Andor Revolution provides a framework for Andor Bioimaging Division's laser spinning disk, live cell confocal microscopy solutions, which combine our own award winning iXonEM+ Electron Multiplying CCD (EMCCD) camera with the renowned Yokogawa CSU 10 and 22 units. Andor has a global distribution agreement with Yokogawa Electric Corporation, to integrate powerful confocal solutions. This partnership brings you unrivalled performance, product understanding and support.Revolution encompasses a range of complimentary components, both hardware and software, that fit seamlessly together creating a complete confocal microscopy solution. A flexible component focus also allows us to provide key pieces of hardware stand-alone.At the core of Revolution systems is Andor iQ, a multi-dimensional imaging software which synchronizes iXonEM+ EMCCD cameras with the CSU 10 or 22 spinning disk confocal and other key hardware components such as Piezo Z100 fast z-stage and our Solid State Laser Combiner, with Acousto-Optical Tunable Filter (AOTF) for rapid laser line selection.Our recommended microscope platform is the Olympus IX81, optimized for live cell imaging, but infinity-corrected microscopes from Leica, Nikon, Till or Zeiss can also be supported.
Andor provides camera-based solutions for low-light live cell microscopy that are primarily based around ultrasensitive and fast EMCCD technology.Andor's cutting-edge Revolution Multi-dimensional Confocal Microscopy solutions are ideal for demanding dynamic live-cell techniques, including:
A neuron image stack recorded with Revolution 488, rendered topographically in Andor iQ. The image represents a height coded image of the specimen, with red being closest to the viewer and blue being furthest away. Data courtesy of Dr Tom Blanpied, University of Maryland –formerly of Duke University.
Whether you have an existing microscope set-up that you need to upgrade for enhanced performance or new applications, or whether you wish to develop a full system from scratch, Andor can help ensure you arrive at the multi-dimensional microscopy solution that best suits your specific needs, in terms of both performance and budget.
ABD can provide a complete Revolution live cell confocal microscopy system, including microscope, tailored to your specific application requirements. The entry-level system is the "Revolution 488", a cost-effective solution incorporating iXonEM+ EMCCD, CSU10 or CSU22 laser confocal spinning disk unit, optional Piezo Z100 fast precision z-stage and a single 488nm laser line, either from an Argon or Solid State source.
For a complete 4/5D multi-channel time-lapse microscopy solution, the "Revolution XD" might be considered which makes use of the solid state Andor Laser Combiner (this model incorporating up to four solid state lasers and AOTF). Incorporation of further gas laser lines is also possible.
Revolution Confocal setup
Electron Multiplying CCD technology provides the ultimate in camera sensitivity. Andor has pioneered the development of digital scientific cameras using this technology.Our award winning iXonEM+ is considered the gold standard across a broad range of light starved applications.
Revolution's confocal dual spinning disk technology provides an ideal platform for high-speed, high SNR imaging, with low bleach rate and low phototoxicity.
Precision Control Unit
Andor's Precision Control Unit (PCU) provides synchronization to all Revolution components, ensuring iXonEM+, CSU, Combiner and Piezo Z100 remain in precise temporal alignment. This minimizes specimen exposure times and allows highest frame rates to be achieved while ensure precision components are stabilized for high quality imaging. Working with Andor iQ, PCU provides an interface for system expansion enabling Revolution to be synchronized with other equipment e.g. electrophysiology, perfusion control or flash photolysis.
Not all computers are created equal! The iQ Workstation is carefully selected to deliver maximum performance to your Revolution System. From internal hardware specifications to external screen presentation, Andor knows what is needed to keep you working effectively. Select this option and allow us to preload your software on proven hardware to ensure first time operation, right out of the box.
When selecting a Revolution system tailored to your application needs, there are a number of component options that must be worked through, including:
Through custom integration of the Revolution core hardware components, the individual techniques in which we specialize range from fast confocal sectioning of fixed specimens, to rapid single wavelength imaging of living cells, through to more complex 4/5-D time-lapse live cell microscopy.All our complete systems or enhancement paths are designed to deliver the industry optimum in sensitivity, speed, synchronization of hardware devices and user accessibility, but with affordability very much in mind.
Offers a cost-effective entry point for personal confocal users. Using a single 488nm laser line (either from Argon source or Solid State source), iXonEM+, CSU and optional Piezo Z100 rapid z-stage, all integrated through iQ software.The 488nm line is ideal for excitation of Fluo calcium dyes, GFP and some voltage sensitive dyes.With Revolution 488, you can study and visualize numerous functional and structural cellular components in 2D and 3D time-lapse (4D) imaging modes, optimized through Andor iQ software. Powerful tools for analysis and tracking are also available. Select either CSU10 or CSU22 depending on absolute speed (CSU22 for >360 frames/sec) and software synchronization requirements.
