86


ONIX live cell imaging platform was used to create a dynamic assay that not only has the potential to simultaneously monitor multiple intracellular components throughout the entire autophagic process without disruption but also allows precise manipulation of culture parameters, thus exposing cells to more physiologically relevant conditions. This platform may be capable of simulating conditions of pulse exposure to drug compounds, and could provide additional information on dose response for compound profiling by revealing rates of autophagosome formation and degradation. It therefore has the potential to help in the discovery of new targets and therapeutic compounds in cancer as well as other diseases.


Conclusion The CellASIC®


References


1. Cuervo, A. et al. (2004). Autophagy: in sickness and in health. Trends in Cell Biology; 14(2); 70-77. 2. Kimmelman, A. et al. (2011). The dynamic nature of autophagy in cancer. Genes and Development 25: 1999-2010.


3. Amaravadi, R. et al. (2011). Principles and current strategies for targeting autophagy for cancer treatment. Clinical Cancer Research 17: 654-666.


4. Neufeld, T. et al. (2010). TOR-dependent control of autophagy: biting the hand that feeds. Current Opinions of Cell Biology 22: 157-168.


5. Cheong, H. et al. (2011). Ammonia-induced autophagy is independent of ULK1/ULK2 kinase. Proc Natl Acad Sci 108: 11121-11126.


6. Mizushima, N. et al. (2010). Methods in mammalian autophagy research. Cell 140 (3): 313-326. 7. Yamamoto A. et al. (1998). Bafilomycin A1 prevents maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes in rat hepatoma cell line, H-4-II-E cells. Cell Structure and Function 23: 33-42.


8. Baumgardner, J. et al. (2003). In vitro intermittent hypoxia: challenges for creating hypoxia in cell cultures. Respiratory Physiology and Neurobiology 136: 131-139.


9. Bambrick, L. et al. (2011). In vitro cell culture pO2 is significantly different from incubator pO2. Biotechnology Progress 27: 1185-1189


Figure 7. Two-colour imaging of transduced LAMP1-RFP/LC3-GFP CHO reporter cells showing autophagosomes (green) and lysosomes (red) throughout the entire hypoxia-induced autophagy assay. Cells were first treated with mild hypoxia (3%) for three hours in the presence or absence of CQ, followed by the removal of the stressors and the treatment of normoxia and the growth medium for another 16 hours. An Olympus IX-71 inverted microscope was used for the entire process, and all images were taken under the 40x objective.


10. Song Y. et al. (2013). Autophagy contributes to the survival of CD133+ liver cancer stem cell in the hypoxic and nutrient-deprived tumor microenvironment. Cancer Letters (in press)


11. Grkovic S. et al. (2013). IGFBP-3 binds GRP78, stimulates autophagy and promotes the survival of breast cancer cells exposed to adverse microenvironments. Oncogene 32: 2412-2420. 12. Hubbi M. et al. (2013). Chaperone-mediated autophagy targets hypoxia-inducible factor-1(HIF-1) for lysosomal degradation. The Journal of Biological Chemistry 288: 10703-10714


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Launch of Fast-Scanning and Super-Resolution AFM System JPK Instruments announces the release of the next generation of NanoWizard®


inverted microscope.


The boundaries for performance of analytical instrumentation are continually being pushed to the limits. In the world of atomic force microscopy, AFM, JPK Instruments have launched a new AFM system capable of delivering fast-scanning and super-resolution on a single instrument platform, the NanoWizard®


ULTRA Speed AFM. The fast scanning NanoWizard® ULTRA Speed AFM is important to users as it enables the tracking of changes


in samples in real time whether the sample be imaged in air or liquid. Scanning at speeds of greater than 100Hz line rate with excellent, true atomic resolution in closed-loop mode is enabled by the enhanced low noise of scanner, position sensor and detection system. The new AFM system uses JPK’s unique QI™ (Quantitative Imaging) mode to provide quantitative material property mapping.


As with previous NanoWizard®


systems, the ULTRA Speed AFM may be fully integrated with an inverted optical microscope thanks to its tip-scanning design and DirectOverlay™ mode for the most precise correlative microscopy. Similarly, the system provides extensive force measurement capability making measurements on single molecules or on living cells thanks to the JPK RampDesigner™ and ExperimentPlanner™. The system is fully compatible with JPK’s unsurpassed range of imaging modes and accessories especially for environmental control of the sample.


Speaking about the announcement of the NanoWizard® ULTRA Speed AFM, founder and Chief Technical Officer, Torsten Jähnke, said that “Once again JPK have set a new standard in terms of resolution


paired with scan speed. We have managed to develop the lowest noise cantilever deflection system which, when put with our latest fast, high bandwidth electronics, is able to deliver the most accurate force control system even on the most delicate of samples, perfect for users studying biological or other soft matter systems. It is the best multipurpose, fast and high-resolution machine on an inverted microscope today.”


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AFM systems delivering fast-scanning and super-resolution on an


Temperature Controlled and X-ray Stages used to Advance Supramolecular Polymer Research


As part of the Supramolecular polymer chemistry group at the Eindhoven University of Technology, Doctorial candidate Berry Bögels and his colleagues are focused on using self-assembly to create smart materials. Specifically, Mr Bögels has been working on developing membranes that can be used in applications such as drug delivery systems, dialysis and biosensors.


In recent years, there has been a concerted push toward the development of artificial materials that can be used to increase the understanding and enable researchers to mimic certain vital biological, stimuli-responsive activities.


Their studies have utilised a variety of Linkam equipment in enabling them to look at the bulk crystalline properties of compounds which can be used to form these nanostructure membranes. The group is using an LTS stage with specially coated ITO cells to analyse liquid crystals, or polymeric materials with a molecular structure consisting of a high degree of order. The LTS stage allows them to view their samples on a polarising microscope whilst simultaneously controlling the temperature of their sample. Mr Bögels has pointed this out as a key feature of the stage: “For the analysis of liquid crystalline materials, a good control of temperature is of upmost importance to be able to form the desired LC-Phase and (especially if) macroscopic alignment is wanted.”


The group has recently acquired a new Linkam HFS X-ray stage which they have also been using in their research. The HFS X-ray stage has been used by the team to study the nanomaterials in an X-ray diffractometer enabling them to view the molecular structures in fine detail. The HFS X-ray stages are specifically designed for this established analytical technique as they have accurate control of temperature under vacuum as well as X-ray compatible windows.


The Linkam instrumentation has helped the group attain the results that they have strived to work for, and it is the robustness and versatility of the stages that the Mr Bögels and his team have found most valuable: “At Eindhoven University, we have already made use of Linkam stages for quite some time. They always seem to work properly, have a very long lifetime and can be used in a variety of different set-ups.”


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INTERNATIONAL LABMATE - JANUARY/FEBRUARY 2014


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