A Ray of Light. Fluorescence Light Sources. Fluorescence Illumination Mercury, Xenon and LED

Transcription

1 A Ray of Light Fluorescence Light Sources Fluorescence Illumination Mercury, Xenon and LED

2 2 MOOD IMAGE 1

3 CONTENTS THE LIGHT OF LIFE Pushing research forward The advent of fluorescence microscopy has enabled all researchers to see greater levels of detail more clearly than ever before. For all processes, no matter how complex, the growing range of dyes and markers has allowed research into life processes to surge ahead, moving microscopy from being static to dynamic. New dyes have extended the range of normal fluorescence microscopy, enabling more indepth observation over extended time periods of days or even months. They have also led to a number of unmatched advancements in Olympus microscope optics, such as the new UIS2 objectives and fluorescence filter sets. The addition of a range of digital cameras, various imaging software platforms and other modules enables Olympus to provide the correct system solutions, whatever the requirements. Arc burner light sources 4 7 Arc burners have been the mainstay of fluorescence illumination in microscopy for a long time and a range of improvements have been implemented to ensure that they are still excellent options for the majority of procedures. Arc burners use mercury (Hg) or xenon (Xe) to provide bright light across a broad spectrum of wavelengths. Recent developments have greatly improved functionality, including alignment-free bulbs and increased lifetime. LED light sources 8 11 LEDs have proved themselves as excellent alternatives to standard bulbs in many lighting situations and have now also entered the microscope fluorescence light source arena with outstanding properties, such as their compact size, stable alignment-free light and extended life. Olympus has two LED systems, one providing routine transmitted light facilities and the other designed for more advanced reflected light processes. Both are easy to control and provide flexible fluorescence. Real-time illumination systems Real-time microscopy needs fast illumination control to capture short-lived events. Therefore Olympus has developed a range of specialised illumination systems which provide bright and stable fluorescence excitation light from arc burners, which are easy to control in tune with your experiments. Your Vision: Our Future Olympus is dedicated to providing the best combination of microscope components to support your work at all levels. We have therefore developed a comprehensive range of fluorescence illumination sources to make the most of any application, whatever the microscope.

4 4 CHAPTER I ARC BURNERS Let there be light Arc burners are accepted as the standard source of fluorescence for microscopy. Olympus has enhanced the design of its burners to provide a number of improvements, including a reduced chance of incorrect use, which provides higher data fidelity, better stability and functionality. These ensure that researchers can concentrate on their work rather than on the light source.

5 ARC BURNER LIGHT SOURCES BURNERS AND BEYOND A UIS2 objectives Lead-free optics Every component of a fluorescence microscope s light path is important to the overall image produced. Probably the single most important optical component, though, is the objective lens, since both the fluorescence illumination and emission pass through it. The new UIS2 objective range from Olympus uses new glasses to not only provide the best resolution and clarity, but also to significantly reduce the autofluorescence, consequently greatly improving the S/N ratio. Furthermore, carefully designed anti-reflection coatings provide broad transmission width and outstanding chromatic aberration correction for reduced focus shift. The UIS2 optical system also features excellent fluorescence filters with similar benefits, which enable steep spectral flanges. These improvements provide the entire light path with a 10 20% increase in efficiency and an accuracy of ±2 nm. With each optical system providing the best characteristics for the research being conducted, Olympus has a range of different fluorescence arc burner solutions to perfectly complement each microscopy system; from lamphouses to fully integrated units. Olympus has continued developing these solutions to offer the best illumination for all protocols. For example, improvements to the collector lens maximise light intensity, and Olympus lamphouses ensure that lamps are simple to fit and easily aligned. The arc burner-based illumination systems available from Olympus also provide other enhanced features to ensure that the fluorescence illumination is taken to a higher level. The X-Cite 120 range and MT_10D modules feature more precise control via the family of imaging and analysis software, advanced electronic power control for longer burner life, as well as integrated intensity (attenuation) and colour filters. Furthermore, the X-Cite 120 range uses a specialised metal halide technology, to further increase lamp life whilst keeping the same wavelength spectrum. Building on the MT10_D system, the MT10 and MT20 systems provide an excellent balance of speed, flexibility and brightness, and can use different burners for expanded research possibilities. For the ultimate in speed, though, the Polychrome V grid monochromator provides the fastest wavelength changes across its spectrum in less then 400 ms. B C Fluorescence filter cubes In various colours Fluorescence lamphouses With Mercury or Xenon burner D Fluorescence light path On a BX51 microscope

