Objectives

THE IMAGING ISSUE

Microscope users are increasingly operating as imaging specialists, working with specific applications. An array of complex peripherals attached to the microscope must deliver integrated reliability and performance. For many, creating such an integrated system is a headache of compatibility issues. For a biologist sourcing cameras, software, microscopes, motorised stages and combinations of dyes and filters can be a distraction from the Life Science studies they wish to pursue. To support the development of such integrated systems, Olympus has a team of technical specialists for cameras, software, and applications, who can advise on creating the optimum configuration. In this issue we look at some Olympus users and what they are achieving in TIRF Microscopy, time-lapse imaging and other areas. We also introduce you to some members of the Olympus Imaging Team. Olympus is introducing new products, including laser cell dissection with the Olympus MicroBeam, new cameras with ever higher resolution, and imaging workstations such as CellR. Take a look inside to learn more about how we can help with your imaging applications - and if you have any questions, call us on 0179 or email microscope@olympus.uk.com.
Professor Guy A. Rutter and Dr Takashi Tsuboi, of the Henry Wellcome Laboratories for Integrated Cell Signalling, University of Bristol are using TIRF to image glucose-dependent translocation of protein kinase C II (PKCII) in pancreatic islet -cells. These cells release insulin when blood glucose concentrations rise, resulting in the clearance of glucose into the tissues. The aim of the research is to understand the underlying molecular mechanisms by which insulin secretion is regulated, and how these may become
introduced from the objective lens (Olympus, Apo X100, numerical aperture = 1.65/TIRF) through a single mode optical fibre and two illumination lenses.To observe the EGFP fluorescence image, a 488 nm laser (Argon ion laser, 50 mW, Spectra-Physics) was used for total internal fluorescence illumination and a long pass filter (515 nm; Olympus) for barrier. The laser beam was passed through an electromagnetically driven shutter which was opened synchronously with camera exposure under software control. Images were acquired every two seconds.
TIRF IMAGING AT HENRY WELLCOME LABORATORIES
defective in type 2 diabetes mellitus. The images show the translocation to the plasma membrane of PKCII fused to enhanced green fluorescent protein, after stimulation of pancreatic -cells with 30mM glucose. Fluorescence was monitored at the cell surface by total internal reflection fluorescence microscopy (TIRF; also known as evanescent wave microscopy). Note that hot spots and a wave of translocation were clearly observed. The scale bar is 5m To assess translocation of PKCII.EGFP the team employed a , TIRF microscope comprising an Olympus IX70 inverted microscope with a TILL IMAGO-VGA CCD camera controlled by TILLvisION software. The incident light for total internal reflection illumination was Professor Rutter and Dr Tsuboi participate in an annual University of Bristol Imaging Course, organized by the University of Bristol Department for Further Education. For further details and dates of future courses please contact Dr Emma Williams at E.V.Williams@bristol.ac.uk For further information on this work contact Prof Guy Rutter at g.a.rutter@bris.ac.uk In addition to two specialist objectives with NAs of 1.65 and 1.45, Olympus provides illuminators, condenser lenses and a range of cameras for TIRF users. A choice of configurations is available for TIRF and users are advised to consult their Olympus Area Manager to help evaluate the best solution.

The Olympus Fluoview confocal system is establishing a growing user base across Europe. First time customers are coming back for a second or even a third system, thanks to the Fluoviews combination of performance with ease of use. For more information go to http://cf.olympuseuropa.com/micro/products/live_science/
For a detailed look at TIRF go to http:/www.olympusmicro.com/primer/techniques/fluorescence/tirf/tirfhome.html

Extra High Resolution

The latest digital camera for microscopists
employs an innovative shifting CCD system together with super-fast hardware processing to give extra high resolution for still images or high speed imaging for easier focusing.
The DP70 user can obtain still images, at a maximum recording size of 4080 x 3072 pixels - an effective resolution of 12.5 million pixels, without interpolation - in approximately three seconds. Alternatively, the DP70 can display live images at 680 x 512 pixels at up to 15 frames per second.
When an image is wholly or partially dark, as is common with fluorescence specimens, the binning function allows preview images to be displayed without delay by combining the light intensity of 2x2 or 4x4 pixel areas. Image control software allows the user to look at the full camera image, then zoom in or move around the image, clipping and saving any part of the image. A scale on the image provides a reference to the specimen size.
Dr Fiona Ross at Wessex Regional Genetics Laboratory in Salisbury, part of the Salisbury Healthcare NHS Trust, is being supported by the Leukaemia Research Fund in association with the UK Myeloma Forum to look for cytogenetic abnormalities in multiple myeloma patients. With more than 700 patients involved in the study, the volume of screening is considerable - so the laboratory is setting up an automated system to facilitate this. We are using FISH (Fluorescence InSitu Hybridisation) to look at around 100 cells for each patient - and can use at least 16 probes on an analysis. Explains Dr Ross.

