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One Frame, Endless Possibilities A new open source microscopy concept opens up the infinite light path Wolfgang Hempell and Simon Reiss
To change the constraints of the traditional microscope system, Olympus has developed an new open source microscopy concept, based on the inverted IX3 microscope frame series. Designed around an accessible infinite light path concept, optical modules can be easily exchanged, moulding the microscope to the diverse requirements of the user. The series suits anything from routine observation to advanced imaging techniques in all live cell applications. Figure 1 shows an image taken using a semi-confocal observation method with a disc-scanning unit. The frames can be easily adapted to apply such observation methods. Concepts in microscopy are constantly changing. Up until a few years ago, microscopy systems were purchased as instruments intended for a limited range of applications, and altering a microscope required a certain level of engineering expertise to dismantle the apparatus and modify the required hardware. This tends to lie beyond
Company Olympus Europa GmbH Hamburg, Germany
Olympus is one of the world’s leading manufacturers of professional microscopy systems for research, clinical and industrial applications, with a European base in Hamburg, Germany. For over 80 years the company has dedicated its in-house expertise to providing complete microscopy solutions for both the life and materials sciences, delivering expert results with intuitive operation. From microscopes for training and routine tasks to high-end imaging system solutions, Olympus has a system to suit every need. www.microscopy.olympus.eu
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Fig. 1 An example of imaging easily achievable on the IX3 frame. An Obelia stained with GFP and DAPI, using a semi-confocal observation method with a disc-scanning unit.
the scope of many microscopists, and therefore future-proofing in microscopy has traditionally been very difficult to achieve. Overcoming these limitations, Olympus developed the IX3 range of inverted microscope frames, optimised for live cell imaging. The frames are built around a swappable deck design, with an architecture analogous to a chest of drawers; interchangeable modules fit into the deck and can be easily slipped in and out of the light path as required (Fig. 2). Since this light path is infinite, inserting complex optical components will not introduce errors resulting from optical aberrations.
The IX3 range can therefore be easily optimised for any application, from routine observation through to the specialised requirements of advanced imaging technologies such as fluorescence recovery after photobleaching (FRAP), photoactivation and high content screening. With such an accessible light path, the IX3 range allows any user to rapidly and effortlessly re-structure the microscope to suit the aims of the experiment. The IX3 range includes three different models. The IX83 is the most advanced system in the range, with fully automated control co-ordinated by the Olympus cellSens imaging soft-
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Trends in Microscopy
Fig. 3 Insertion of the encoded intermediate magnification changer module into the two-deck IX83 frame. Adding this module into the light path allows control of 1×, 1.6× and 2× magnification settings, and automatically saves these settings alongside image data.
Fig. 2 The IX83 inverted microscope frame. With a fully accessible infinite light path, anyone can easily change the function of the microscope by swapping in one or two interchangeable modules. It also contains a specialised port for the Olympus Z-drift compensation unit, for precise image focus.
ware. The IX73 is instead optimised for manual and semi-motorised flexibility, and both models are available as either a one-deck or a two-deck system, where each deck is capable of holding a single module. The IX53 is available as a single deck system for cost-effective routine sample analysis.
Building blocks A range of optical modules are currently available, and this collection is sure to expand in the future as the concept of the open source microscope takes hold. Multiple combinations of modules can be chosen in such a way as to allow users to mould the components to best suit their workflow. For precise control of magnification settings in any experiment, the user can insert the encoded intermediate magnification changer module into the light path. This allows the control and recording of magnification settings between 1×, 1.6× and 2× which are directly encoded within the cellSens software, and saved alongside the captured image. An example of how this module is employed in brightfield microscopy is shown in Figure 3. In fluorescence applications, the user can insert the fluorescence filter turret module into the infinite light path, which is available either as a motorised or encoded unit. For example, multichannel imaging of HeLa cells (Fig. 4) can be achieved using separate filter positions. The turret can incor-
porate up to eight cubes which can be easily switched without the use of tools. Within the two-deck frames, this can be combined with the magnification changer as shown in Figure 5, for automatic communication of magnification settings in fluorescence experiments. Alternatively, adding two fluorescence mirror turrets allows the simultaneous use of two light sources, ideal for applications such as photoactivation, which requires distinct light paths and filters for fluorophore activation and imaging. Building upon these possible set-ups, the third unit currently available for the IX73 and IX83 is a right side port module
with a C-mount, which allows access to components such as detectors and light sources that can be directed into the infinite light path (Fig. 6). This allows the use of an additional laser for FRAP experiments. Furthermore, when used alongside the fluorescence filter turret module, the connection of an extra camera enables the construction of a system optimised for dual camera observation, such as fluorescence and colour images of specimens. This is ideally suited for the brightfield observation of coloured specimens in parallel with fluorescence microscopy. Figure 7 shows an example of an application in pathology: a coloncarcinoma can be visualised by fluorescence with a monochrome camera attached to the right side port module, and by brightfield using a colour camera on the standard camera port.
Fig. 4 Fluorescence capture of HeLa cells is achieved here using separate filter positions, and merged to form a multichannel image. The fluorescence cube turret module enables multichannel images – mitochondria in green, endoplasmic reticulum in red and cell nuclei in blue.
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Fig. 5 The two-deck IX3 frame with fluorescent cube turret module. This module can be included for fluorescence applications, with up to eight fluorescence cube positions that can be controlled manually with the encoded version, or automatically with the motorised version.
Fig. 6 Insertion of the right-side port module in the two-deck IX3 frame. This right side port module provides access to a variety of light sources and detectors.