6 6 A X-Cite 120PC Metal halide fluorescence illumination system X-Cite 120 A The X-Cite 120 XL range will provide the same fluorescence spectrum and similar intensities as a standard 100 W mercury burner, but will ensure an additional level of consistency and safety, making them excellent options for a broad range of requirements. Furthermore, the units are thermally uncoupled from the microscope, with a liquid light guide delivering the light through collimators to the Olympus BX2, IX2, SZX2 or MVX microscopes. B X-Cite interface In software Three times better B The three versions of the X-Cite 120 XL range provide each user with the correct solution for their fluorescence illumination requirements. Each of the three brings the same level of consistency and safety, but with different levels of controllability. The entry level model offers a consistent intensity level and is controlled directly from the unit. The X-Cite 120 XL Iris model adds to this with five intensity levels set by an iris system (0%, 12%, 25%, 50% and 100%), enabling finer control for sensitive and bright samples. The X-Cite 120 XL PC combines all these features with PC-based control via an RS232 interface. Unique longevity C The X-Cite 120 XL range uses alignment-free metal halide burners, making them much easier to handle and move between different microscopes. By using metal halide technology, much of the tungsten eroded from the electrodes during burning, which is normally deposited on the inner bulb surface, is recycled back to the electrodes. This slows down the widening of the arc gap, which in turn decreases the rate of intensity reduction. This, coupled with the electronic control gear (ECG), which ensures that as the gap between the electrodes grows the correct voltage is used to generate a consistent arc, greatly extends the life of the burner and guarantees at least 2,000 hours of use. C Pre-aligned burner For X-Cite 120 Typical lamp degradation for X-Cite 120 XL Degradation 90% 80% 70% 60% D Collimator For X-Cite % 40% 30% 20% 10% 0% ,000 1,200 1,400 1,600 1,800 2,000 Elapsed time (hours) Safety first The unique IntelliLamp system monitors and maintains the optimum lamp temperature to ensure consistently safe operation and even prevents the lamp from being turned on if it has not had time to cool down. Furthermore, it automatically tracks the lamp s usage time importantly, this information stays with the lamp, even if the lamp is moved to a different unit. The ECG system also ensures that once the voltage used to maintain the arc reaches a certain level it is shut down safely. This greatly decreases the risk of dangerous lamp explosions.

7 ARC BURNER LIGHT SOURCES MT10_D A The Olympus MT10_D features all the components necessary for advanced multicolour fluorescence excitation illumination. It includes the burner compartment, an 8-position filter wheel, 7-level light attenuator and high-speed shutter. This versatile all-in-one system can be controlled conveniently via the manual controls or smoothly integrated into the different Olympus imaging software programmes. A MT10_D Advanced fluorescence illumination Convenient and easy A The intuitive manual control can be easily configured and incorporates a digital status display which makes all of the functions very convenient and straightforward. Further unique tool-free features simplify other processes such as excitation filter changes, lamp exchange and intensity optimisation via an integrated photodiode, all of which also reduce downtime during maintenance. Fast and flexible Modern cell labelling techniques enable researchers to use multiple fluorescence dyes and proteins to provide simultaneous observation of different cellular structures and physiological parameters. This is made possible by the MT10_D due to the fast internal 8-position filter wheel, which switches between different filters in just 85 ms. The 7-level attenuator provides quick adjustments in light intensity and the highspeed shutter can be switched off and on in less than 5 ms, providing fine control over the light entering the microscope optics. B 150 W Burner Xenon or mercury-xenon Bright and stable B C The peerless Olympus MT10_D can use either a 150 W Xenon or a 150 W mercury-xenon burner, depending on the wavelength requirements of the research work. The advanced electronics ensure that both burner sources provide bright stabilised light over long periods of time, ensuring the utmost data reliability and consistency. Light is transferred to the microscope via a quartz fibre coupling, which guarantees the highest levels of light efficiency. Control and upgrade The MT10_D can be controlled from a PC using the family of advanced software packages via RS232 or USB hardware communication interfaces. The MT10_D can be upgraded to the MT10 or MT20 real-time illumination systems to provide even better parameters for live-cell imaging. For more about the MT10 or MT20 illumination systems, see chapter C Relative intensity (%) Highly stable light output Comparison with standard Hg lamp houses: light output normalised to the average intensity (100%). MT10/20 0 Hg-Xe ebu burner Standard ard Hg burner Time (min)

8 8 CHAPTER II LED LIGHT SOURCES The future is bright and colourful LEDs have been around for many years, finding new applications as the technology advances. As a result, LEDs can now be used as alternative sources of fluorescence illumination for microscopy. Importantly, the most recent advances have made them an even more attractive solution: from extremely long lifetimes with very little drop in intensity, to high-speed electronic control and completely stable illumination. LEDs are also very safe and reduce the requirement for intensity filters. All in all, LEDs offer a new level of functionality, bringing fluorescence microscopy within reach of an even wider range of applications.