We are using the Olympus IX71 as a conventional fluorescence microscope to undertake visual analysis, but we want to work it full time in creating the images. We are evaluating Applied Imagings SPOT system - an automated scanning platform for the documentation and analysis of interphase FISH slides. Every image captured is stored in the onboard database, so we can leave the system operating overnight and quickly scan through the images
Olympus BioSystems is a new company established to develop integrated imaging solutions in Life Sciences. Based in Planegg, near Munich, Olympus BioSystems brings together the expertise of software and hardware specialists, researchers, and of course Olympus. With these partnerships it can deliver optimised application-based systems for specific experimental tasks in cell research.
Within the MRC Centre for Synaptic Plasticity in the Department of Anatomy at the University of Bristol, Neil Bannister and John Isaac make patch-clamp recordings from spiny stellate neurones within layer 4 of mouse somatosensory (barrel) cortex. This is the part of the brain that processes information from the animals whiskers. It is highly developed in rodents and serves as a good model for how all mammals, including humans, process sensory information. The team use an Olympus BX51WI microscope equipped with water-immersion DIC optics to visualise barrels using low 4x magnification and to identify individual spiny stellate neurones, under high 40x magnification, in order to record from them. The work is part of an investigation into the mechanisms by which synapses change strength during development in the barrel cortex. This developmental synaptic plasticity is thought to be the process by which the brain wires itself up in response to experience during development.
Complete fluorescence imaging with
The first system from Olympus BioSystems is the new CellR - a complete, self-contained imaging station for biological fluorescence microscopy. The workstation brings together an all-in-one fluorescence illumination system, the finest CCD cameras, real-time image acquisition, and peripherals including stages, z-drives, emission filter wheels and micro-injection. All are managed and synchronised from the central control unit. CellR supports the representation and analysis of processes in living cells with maximum precision. The CellR imaging station is tailored for fast time-lapse acquisition, multi-colour imaging, ion ratio, z-stack, TIRF and FRET applications.

SENSORY PERCEPTION

Low magnification (4x objective) of barrel cortex from a 4 day old mouse. The distinctive barrel formations are visible within layer 4 (arrows) using DIC optics. A glass microelectrode has been inserted into the slice (within one of the barrels).
High power magnification using a 40x objective. DIC optics enables us to visualise the cell bodies of spiny stellate neurones which populate layer 4. A glass microelectrode has been positioned up against one of these cells to enable us make a whole-cell patchclamp recording from it.
Imaging of Astrocyte Calcium Signalling
Arthur Butt and Greg James, of the Centre for Neuroscience Research at GKT Guys Campus, Kings College, London, are looking at the role of adenosine triphosphate (ATP) in glial calcium signalling. A single application of ATP, a neurotransmitter in the brain, can be seen to produce calcium signalling in glial cells of the isolated optic nerve of adult rats. To observe this, the researchers loaded the nerve with the calcium sensitive dye Fura-2 and excited at 340:380 nm using a Cairn Optoscan. The nerve was viewed on an Olympus BX51WI microscope through a x20 0.95 NA water immersion single position objective and using an intermediate magnification changer. Images were captured via an intensified CCD camera (Cairn Research, Kent) and sequences analysed using Axon Imaging Workbench software. (Axon Instruments/Indec, USA). The individual glial cell bodies are visible and the changes in fluorescence intensity from blue to red are a measure of increase in cytosolic calcium concentration. The images are taken at increasing time intervals after application of ATP and the traces represent the response of the 4 cells identified in the upper left image.The results show that ATP evokes calcium elevations in optic nerve glia in situ, supporting a role for this neurotransmitter in glial calcium signalling.

v F/ F

CYTOLOGY COMBINATION
Laser Scanning Cytology (LSC) combines flow cytometry with image analysis to give statistical data on large numbers of cells using automated fluorescence microscopy techniques. It extracts measurements from thousands or millions of cells with fluorescence and laser light scattering techniques, and then analyses that data. Compucytes exclusive LSC technology combines an Olympus IX71 microscope with multiple laser excitation and scattering, image acquisition and analysis and detailed cytometric data analysis. Applications include: s Studying cultured cells on the individual and colony level simultaneously. s Studying the interactions between cells of different types. s Re-analysing the same cells under varying conditions and performing kinetic experiments with merged data sets. s Detecting molecular constituents in the surrounding environment and correlating their presence with cell processes.

