The capability to perform rapid, multi-dimensional imaging of living cells or model organisms is becoming increasingly necessary in many areas of modern biological research. Much of fundamental biological phenomena (spanning development, repair, cancer, cell cycle, signalling, motility, intracellular transport and viral pathways), involve microscopic structures moving rapidly through 3D spatial dimensions.In order to capture these intricate, interactive processes effectively in "real time", we are required to perform rapid time-lapse imaging of the cell through all three spatial dimensions i.e. effectively 4D imaging.
Furthermore, since we commonly want to follow the motion and interactions of multiple intracellular structures, this often requires us to either simultaneously or very rapidly switch between, the excitation and imaging of several spectrally separated fluorophore labels i.e. 5D! Underlying all direct imaging studies of living cells or organisms, is the desire to preserve the live subject for as long as possible, through minimization of both phototoxic cell/tissue damage and photobleaching of the incorporated fluorophores. Techniques that may be employed for 5D cell imaging include spinning disk confocal microscopy, deconvolution widefield and structured illumination, all of which can benefit from using Electron Multiplying CCD technology. e.g. iXon EMCCD Camera.
Image from 4D time-series of live neuron labeled with quantum dots. Courtesy Dr. Tom Blanpied, UMD.
Andor's EMCCD technology is the ideal detector for incorporation into your ultrasensitive 4/D microscopy set-up, whether as a key component in our Revolution confocal live cell imaging system, or as a "stand-alone" EMCCD + iQ imaging software solution. The extraordinary signal-to-noise (S/N) ratio offered is significantly greater than that afforded by conventional point scan detectors. Furthermore, since it is a rapid-readout imaging device the EMCCD yields rapid frame rates, ideally suited to rapid acquisition of z-stacks and time-lapse sequences.The spectral dimension is accessed either through fast switching between multiple fluorophores, or even splitting of the emission signal simultaneously onto different areas of the sensor. There is also the possibility to perform true "spectral imaging" covered elsewhere in this catalogue. This is covered elsewhere within this section. Through minimization of excitation powers, the rates of dye photobleaching and cell phototoxicity are significantly reduced. iXon3 EMCCD Camera and Luca electron multiplying CCD imaging platforms each displays single photon sensitivity combined with high Quantum Efficiency (QE) at multi-MHz rapid readout speeds.
Quantum Efficiency and Fluorescent Dyes relevant to 4/5D Microscopy
Andor's pioneering iXon3 EMCCD Camera is a revolutionary range of CCD cameras that provides ingle photon Detection sensitivity, highest QE, and -100°C Thermoelectric (TE) cooling at rapid frame rates, utilizing Andor's pioneering and award-winning EMCCD technology.
Andor's Luca electron multiplying CCD is the latest EMCCD innovation, a highly cost-effective option making EMCCD available to every laboratory carrying out 4/5D microscopy.Operate "gain off" for conventional CCD operation under brighter conditions or turn on the EM gain when the photons become scarce!LucaEM DL658M provides single photon detection sensitivity and 60% QE at 30 full frames/sec, in a cooled USB 2.0 platform.
Selected images through a z-stack showing effects of deconvolution with iQ ClearView.
Widefield (+ optional deconvolution or structured illumination) and confocal fluorescence microscopy are both common techniques for 4/5D microscopy of living or fixed cells or tissues. For techniques such as confocal fluorescence microscopy (designed to deliver reduced fluorescence background) the high-end ultrasensitive iXonEM+ EMCCD platform is invariably recommended, delivering absolute minimal levels of darkcurrent from TE cooling down to -100°C, QE up to >90% and pixel readout rates up to 35 MHz. For imaging techniques that are fundamentally restricted by background focus, such as widefield fluorescence microscopy with deconvolution or structured illumination, the level of darkcurrent at shorter exposure times can be less critical, being masked by the photon background level. Consequently, either iXonEM+ or LucaEM cameras can be used to deliver enhanced EMCCD performance at high imaging rate, the choice depending on a number of factors such as:
Butterfly Red Autofluorescence. A wing from a butterfly (deceased of natural causes!) was mounted on a 3"x1" slide in glycerin and a cover slip. Fast Z stacks were acquired using Andor iQ and iXonEM+ DU-897 back illuminated, running on an Olympus BX61WI and DSU slit scanner, excited with a Sutter DG4 illumination system. Stacks of 105 images with 0.3 µm spacing were acquired with an exposure time of 100 ms and then deconvolved with iQ ClearView. This image shows a Maximum Intensity Projection (MIP) image rendered with a red look-up table.
iQ live cell imaging software is a state-of-the-art multi-dimensional imaging package, ideal for even the most complex imaging protocols.
This flexible and intuitive platform allows tight synchronization between devices such as EMCCD, z-stage or fast switchable excitration sources, enabling unmatched frame rates through multiple dimensions with minimal light loss and minimal photodamage to the cell. A comprehensive array of multi-dimensional processing, analysis and visualization functionality comes as standard.
Andor has developed a modular approach for the Andor iQ software. So depending on your precise 4/5D application, we have bespoke solution to match your needs, making it the most cost effective yet high-performance software solution available.
Andor's Revolution Live Cell Confocal Microscopy solutions are ideal for demanding dynamic 4/5D applications, including:
Andor's effective and unique implementation of EMCCD means that many of the traditional challenges of live cell microscopy are overcome, enabling reduced phototoxicity and photobleaching, faster frame rates and lower fluorophore concentrations.
Based around ultrasensitive EMCCD technology, the Revolution systems offer perfect synchronization of camera with other key hardware components such as the CSU laser confocal spinning disk unit and Nano Z100 fast stepper stage, each integrated seamlessly by Andor iQ multi-dimensional imaging software.
This effective and unique synergistic combination of EMCCD and CSU confocal spinning disk unit means that many of the traditional challenges of live cell microscopy are overcome, enabling reduced phototoxicity and photobleaching, faster frame rates and lower fluorophore concentrations.
Revolution 488 is the entry-level single-line 488nm confocal system, enabling high-performance 4D microscopy, optimized for GFP imaging.
A standard mouse-gut specimen from Molecular Probes, labeled with Alex488 was imaged with Andor Revolution 488 to illustrate confocal optical sectioning. Image stacks of 56 sections were acquired at 0.25 um steps using the PZT100 (every second images is shown here), iXon 887BV with 20 ms exposure.
Revolution XD is Andor Bioimaging Division's multi-line 5D ultrasensitive confocal solution for fluorescence imaging of two or more fluorophores, ideal for real time 5D colocalization and FRET
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