Microscopy & Microtechniques 87 Report on the Use of AFM and Single-Cell Force Spectroscopy


The interdisciplinary Nanoscience Center (iNANO) was formed by various research groups at Aarhus University together with groups from the Faculty of Science at Aalborg University. iNANO comprises facilities for the synthesis of nanostructured and nanopatterned 0D (i.e. nanoparticle), 1D, 2D and 3D materials.


The group of Dr Rikke Meyer works at the interface between microbiology and nanoscience in the quest to understand how bacteria form biofilms and how this may be prevented. AFM and optical microscopy are used to visualise bacterial cells and to study the interaction forces between cells and an abiotic substrate.


The motivation for using AFM in Dr Meyer's research was firstly to obtain detailed images of bacterial cells without extensive sample preparation. Furthermore, as she is interested in the interactions between bacteria and abiotic surfaces, she and her team use AFM force spectroscopy to quantify these interaction forces.


AFM is one of several techniques used in these studies. These also include brightfield microscopy, fluorescence microscopy, confocal laser scanning microscopy, scanning electron microscopy and transmission electron microscopy.


Dr Meyer commented on her research and reasons behind her choice of AFM: "The coupling with optical microscopy is no doubt the feature that was most important for me in deciding to go with an AFM from JPK. As a microbiologist, I work with very heterogenous samples and it is not feasible to use AFM imaging to locate the field of interest, as large areas of the sample are often visualised to locate a site of interest. In the combined system, we can use the optical image to locate cells of interest before engaging the AFM for imaging or other measurements."


She continued: "AFM has mostly been used to study bacterial cells that are isolated in pure culture. However, the vast majority of the bacterial species we know to date have not been isolated and can only be studied in situ. Fluorescence labelling allows a rough identification of bacteria directly in the sample and fluorescence imaging can thus be used to locate cells of interest before AFM imaging begins. The combination of AFM with optical imaging is thus particularly important for the analysis of bacteria in environmental samples."


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Report on Nanoparticle Tracking Analysis use for Environmental and Biomedical Nanoparticle Monitoring


Ilya Kurochkin is Professor and Head of the Laboratory of Postgenomic Chemistry in the Department of Chemistry at the Lomonsov Moscow State University. His research requires characterisation of size distribution, concentration and to identify the presence of certain epitopes in two areas: exosomes for the prediction of adverse outcomes in patients with chronic heart failure and for the characterisation of gold, silver and metal oxide nanoparticles in the development of new electrochemical biosensors and nanobioanalytical systems based on nanoplasmonic structures.


In the research, it is important to be able to make a quick calculation of the number and estimate the size distribution of the exosomes in the biological fluids. The use of plasmonic nanoparticles will allow multiplex analysis of a large number of target proteins in exosomes. These measurements provide information about a specific protein composition of exosomes, and thus make it the basis of a new nanobioanalytical platform – ‘human exosome profile’.


To achieve this, Professor Kurochkin chose Nanoparticle Tracking Analysis, NTA, from NanoSight as his preferred technique as he needed accurate information on the size and number of his nanostructures in the presence of much larger particles. Prior to NTA, he used a combination of techniques including dynamic light scattering, atomic force microscopy and electron microscopy. As he noted, "The main benefit of the NTA is the ability to accurately measure size distribution for nanostructures in the presence of much larger particles directly in test solutions.


The fact that the intensity of light scattering is proportional to the particle diameter to the power of 6, DLS (which captures the signal from all the particles in the sample mixture) does not give an accurate measurement of a particle with a small diameter in the presence of particles with a larger diameter. NTA captures the trajectories of individual particles and therefore the ratio of the particle size does not affect the accuracy of the analysis. Thus, the fact that NTA measures each particle separately is very important.


I am particularly excited that I can measure my exosomes with specific antigens using fluorescent tags and plasmonic nanomarkers (gold or silver nanoparticles). Using NTA perfectly overcame the earlier experimental problems of my work. Measurements are rapid and by counting particle by particle, I achieved the level of accuracy I was seeking."


MORE INFO. 247 Launch of New Cryo-Correlative Cooling Stage


Linkam are pleased to announce the development and launch of a stage for cryo-correlative light/electron microscopy (cryo-CLEM). It was designed in collaboration with scientists at the Leiden University Medical Centre (LUMC) led by Bram Koster and Erik Bos. Cryo-CLEM is the correlation of images captured with a cryostage on a fluorescent light microscope and images of the same sample observed with Transmission Electron Microscopy (TEM).


Dr Lucy Collinson of the London Research Institute (LRI) and Cancer Research UK (CRUK), is using the correlative stage as part of a workflow that starts with cells expressing cancer-related genes and ends with imaging in synchrotrons located in Oxford, Berlin, and Barcelona. Within the synchrotrons, the scientists are able to image the cells using a cutting-edge technique called 'soft X-ray tomography'. The LRI focuses on three themes of research: the biology of tissues and tumours, cellular regulatory mechanisms and genomic integrity & cell cycle.


The correlative stage can hold samples at a stable -196°C enabling scientists to study TEM grid samples at 100x magnification, identifying areas of further interest, and facilitating the movement of these analysed grids to the TEM. With automated liquid nitrogen control, heated optics and a


digital display the unit is a compact and efficient system for this work.


In particular, Dr Collinson is working with Dr Sharon Tooze, Head of the Secretory Pathways Laboratory at the LRI, to study cells that are undergoing autophagy, which is a normal process involving degradation and recycling of unnecessary or dysfunctional cell components


With Dr Liz Duke, a Principal Beamline Scientist at the Diamond Light Source synchrotron in Oxfordshire, they have developed a process for imaging these fluorescent proteins in cells as close to their living state as possible, considering that they must place them in a vacuum to take very high magnification images. They grow the cells on very thin carbon films attached to a 3mm diameter gold grid and freeze the cells in liquid ethane at –174°C.


The correlative stage is used to image the fluorescence in the cells while they are still frozen. The frozen grids are then shipped to the synchrotron, where they put them into the soft X-ray microscope so that they can image the structure of the entire cell in 3D.


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INTERNATIONAL LABMATE - JANUARY/FEBRUARY 2013


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