Belfast, UK – CD44 is recognised as an important cell surface receptor involved in the adhesion of circulating leukocytes to endothelium, a process that is critical for their trafficking in the vasculature. Now, a Japanese group of researchers using an ultra-sensitive Andor Neo sCMOS camera in their correlative microscopy set-up has demonstrated that microvilli projections around the cell surface mediate this adhesion.
Although fluorescence imaging is a powerful approach to studying the cellular distribution of membrane receptors, it is limited in its ability to display fine structural information. Therefore, the team mapped the distribution of CD44 at the lymphocyte cell surface in liquid by correlative immune-fluorescence optical and immuno-electron microscopy using a combined fluorescence and atmospheric scanning electron microscope. Once CD44 cell surface labelling was confirmed with the images from the Neo sCMOS detector, atmospheric SEM revealed the microvilli on the cell surface with the localisation of label on the microvilli.
"Dual labelling was employed using an anti-CD44 monoclonal antibody and a secondary antibody conjugated with Alexa Fluor 488 and 1.4 nm positively charged Nanogold particles," says Toshiyuki Murai, lead author of the paper. "We also demonstrated that treatment of cells with cytochalasin D resulted in the loss of the microvilli projections and the simultaneous nullification of CD44-mediated adhesion to its ligand hyaluronan. These results suggest the functional relevance of microvilli in CD44-mediated rolling adhesion under shear flow."
The correlative light and SEM imaging was accomplished by culturing the mouse lymphocytes in a petri dish to which a 100 nm silicon nitride window was fixed. This window is transparent to the electron beam, which is projected from beneath the dish by the inverted SEM, while the upright fluorescence microscope excites the sample from above through the objective lens.
"Atmospheric or Environmental SEM enables life scientists to examine biological specimens in exquisite detail, far beyond the optical diffraction limits associated with optical microscopy," says Orla Hanrahan, product specialist at Andor. "It can be used to observe mammalian cells, bacteria, and protein crystals, for instance, and its impact in correlative microscopy studies of cellular structure and function cannot be underestimated. For these studies, Andor's Neo 5.5 megapixel sCMOS is the perfect, ultra-sensitive, high-resolution partner for fluorescence imaging."
Andor's Neo 5.5 megapixel sCMOS camera is a unique -40°C vacuum cooled platform to drive lowest possible dark noise. Neo 5.5 has low noise and excellent resolution with a 5.5 megapixel sensor with 6.5 ?m pixels and a 22 mm diameter. Ideal for cell microscopy, astronomy, digital pathology, and high content screening, Neo 5.5 delivers an unmatched 30 fps sustained or up to 100 fps burst mode to its internal 4 GB memory. The Rolling and Global shutter flexibility further enhances application flexibility, Global shutter in particular offering an ideal means to simply and efficiently synchronize the Neo with other 'moving' devices such as stages or light switching sources and eliminating the possibility of spatial distortion when imaging fast moving objects.
Toshiyuki Murai, Mari Sato, Hidetoshi Nishiyama, Mitsuo Suga and Chikara Sato. Ultrastructural Analysis of Nanogold-Labelled Cell Surface Microvilli in Liquid by Atmospheric Scanning Electron Microscopy and Their Relevance in Cell Adhesion, Int. J. Mol. Sci. 2013, 14, 20809-20819; doi:10.3390/ijms141020809
Andor is a global leader in the pioneering and manufacturing of high performance scientific imaging cameras, spectroscopy solutions and microscopy systems for research and OEM markets. Andor has been innovating the photonics industry for over 20 years and continues to set the standard for high performance light measuring solutions, enabling its customers to break new ground by performing light measurements previously considered impossible. Andor's digital cameras, are allowing scientists around the world to measure light down to a single photon and capture events occurring within 1 billionth of a second.
Andor now has over 400 staff across 16 offices worldwide, distributing products to over 10,000 customers in 55 countries. Andor's products are used in a wide range of applications including medical research to further the understanding of heart disease, cancer and neuronal diseases such as Alzheimer's and Parkinson's disease. Andor also has applications for forensic science and astronomy. Through continuous dialogue with customers and strong teamwork, Andor continues to innovate ground-breaking products that improve the world in which we live.
More information is available at www.andor.com.
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Oxford Instruments designs, supplies and supports high-technology tools and systems with a focus on research and industrial applications. Innovation has been the driving force behind Oxford Instruments' growth and success for over 50 years, and its strategy is to effect the successful commercialisation of these ideas by bringing them to market in a timely and customer-focused fashion.
The first technology business to be spun out from Oxford University, Oxford Instruments is now a global company with over 2300 staff worldwide and is listed on the FTSE250 index of the London Stock Exchange (OXIG). Its objective is to be the leading provider of new generation tools and systems for the research and industrial sectors with a focus on nanotechnology. Its key market sectors include nano-fabrication and nano-materials. The company's strategy is to expand the business into the life sciences arena, where nanotechnology and biotechnology intersect.
This involves the combination of core technologies in areas such as low temperature, high magnetic field and ultra high vacuum environments; Nuclear Magnetic Resonance; x-ray, electron, laser and optical based metrology; atomic force microscopy; optical imaging; advanced growth, deposition and etching.
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