9 LED LIGHT SOURCES A NEW KIND OF LIGHT A With Olympus microscopes already pushing the boundaries of fluorescence microscopy with excellent optical systems, the introduction of light emitting diode (LED) -based fluorescence light sources further expands the possibilities. LEDs use solid-state electronics to emit light within very tight wavelength bands. This enables more precise illumination of fluorescent dyes with reduced bleed-through and also simplifies control since one LED can be used for each specific illumination wavelength range. LEDs are therefore well placed to maximise the impact of new fluorescent dyes. Furthermore, LEDs offer a very long lifetime (guaranteed minimum of 10,000 hours) with excellent lumen maintenance. This means that during an experiment (however long), the light intensity will hardly change, which means that LEDs provide highly reproducible results and the ability to be fully quantitative. This is also useful for screening processes where illumination is key to consistency. LED lamp An important aspect, especially in fluorescence live-cell imaging applications, is the protection of the specimen. The tight wavelength bands emitted by LEDs make it easier to avoid the dangers of overexposure to light: phototoxicity of the specimen and photobleaching of fluorescent dyes. Moreover, LEDs do not emit heat in the form of light, thus the risk of specimen damage by IR radiation is reduced. B Typical construction Of a high-performance LED Gold wire Lens Body LED chip Arc burner units use shutters to control when samples are illuminated, whereas LEDs can be switched on and off very quickly, which provides more controllable illumination windows (into the microsecond range) and therefore also reduces unwanted illumination. LED-based light intensity is controlled uniformly by varying the voltage and therefore there is no need for neutral density filters as with arc burners. With fewer mechanical components such as shutters and filter changers, and the flicker-free nature of LED light, image stability and ease of use are both improved. Cathode lead Heatsink slug Anode lead Silicon sub-mount chip LEDs are mechanically very stable and also pose no threat of explosion or exposure to poisonous gases, which is a great benefit, especially for inexperienced users. Environmentally, LED-based fluorescence illumination systems offer lower power consumption due to the increased efficiency of LEDs, reduced reliance on mechanical components and there are no disposal issues. This also means that they have lower running costs.

10 10 A FluoLED CX41 with multi-fluorescence version FluoLED The FluoLED system brings both flexibility and value to LED-based fluorescence, making it perfect for routine and educational requirements, but with the added benefit of in-field operation. The system has been specifically designed for use with transparent specimens, such as bacteria and thin sections, which require transmitted fluorescence illumination. The system provides very good signal-to-noise ratios (S/N) with high intensities and is very easy to retrofit onto the Olympus CX upright microscope range. B Single fluorescence unit Illuminator with LED cassette All-rounder A The entry-level FluoLED fluorescence excitation illumination system introduces additional flexibility to fluorescence microscopy. It is designed to fit Olympus CX microscopes making it ideal for educational purposes. Further extending this application area is the ability to power the LEDs by battery or even solar power, ensuring that fluorescence microscopy can be conducted in field where samples are fresh. Straightforward B C D The FluoLED system is available in three versions, all of which attach directly to the microscope to provide transmitted fluorescence, without affecting the use of the normal transmitted brightfield illumination: the FluoLED EasyBlue (480 nm) provides a single wavelength of fixed intensity for the most cost-efficient solution. The FluoLED Single provides users with the capability to use one of seven interchangeable LED cassettes from 365 nm (UV) to 630 nm (red) and control the intensity of the light. The FluoLED MultiFluo enables up to three of the interchangeable LED cassettes to be installed and controlled on the microscope at once via a three-channel electronic driver. C Multi-fluorescence unit Illuminator with three LED cassettes Simple to use A With no lamp alignment requirements or the need for excitation filters, the simplicity of the FluoLED systems makes them easy and intuitive to use. An integrated UV-blocking filter and a filter slider, which holds the emission filters for signal detection, ensure that the systems are safe for the user s eyes. D FluoLED LED cassette A long time going D With a minimum lifetime of 30,000 hours and a 24-month warranty on each LED cassette, the FluoLED systems are not only cost-effective to purchase, but also very efficient to run. The low power usage also ensures that fluorescence microscopy is not a drain on electrical supplies when they are scarce, and that they can be used in the field from non-mains sources (excluding the MultiFluo).