The Revolution XD is the workhorse high-performance live cell 4/D confocal system, and makes use of the Revolution Laser Combiner with up to 4 solid state lasers, or a mix of solid state and gas lines, matched to your fluorophores. An integrated AOTF, controlled through Andor iQ software, is employed within the Laser Combiner unit to control and balance laser powers, and also to rapidly switch between individual lines for measurements such as FRET or co-localization.Revolution XD is recommended for laboratories and facilities where routine changing of excitation wavelengths is commonplace, or for multi-wavelength experimental protocols.CSU22 is the recommended Revolution XD confocal unit (it can however be equipped with CSU10 on request), enabling automated switching between fluorophore-specific filters and software synchronization of disk speed to any camera exposure time.
Revolution can be customized with further components to enable:
The Revolution XD can be fitted with the Cairn ResearchOptoSplit II or Optical Insights DualView emission splitter units, which project two spatially equivalent images onto adjacent sections of the image sensor – each image corresponding to emission of a particular fluorophores (donor and acceptor).This addition, in conjunction with the AOTF laser combiner and functionality within iQ software to process wavelength separated images, means that high S/N FRET data can be acquired, processed and analyzed efficiently.This configuration can also be applied to co-localization studies.
Fast confocal and fast switching widefield capability can be combined on one system. Widefield imaging of the ratiometric calcium dye Fura 2, is achieved with the Till Photonics PolyChrome V or Cairn Optoscan rapid excitation wavelength switcher, synchronized through Andor iQ to the iXon+ EMCCD frame transfer camera, for ultra-fast image pairs.Switch easily to confocal mode for rapid imaging of non- ratiometric dye Fluo-4, or for fast confocal imaging of ratio- emission dyes, e.g. Indo –1.Furthermore, the Andor PCU can offer high-precision synchronization of electrophysiology and fluorescence measurements. Benefit from extremely fast frame rates (up to 1000/sec with the CSU22 and iXon+) and perfect synchronization of all components.
The Revolution 488 or XD systems can be further enhanced to enable whole cell flash photolysis, through integration of Cairn Research's Opto - Flash Photolysis system. Synchronization with the imaging system is performed readily through the Andor Precision Box and iQ software.
Using an ultrafast iXonEM+ DV860 back illuminated camera and 1MHz Cairn LED controller the realms of ms biology can be accessed. The iXonEM+'s exquisite sensitivity and synchronized LED illumination allows frame rates up to 1kHz to explore fast processes in biological systems. Dyes such as RH795 (Molecular Probes) allow monitoring of action potentials and global field changes in neuroscience and cardiology.
All Revolution systems can be combined with DIC microscopy. This capability enables confocal fluorescence images to be over-layed onto to DIC images of the cell, enabling fluorescence events to be spatially referenced.
The powerful technique of time-lapse multi-dimensional confocal microscopy can be applied across a diverse range of cell function, pathogenic and model organism developmental studies.
The Andor Revolution ultrasensitive, high-speed confocal systems, enable GFP, CFP, YFP and RFP to be studied with minimal photobleaching and shorter exposures/faster frame rates, through multiple dimensions. Access the capability to do z-sectioning, CFP-YFP FRET, co-localization, or simply very fast tracking of GFP labelled molecules.
Revolution 488 is Andor single-line 488nm confocal solution for fluorescence imaging of GFP with unparalleled sensitivity, speed and resolution. GFP is a commonly used and extrordinarily effective probe into the inner function of cells and organisms. The GFP chromophore, consisting purely of amino acids, renders it well suited to genetic engineering and protein expression studies, and it is often used to monitor localization and dynamics of cellular components and functions.
Prepared for imaging on Revolution 488 by Dr Mark A Smith.
Description of figure above: This embryo is engineered to produce GFP actin, which glows throughout its form. Imaged with a 60x 1.4 NA oil immersion Olympus objective, a 3D stack was gathered every 10 seconds for 50 minutes (300 stacks). Each stack consisted of 41 frames with a 1 um spacing. Exposure time per frame was 50 ms with EM Gain. Very low photo bleaching was observed. In the movie we see the "string" close. This closure is the fusion of two epithelia sheaths, which form the skin of the insect. The closed parts of the sheaths are seen as the bright linear structure on the mid right of the image, following along this line NWW, you will see the two strands of the string. As closure occurs like a zipper, we can see cells on either side of the divide creating projections, and gripping onto their opposite number when they come into proximity. They literally pull themselves together. The epithelial cells in the sheaths do not divide, but are stretched to complete the structural change.
