifi c represents the products of Kleindiek Nanotechnik in the UK & Irish markets. The company provides a comprehensive online catalogue and price list on all accessories and consumables for microscopy. 28203pr@reply-direct.com


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