11 LED LIGHT SOURCES precisexcite The precisexcite is a peerless LED fluorescence light source, featuring the latest advances in the LED technology and delivering the most comprehensive range of wavelengths with the highest available intensities. A precisexcite Easy adaptation to all Olympus fluorescence microscopes Technically advanced A It is sometimes said that light-emitting diodes do not produce heat. But they do! Even the best LEDs available today transform most of the applied electric current into heat and not into light. And LEDs don t emit heat in the form of IR light. Efficient cooling is therefore a must to avoid detrimental effects on LED performance such as reduced intensity, spectral shifts and reduced lifetime. The precisexcite is the only LED illumination system on the market to use specially developed, proprietary LED array design and a Peltier cooling system which enable it to attain intensities not possible on other systems. This also helps to increase the life and stability of the LEDs to provide in excess of 10,000 hours of use with no more than a 7.5% decrease in light intensity. Rapid modularity B The LEDs are provided as LED array modules (LAMs) which are easily interchangeable and enable up to three different wavelengths to be used. The precisexcite is supplied with LAMs for 400, 465 and 525 nm wavelengths perfect for DAPI, FITC and Rhodamine dyes, for example. The LAMs are automatically recognised by the precisexcite so changing between different modules is both quick and easy. Each LAM has a three-year guarantee and it is therefore possible to reduce running costs to an absolute minimum. B Installation and control The output from the precisexcite is designed for reflected light microscopy and is delivered to the microscope via a liquid light guide, which only needs to be aligned once at installation. The combination of the advanced LED system, liquid light guide and collimator ensures that the precisexcite provides even illumination across the entire field of view. The use of LEDs means that there is little need for intensity filters since the unique electronic control system enables intensity settings from 0 100% in 1% increments. precisexcite LAM: The brightest fluorescence LEDs currently available C The precisexcite can be controlled in two ways. The remote pod provides simple button functionality along with a clear screen. For a more integrated system, the precisexcite can be fully operated via the Olympus family of software programs. This also extends the functionality, enabling the use of TTL pulses to provide extremely fast on/off switching, which also removes the need for a shutter in the light path. The software also enables the user to switch between the wavelengths in under 10 ms, as well as store and reuse the parameters used for illumination. This, along with the excellent stability of the light source, even over long periods of time, ensures that any data generated is consistent and repeatable. C precisexcite hand switch Intuitive illumination control

12 12 CHAPTER III REAL TIME Don t make your research wait At the molecular level, a second is a long time and therefore if you are looking for subtle changes over time in a cellular system, then you will need to record in real time see the changes as they happen, record more data and get the whole picture. Achieving this re quires some dedicated equipment such as high-speed illumination sources with computercontrollable components. The Olympus range of real-time illumination sources takes this a step further with exquisite precision, excellent reliability and an easy-to-use interface.

13 REAL TIME THE NEXT LEVEL IN CELL IMAGING A Advanced live-cell imaging applications, such as multicolour time-lapse imaging, Z-sectioning, multidimensional imaging, ion imaging, FRET, TIRF, etc., need illumination systems with bright light for efficient illumination, fast switching to different wavelengths, high-speed shutters for maximum specimen protection and high precision for analysis. The Olympus MT10 and MT20 light sources provide highly stabilised and easily controlled light from either a 150 W xenon or 150 W mercury-xenon burner. To maximise the potential of these unique units, they are fully integrated with the Olympus imaging systems and respectively. Screenshot of software MT10 B The MT10 (and ) is designed for fast events and incorporates an 8-position filter wheel which can be moved between neighbouring filters in just 85 ms. Intensity can be controlled even more quickly via the 7-position attenuator (4 100%) and the high-speed shutter can be opened or closed in under 5 ms. The system also incorporates a system coordinator (PC control board) which provides 10 ms temporal resolution (camera 1 ms) with a timing precision of 15 ms. The combination of the MT10, system coordinator and software makes even the most complex data acquisitions straightforward and repeatable. B MT10 Fluorescence illumination system MT20 C The MT20 (and ) is designed especially for high-speed live-cell imaging and uses a faster filter wheel than the MT10 which moves between each of the 8 neighbouring filters in just 58 ms. The expanded attenuator set includes 14 positions offering intensities between 1 100%. The shutter is also much quicker, with on/off times of less than 1 ms. The more powerful real-time controller PC board ensures, via parallel command execution, temporal resolutions of 1 ms and precision in the microsecond range. C MT20 Fluorescence illumination system for high-speed imaging Experiment Manager With both the and software programs, the powerful Experiment Manager provides precise planning and execution tools via an intuitive drag-and-drop system that enables easy assembly of command icons such as image acquisition, Z-stack or time loop. Spectral microscopy D For the ultimate in speed, the system can be used with the Polychrome V monochromator-based system, which can provide wavelength changes of up to 400 nm/ms. The Polychrome V uses a 150 W xenon burner with 3,000 h lifetime and provides UV to near-infrared wavelengths. An optional 10-level attenuator enables fast setting of different excitation light levels. D Polychrome V Ultimate wavelength switching speed