Revolution 488 for GFP provides you with the following features & benefits when working with GFP:
The Revolution 488 is entirely upgradeable to Revolution XD at a later date should your experimental needs evolve, e.g. extension to multi-color CFP, YFP, D, Red Probes, or for study and analysis of FRET pairs.
Revolution XD for Multiple GFs - Andor's multi-line 4/5D ultrasensitive confocal solution for fluorescence imaging of two or more of the fluorescent proteins (and other combinations of common dyes), ideal for real time colocalization and FRET analysis, down to the single molecule level.
Andor's highly flexible Revolution confocal systems can be optimized for various types of ion signalling experiments. Monitoring these changes can be important for understanding signalling and functional pathways in cellular systems. Fast confocal and fast switching widefield capability can be combined on the one automated microscope. The Andor PCU can deliver high-precision synchronization of electrophysiology and fluorescence measurements. Using PCU, one can benefit from extremely fast frame rates (up to 1000 sec-1 with the CSU22 and iXonEM+) and perfect synchronization of all components. Buffering of calcium signal can be significantly reduced through use of lower dye concentrations.
Revolution 488 is Andor Bioimaging Division's single-line 488nm confocal solution for highly rapid, highly sensitive fluorescence imaging of the Fluo dyes, a family of indicators used to monitor rapid changes to intra-cellular calcium ion (Ca2+) concentrations. Monitoring Ca2+ concentration changes can be fundamentally important for understanding signaling and functional pathways in cellular systems.
*Approximate values estimated from published values
a Relative to EGFP
b Rapid photobleaching
Pair of HeLa cells loaded with Fluo4. A pair of HeLa cells, loaded with Fluo4, was observed shortly after mitosis, using Revolution 488 at 10 fps. The cells were stimulated with histamine and showed a slow, but substantial rise in Ca2+signal.Courtesy of Professor Rainer Duden, Royal Holloway College, University of London.
More detail on Ion Signalling application solutions can be found in a separate area of the Biology section.
Andor's Revolution ultrasensitive, rapid confocal systems, enable voltage sensitive dyes to be imaged with shorter exposures/faster frame rates, through multiple dimensions if desired. Revolution can be readily employed to study muscle contraction, nerve-impulse propagation, cell signalling and ion- channel gating, through observing increases and decreases (hyperpolarization and depolarization) in membrane potential. Voltage sensitive dyes are also used for visualizing mitochondria and for assessing cell viability.
The unparalleled speed and sensitivity of the synchronized CSU and iXonEM+ components renders Revolution a highly effective solution within neuroscience. For example, Revolution 488 with CSU22 and the iXonEM+ DV860 can be used to monitor depolarization of DiSBAC42(3) at imaging rates up to 1000 frames/sec. Using Revolution XD with FRET option, one can also perform FRET-ratio analysis of common voltage sensor probe pairs, such as DiSBAC42(3) / CC2-DMPE.To add further flexibility and explore super fast imaging (up to 1000 fps) of dyes like RH795, this system can be extended incorporating Cairn LED source and Andor’s iXonEM DV-860 back-illuminated.
Cultured neurons were loaded with DiSBAC2(3), a voltage sensitive dye and visualized with the Revolution 488. Exposure time of the iXon 887BV was 100 ms and EM Gain 200. The figure shows a film-strip from Andor iQ of maximum intensity projection time series. Time point 4 is taken almost simultaneously with the addition of KCl, which depolarizes the cells and shows a near instantaneous rise in DSBC signal. DF of almost 45% is observed in this example.Courtesy of Dr Iain Johnson, Invitrogen, Eugene, OR, USA.
The Revolution XD is ideal for dynamic confocal imaging of FRET Pairs, enabling elucidation of quantified molecular dynamics, such as protein-protein interactions, protein-DNA interactions, and protein conformational changes. High S/N multi-dimensional movies can be built through combination of CSU, iXonEM+, iQ software and Andor Laser Combiner with matched solid-state laser lines and AOTF for laser balancing/rapid switching.A Cairn Research OptoSplit II or Optical Insights Dual View emission wavelength splitter is placed between the CSU and camera for simultaneous quantitative imaging of donor and acceptor. One can also opt to perform rapid optical sectioning with a Piezo Z100 stage, for 5D imaging - up to 100 sections sec-1. With Revolution XD, you can image and quantify molecular interactions in intact cells, tissues, and whole organisms, with greater sensitivity and efficiency than any other
FRET solution on the market.
