86 Report on the use of Tip Assisted Optics to Characterise Biomolecular Hydrogels


CIC biomaGUNE is a non-profit research organisation created in 2006 to promote scientific research and technological innovation at the highest levels in the Basque Country. Dr Ralf Richter leads Laboratory 3 in the Biosurfaces Research Unit applying a number of techniques for surface nanostructure characterisation and biofunctionalisation to guide the assembly of molecules down to the nanometer-scale. For this characterisation, his group has developed a toolbox of biophysical in-situ techniques including a quartz crystal microbalance with dissipation monitoring (QCM-D), atomic force microscopy (AFM), reflection interference contrast microscopy (RICM), ellipsometry and fluorescence methods. The current research work is centred on biomolecular hydrogels, a broad class of materials that are produced in the human body or by other bioorganisms.


Dr Richter's group develops methods to re-create these specialised natural hydrogels through the controlled assembly from their molecular components in vitro. Functionalised surfaces are used to guide the self-assembly process.


While AFM is used to obtain nanoscale topographic information about the self-assembled architecture of the materials created, AFM is also used to quantify the mechanical properties of these hydrogels. Dr Richter explained: "We employ colloidal probe AFM. When analysing polymer films, a fundamental problem in colloidal probe AFM experiments is to determine the distance at closest approach between the probe and the substrate on which the film is deposited. In a study published earlier this year (Attili & Richter, Langmuir, 2012, 28:3206) we have overcome this problem by combining optical interferometry (RICM) and AFM in situ. With the combined setup, forces and absolute distances between substrate and probe can be measured at the same time. Thanks to its tip-assisted optics (TAO™) module and the integration of high-resolution optical microscopy, the combination of RICM and AFM can be readily setup with the JPK NanoWizard®


system. By using our toolbox of techniques, we are able to combine their results to produce a very detailed picture about the physico-chemical properties and dynamics of the films of interest."


He continued: "I have worked with NanoWizard systems since 2005. The combination of high resolution optical and atomic force microscopy is critical for the combination of AFM and RICM. Here, the tip-assisted optics (TAO) module is also very useful. It keeps the colloidal probe in the centre of the optical image, while probing different spots on the same surface."


MORE INFO. 243


Nanoparticle Measuring System Used for Vaccine Characterisation at Lomonosov Moscow State University


The MSU Department of Virology headed by Professor Joseph Atabekov is studying the in vitro assembly of compositions, consisting of artificial plant virus particles and antigens, potentially attractive for vaccines development. Artificial plant virus particles are spherical particles (SPs) generated by thermal denaturation and structural remodelling of helical plant tobacco mosaic virus, a rod-shaped virus with a diameter of 18nm and a modal length of 300nm. It has been found that upon thermal denaturation of TMV, viral RNA is released and becomes degraded whereas viral coated protein is assembled into spherical particles. The size of SPs depends on the initial TMV concentration and particles from 50 to 800nm may be obtained. The group of Professor Olga Karpova has shown that SPs based on TMV are stable and may adsorb a diversity of proteins. Thus, SPs represent a new type of biogenic nanoplatform attractive for binding antigens and antigenic determinants of different pathogens.


Describing the choice of NTA from NanoSight for this work, Dr Nikitin said "It permits us to analyse and control the size, state of aggregation and concentration of artificial plant virus particles and small spherical plant and animal viruses. Furthermore, NTA allows us to see the formation of immunogenic complexes by using the indirect immunofluorescence or immunogold staining methods. The technique provides us with the opportunity to obtain simultaneous information concerning nanoparticle size, state of aggregation, concentration and antigenic specificity in liquid. This is particularly important for vaccine characterisation and standardisation.


"Previously, we had used transmission electron microscopy (TEM) and dynamic light scattering (DLS) for sizing SPs, isometric viruses and virus-like particles. To detect the formation of immunogenic complexes we use immunogold TEM and immunofluorescence microscopy. For us, the main advantage of NTA over these microscopic methods is that there is no need to fix and dry the object on a supporting film which could lead to morphological deformations and aggregation of nanoparticles. NTA provides the means for analysis of samples in liquid in real-time and makes measurements particle by particle.”


MORE INFO. 244


Report on the use of Warm Stages in UK Fertility Laboratories to Assess Sperm Viability


Animal fertility assessment is crucial for animal husbandry in the UK: farmers need to know that their breeding animals are healthy before the breeding season so that lambing and calving can be organised effectively. Linkam warms stages are designed to hold a specified temperature to +/- 0.1°C from ambient to 60°C. They are an accurate temperature control platform for inverted or upright microscope applications where it is crucial to maintain at 0.1°C stable temperature in the sample whether mounted in a petri dish or on a microscope slide.


Sperm motility is temperature dependent. To establish in-vivo fertility, observations need to be carried out at body temperature to mimic conditions within the body: for humans, bulls and rams is exactly 37°C. The use of a precise, accurate heated warm stage is an essential part of the assessment. This is to ensure the assessment is conducted at a stable and specific temperature.


Within the design of this Linkam warm stage, a platinum resistor temperature sensor is used for higher accuracy and stability. A sophisticated CAD designed bi-filar heating element covers the entire working surface which provides a uniform temperature distribution in the sample slide.


A sample is placed onto a clean glass slide and covered with a coverslip to provide a chamber. The weight of the cover slip spreads the sample evenly. Scientists initially look for aggregation or agglutination, and the presence of non-spermatozoa cells. The preparation is then observed at 400x magnification with a phase contrast microscope to observe motility. Each Linkam-designed warm plate is incredibly thin, as little as 0.5mm. This ensures that high resolution objective and condenser lenses can be used. Approximately 200 spermatozoa are counted and categorised to determine the percentage of each category. Spermatozoa can be classified as progressively motile, non-progressively motile or non-motile. The percentage of progressively motile sperm is important to fertility assessment.


Linkam warm stages are found in cell biology labs, veterinary hospitals, hospitals and IVF clinics all over the world. The Linkam warm stages provide a simple, accurate, low cost temperature controlled platform that can heat/cool samples from ambient to 60°C.


MORE INFO. 245


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