Support for Alzheimers Research

TOP TIPS

for phase contrast set up
Requirements Phase contrast condenser with relevant annuli (ring slits) Phase contrast objectives to match Centring telescope or Bertrand lens Green filter (optional) Unstained specimen, e.g. cheek cells Setting up 1. First check that Koehler illumination is set up, using a stained specimen if necessary. 2. Focus on the unstained specimen in brightfield using a phase contrast objective. Closing the aperture stop may help visualise the specimen. 3. Bring the relevant phase annulus into the light path. This is usually done by rotating part of the condenser, or by moving a slider in the condenser. Check that the annulus matches the objective, e.g. PH1 or 10X. 4. Either remove an eyepiece and install the phase telescope in its place, or bring the built-in Bertrand lens into play. Focus this device to provide a sharp image of the circles. 5. Locate the phase centration screws on the condenser (not the condensers centring screws) which may be permanently fitted to the back of the condenser (they will need to be pushed in to operate them) or stored separately, perhaps in the microscope frame. 6. Rotate the phase centration screws while observing through the telescope. Move the bright circle (the light from the annulus) until it aligns exactly with the grey circle (the phase plate in the objective). Note that if the circles are different in size you have selected the wrong annulus for that objective. 7. Repeat the centration process for each phase objective in use. 8. Remove the telescope and replace the eyepiece or take the Bertrand lens out of the light path. Other points
Over the past fifteen years we have established a library of over 700 brain specimens, which is the largest in the UK possibly the world.
An Olympus CH microscope has been donated to Dr Patrick Kehoe of the John James Laboratories at Frenchay Hospital in Bristol to support the groups work in the study of Alzheimers Disease and other dementias. The John James Laboratories form one of three units headed by world renowned geriatrician Professor Gordon Wilcock of the University of Bristol, Department of Care of the Elderly. Collectively the units are known as the Bristol Dementia Research Group (BDRG). The units are partly funded by charitable donations through BRACE (Bristol Research into Alzheimers and Care of the Elderly). This Bristol-based registered charity (http://www.alzheimers-brace.org/) was formed in 1987 to help support the research programme of the BDRG and to raise public awareness of dementia and the need for research. Over the past fifteen years a library of over 700 brain specimens has been established at the John James Laboratories, which is the largest collection in the UK and possibly in the world. This is a valuable resource in researching into potential causes of the disease.

In Alzheimers sufferers, their brains are found to contain senile amyloid plaques and neurofibrillary tangles, and these plaques are clearly identifiable under the microscope when targeted with a silver stain. But it is not known whether these are causative of memory loss or are themselves a consequence of other changes. One aspect of the work at the Laboratories involves looking at processes in the brain in order to identify the proteins involved, then studying the particular genes responsible for each protein to see if any mutations or polymorphisms affect the performance of the protein. DNA extracted from a brain sample the size of a pea is sufficient for several years of analysis. The multidisciplinary team can also look at DNA damage in individual cells in fresh blood using a technique called a Comet assay the altered metabolism in Alzheimers patients brains, combined with the aging process, generates a high level of reactive oxidant products which cause DNA damage.
s A green filter will help give a sharp image. s Phase contrast objectives are identified with the engraving Ph, PL, Phaco or similar lettering. Olympus also uses green engraving on objectives. s Cheek cells make good specimens for setting up. Scrape out some cells ( using, e.g. a coffee stirrer) and mount them in saliva on a slide and coverslip. s The illumination will need to be increased for phase contrast, so take care to reduce it again when viewing in brightfield. s Many phase condensers have a darkfield position. Take the opportunity to see what the images look like. Notice how well edges and particles (such as bubbles, dust and the edge of the coverslip) show up.