Quantum Efficiency and Fluorescent Dyes relevant to Live Cell Confocal Microscopy
At the level of single cell visualization, cell motility envelopes a broad area of study including the mechanisms of cell migration, chemotaxis, axon guidance and motility of dendritic spines. Of interest are whole cell movement, cell polarity, adhesion, membrane ruffles, protrusion of lamellipodia and filopodia, morphogenesis and also the involvement of the cytoskeleton, particularly at the leading and trailing edges of locomotion.
A powerful and flexible system solution for cell motility studies is Revolution 488 or Revolution XD (depending on fluorophore requirements), incorporating the fast piezo stage for rapid volumetric monitoring of the motile cell environment or cytoskeleton dynamics, and adapted also for standard widefield fluorescence microscopy. It is important to have a capability to monitor beyond a single narrow confocal plane of excitation, since movement of cell or within the cell is not restricted to the 2D spatial dimensions. The Revolution extended for combined widefield fluorescence microscopy, enables ready switching between these two modes of fluorescent operation, enabling rapid acquisition of data from an extended focal plane. For each mode, the iXonEM+ EMCCD technology offers improved temporal and spatial resolution, with high S/N and minimal photobleaching. iQ software is ideal for acquisition and comprehensive analysis of cell motility image series. iQ contains kymograph functionality enabling multi-dimensional rendering and analysis of cell movement. Furthermore, the iQ Tracker module can be employed for advanced automated tracking and display of motile cells.
Revolution systems are ideal for high resolution imaging of movement through the intracellular environment, including Endo-exocytosis, Vesicle tracking, Protein transport, Virus transmission and Nuclear-cytosol membrane mechanisms.For example, intracellular trafficking of vesicles can be followed in real time through multiple dimensions with Revolution 488 or Revolution XD, both incorporating the Piezo Z100 stage for fast z-stacks. Sharp intracellular resolution and high S/N contrast, ensures fine elucidation and tracking of small vesicles from the moment of formation. Complete synchronization of components through iQ means that rapid z-stacks can be recorded across multiple fluorophore channels, with minimal photon loss. IQ can then be used to produce 3D rendered, color merged and animated sequences, analyse and display the data through multiple dimensions, and perform automated tracking of vesicle motion.
Total internal reflection microscope (TIRFM) is an alternative means of illuminating a very narrow optical section at the interface between a cover slip and specimen. TIRFM is used to reveal structure at the cellular surface living specimens with extremely high contrast. TIRFM can be especially useful in the field of study and benefits from the speed and sensitivity of Revolution components. Illumination is supplied by Andor’s laser combiner and controlled through the PCU and Andor iQ.
Multi-colour fluorescence microscopy is frequently useful to display and determine the extent of pixel (or 3D voxel) overlap of two or more fluorophores, since this can relate directly to the spatial overlap of two or more types of specifically labelled cellular component or protein within the cell at a given point in time. As such, colocalization is a parameter that enables verification, spatial determination and quantification of their intracellular molecular interactions.
Revolution XD is the core system for recording rapid, high S/N, multi-dimensional images of multi-labelled cells. Analysis functions in iQ software enable one to perform intensity and area measurements for the individual color channels as well as coincident statistics for actual and percentage area of colocalisation between RG, GB, RB and RGB, as well as integrated intensities of RG, GB, RB and RGB.
Fast intra cellular trafficking events captured at high temporal resolution in a region within a fibroblast cell. The montage shows a series of maximum projected images from 8 Z sections with a 0.8 microns Z spacing. Each stack of images took 0.7 seconds to capture and this was repeated over 90 seconds.The image series shows two endosomes, the larger one being approx. 2 microns in diameter, that undergo fusion. This is typically a very fast biological event and requires high speed and high resolution imaging capability. Data courtesy of Frode Skjeldal, who works in Prof. Oddmund Bakke’s laboratory in the department of Molecular Biosciences at Oslo University.
One frame from a time-series in which a mammalian cell is engaged in mitosis (cell division). The GFP-labeled cell centromeres are separated by a "spindle" and chromosomes are "driven" to poles of the complex.Acquired with Revolution 488. Data courtesy Dr Ted Salmon, UNC, Chapel Hill.
Long term 4D imaging of Caenorhabditis elegans embryo as it undergoes early cell division. Z sections were taken with a step size of 1 micron. The stack took 5 seconds to acquire and an interval of 5 seconds between stacks was used.This series of maximum projection images is made up from 2716 frames that were acquired over a time period of 36 minutes. Virtually zero photo-bleaching is apparent and the embryo undergoes two cell divisions demonstrating very limited phototoxic damage to the living specimen. Data courtesy of Nathalie Le Bot, who works in the Ahringer lab at the Gurdon Institute, University of Cambridge.
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