Fig. 8 The Olympus Z-drift compensation module is inserted into the specialised deck of the two-deck IX83 frame. This module employs IR light to ensure accurate sample focus with either a one-shot or continuous mode for time lapse experiments.
Automated focus adjustment
In addition to the ZDC, the square architecture of the IX3 frame itself provides increased rigidity that reduces the impact of both vibration and heat. It maintains position along the axes to enable reliable time-lapse imaging.
High performance optics are delivered by Olympus UIS2 infinity-corrected objectives, engineered to provide sharp, bright images, while the proprietary fly-eye fluorescence illuminator generates even, vivid illumination across the specimen. This enables a much wider field of view than previously possible with even illumination that fills the detectors of most cameras, even those with larger chips, creating a field number of up to 22 on the camera port.
In addition to modules used to diversify the function of the microscope system, the IX83 frame also includes as standard a specialised port for the Olympus Z-drift compensation (ZDC) module (Fig. 8). The ZDC uses low phototoxicity IR light to maintain precise focus, and uniquely includes two modes, for either one-shot or continuous focus under all circumstances. The one-shot mode allows several focus positions to be set, enabling efficient Z-stack acquisition for example in multi-position experiments. Continuous mode is instead suited to fast time-lapse capture, avoiding focus drift due to temperature changes or the addition of reagents. The ZDC module achieves an accuracy of the focus within 70 nm which is of particular importance for time-lapse experiments with comparatively thick specimens. It operates independent from any software, which ensures precise focus adjustment even when the frame is completely customized to an individual experimental setup.
Ergonomic design The IX3 frames provide several further enhancements to optimise user experience. These include a new motorised stage that provides precise and fluid control, making sample positioning at high magnifications an easy task. The frame under the stage has been ergonomically designed, utilising a removable drip tray that helps to prevent contamination and simplifies maintenance of the optical system. IX3 is compatible with the Olympus Real-Time Controller, offering high-precision imaging via microsecond synchronisation of filter wheels, shutters, light sources and cameras for advanced studies using high-speed light sources and triggered cameras, with the IX83 being capable of controlling up to six filter wheels and four shutters.
Customised microscopy: one frame for any study The benefits of the IX3 open source frame design are far-reaching, and set to revolutionise the way we perceive microscopy. In situations where multiple users depend upon a single microscope to perform a diverse range of applications, the open source frame concept really comes into its own. In a shared microscopy suite for example, each user must compromise with a single all-purpose instrument. In place of this, an IX3 system with a choice of different modules would allow each
Fig. 7 An example of dual camera use via the right side port module. Analysis of pathology sections: a colon carcinoma has been imaged using a colour camera for brightfield, and a monochrome camera for fluorescence.
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user to quickly swap in their required modules at the start of the session, and the microscope is instantly optimised for the experiment at hand. It is equally easy to imagine how users of advanced and complex technologies can benefit from such a fully accessible light path. The IX83 is meant to serve as a basis for a variety of high-end imaging systems, such as confocal laser scanning microscopy or high content screening.
Infinite possibilities The scan^R 2.4 high content screening station is one example of the growing collection of microscopy systems based upon the IX3 frame. The recently updated scan^R 2.4 station can be incorporated into the the fully automated IX83, combining the benefits of modular frame design with increased scan speed. The system delivers quantitative expert results in a vast array of high content screening of cell-based assays, from gene expression to bacterial infection assays and is optimised for many different assay formats, including multi-well plates, slides and custom-built arrays. In addition to those systems designed by Olympus, the possibility of integrating optical modules designed by third party vendors such as Prior Scientific, Sutter Instruments and Rapp OptoElectronics, opens up many additional applications of the IX3 frame. Prior Scientific has already released their high-speed filter wheel module designed specifically
for the IX3 range, which is mounted into the deck using a support known as the Breadboard platform. Utilising this platform along with custom components, users can optimise the microscope according to their specific needs, benefitting from the open source concept.
Summary The unique IX3 frame design aims to change microscopy, as systems of integrated components can be moulded and optimised for the highest performance in any application. Access to the infinite light path allows rapid and effortless switching
between applications such as brightfield, fluorescence and dual camera imaging, without specific engineering knowledge or tools. This open source concept allows the IX3 microscope systems to grow alongside the evolving demands of any life science research project. Perhaps one of the most intriguing aspects of this concept is handing over system design to the end user. How will modules be developed by the microscopy community, to push the boundaries of technology forward and advance the speed at which microscopy moves into the future? DOI:10.1002/opph.201300002
Authors Wolfgang Hempell
studied photoengineering at the Cologne University of Applied Science, with a particular interest in microscopy and the development of sensors for quality control of optical lenses. He joined Olympus Europa in 2001 and has spent the last decade working on the development and marketing of innovate microscopy systems for the life sciences. He is currently section manager at Olympus responsible for the product management of imaging and microscopy products.
Dr. Simon Reiss
studied biology at the University of Heidelberg and gained his Ph.D. in infectious diseases. His main focus was on targeting human proteins in order to disrupt Hepatitis C viral replication. He continued his scientific research as a postdoc at the university before joining Olympus Europa at the beginning of this year. He is currently working as a product manager for the imaging and microscopy division at Olympus involved in the development and marketing of the IX3 microscope systems.
Wolfgang Hempell, Dr. Simon Reiss, Olympus Europa GmbH Tel: +49 40 2 37 73-0, E-mail:
[email protected], www.microscopy.olympus.eu
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