Control

Quantitive image analysis of in vitro angiogenesis
Immunostained endothelial microtubules

Digitised images

Quantitation
The images show immunostained endothelial microtubules in control and test samples ( top left and right ) and the digitised images ( below ) from which measurements are taken.
Dr. Suzanne Eccles of the Cancer Research UK Centre for Cancer Therapeutics, part of the Institute of Cancer Research in Sutton, Surrey is using automated image analysis to test novel compounds for anti-angiogenic effects.

There is considerable interest in the strategy of combating cancer not only by attacking tumours directly, but by choking off their blood supply with relatively nontoxic drugs. These angiogenesis inhibitors can destroy the fine network of blood vessels that tumors must have to grow and metastasize. Several pharmaceutical companies and research institutes are racing to make and test a variety of angiogenesis inhibitors. At the Centre for Cancer Therapeutics, in one of the functional assays used, endothelial cells are plated onto Matrigel with or without test compounds and allowed to differentiate into tubules which are visualised with anti-CD31 antibody.
Using an Olympus IX70 and Image-Pro Plus software, digital images are produced of the tubules and thresholds set to eliminate noise. A macro calculates angiogenesis area and/or perimeter and control and test wells are compared. This enables the Centre to quantitatively measure the effectiveness of various compounds in inhibiting the growth of blood vessels. A motorised Ludl stage allows automated readings in 24 well plates so that a number of compounds can be tested simultaneously with minimal hands-on involvement for the researchers.
Image Processing and Optical Technology, more commonly known as IPOT, will take place at Birminghams NEC on February 12th and 13th. Once again it will be an opportunity to see a broad selection of technology from companies such as Olympus, including cameras, software, stages and other innovative imaging equipment. For Olympus the main focus will be the BX61 motorised microscope. With in-built motorisation and developed to interface with peripherals such as motorised stages under software control, it is ideal for the most demanding applications in semi-automated microscopy. Last year, Olympus microscopes were present not just on our own stand but supporting a number of other companies in demonstrating their microscopy tools - showing just how strong Olympus co-operation within the industry is in developing complete imaging solutions. Come and have a look you will find a warm welcome and helpful advice on Stand No. B10 and B9 in Hall 12 of the NEC.
Dr Liliana Neaga, Head of Pathology at the Salaj District Hospital, Romania, is seen with an Olympus microscope used to facilitate clinical meetings between doctors of different disciplines. Two microscopes in Cytology and Histopathology are also from Olympus. Peter Colquhoun, director of Medical Support in Romania is visiting the hospital in May this year. He will be delivering another microscope donated by Olympus for the Serology and Immunology Laboratory.
NEW SUPPORT FOR IMAGING APPLICATIONS
Olympus is expanding its team
with the introduction of new members with valuable experience.
Peter Call has worked in cytogenetics, immunology and microscopy for several years. As an ex-Chief MLSO at Stoke Mandeville Hospital and having gained valuable experience within the industry, he is ideally placed to advise and service customers in the south of England. Raj Hathiwala was with Shell Research for 11 years before joining a digital imaging company. Now, his role with Olympus is to support the imaging needs (including digital cameras and software) of hospitals, university departments and the industrial sector in the North. Alan Kidger has a background in supporting advanced software applications in microscopy image analysis. As a graduate in Applied Biology and with experience as a Bioanalyst for a pharmaceutical company, customers in Eastern England will appreciate his specialist knowledge. Andy Billington joins from a strong imaging background and will provide office-based support.

Win one of three superb Olympus Superzoom cameras - in just two minutes!
Instead of our usual competition, in this issue wed like to know what you think about Objectives. If you can spare a couple of minutes to complete the questionnaire on the reply card the first three drawn out of the hat will receive an Olympus Superzoom 105G compact 35mm camera with 3 X zoom lens, 6 flash modes and self-timer. Your feedback is essential for us to continue supplying useful and interesting information to all microscope users and dont forget you can use the reply card to request further information on Olympus microscopes and imaging systems. To be in the draw, reply cards must be received by 12th May 2003.

CONGRATULATIONS

Hats off to Keith Davidson, a research scientist at the Babraham Institute, the lucky winner of our latest competition. Tina Howe of Olympus is shown presenting Keith with an Olympus C-3020 ZOOM digital camera.
To request further information:
Return the response card, telephone: 0179, fax to 4677, email microscope@olympus.uk.com Olympus Optical Co. (UK) Limited, Dean Way, Great Western Industrial Park, Southall, Middlesex UB2 4SB www.olympus.co.uk