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15 SYSTEM CHART

16 Light sources specifications Arc burner light sources Mercury lamphouses Xenon lamphouses X-Cite 120 X-Cite 120 Iris X-Cite 120 PC Type Mercury Xenon Metal halide Metal halide Metal halide Wattage 50/ Lifetime (h) 100/300 1,000 2,000 (guaranteed) 2,000 (guaranteed) 2,000 (guaranteed) Operation Manual Manual Manual Manual Manual/PC Intensity control Additional ND filter Additional ND filter Additional ND filter Iris with 4 positions plus Iris with 4 positions plus shutter function shutter function Shutter: Manual Via slider in illuminator Via slider in illuminator Via slider in illuminator Via iris Via front panel/foot switch Online Online via GUI Automatic Via SW Shutter speed NA NA NA NA 200 ms Filter positions NA NA NA NA NA Switching speed NA NA NA NA NA Control interface NA NA NA NA RS232 Real-time system NA NA NA NA No Microscope BX, IX, MVX, SZX (100 W) BX, IX, MVX, SZX BX, IX, MVX, SZX BX, IX, MVX, SZX BX, IX, MVX, SZX CX and CKX (50 W) MT10_D MT10 MT20 Polychrome V Type Mercury or mercury-xenon Mercury or mercury-xenon Mercury or mercury-xenon Xenon Wattage Lifetime (h) 1,000 1,000 1,000 3,000 Operation Manual/PC PC PC PC Intensity control Attenuator with 7 levels Attenuator with 7 levels Attenuator with 14 levels Attenuator with 10 levels (optional) Shutter: Manual Via hand switch Online Online via GUI Online via GUI Online via GUI Online via GUI Automatic Via SW Via SW Via SW Via SW Shutter speed < 5 ms < 5 ms 1 ms < 1 ms Filter positions Continuous wavelength selection ( nm in 1 nm increments) Switching speed 85 ms 85 ms 58 ms Up to 400 nm/ms Control interface R232/USB Independent plugin CPU board Independent plugin CPU board Voltage ( 10 V to + 10 V), RS232, trigger in and out Real-time system Yes Yes Microscope BX, IX, MVX, SZX BX, IX, MVX, SZX BX, IX, MVX, SZX BX, IX, MVX, SZX LED light sources FluoLED EasyBlue FluoLED Single Fluorescence FluoLED MultiFluo precisexcite Illumination method Transmitted fluorescence Transmitted fluorescence Transmitted fluorescence Reflected fluorescence Lifetime (h) 10,000 (minimum) 10,000 (minimum) 10,000 (minimum) 10,000 (minimum) Wavelength 480 nm fixed One Up to three Up to three Available LED modules Blue (fixed) UV/royal blue/blue/cyan/ UV/royal blue/blue/cyan/ 10 different LAMS green/yellow/red green/yellow/red From 400 to 635 nm Intensity control Fixed One-channel electronic driver Three-channel electronic driver 0 100% in 1% increments via pod or SW Cooling NA NA NA Peltier Control interface NA NA NA RS232/Ethernet/USB Battery pack for in-field use Optional Optional NA NA Microscope CX21/31/41 CX21/31/41 CX31/41 BX, IX, MVX, SZX The manufacturer reserves the right to make technical changes without prior notice. Art. code: E Printed in Germany 04/2008 Postfach , Hamburg, Germany Wendenstrasse 14 18, Hamburg, Germany Phone: , Fax: microscopy@olympus-europa.com