Techniques for the study of intracellular ions are used widely in biology, for example, to identify spatial variations in calcium levels within living cells, to measure the concentrations of intracellular ions including cell pH, and to monitor how these concentrations change with time. Monitoring intracellular ion changes is vital for our understanding of signaling and functional pathways in cellular systems, central to many fundamental processes such as muscle contraction as well as synaptic nerve signal transmission. Such measurements have been central to a range of research programs, including drug discovery research. Calcium channels are representative of an important class of cell membrane molecules known as ion channels. As these open or close, e.g. in response to extracellular messenger molecules, intracellular ion concentrations can also change, altering how the cell behaves. Therapeutic agents that act directly on these ion channels may provide effective treatments for many diseases. For example, drugs that target calcium channels, are useful in treating a variety of cardiovascular disorders.Fluorescent dyes are designed to have affinity to specific ions; for example, FURA-2 is specific to calcium ions. When they bind to an ion, their absorption or fluorescent properties are altered. The principle of ion concentration determination is such that, when the free ion concentration changes, the equilibrium between free indicator dye and ion-bound dye also changes, resulting in a change in the photophysics of the indicator dye.Ratiometric microscopy is one approach for such measurements - by quantitatively interpreting the changes in fluorescent properties by looking at both bound and unbound dye ratio, the concentration of the ion being investigated can be measured. However, there are some common non-ratiometric dyes also, such as Fluo-4.
Selected fluorescence images, taken with the Revolution 488, from a 1500 frame kinetic series at 30 frames/sec, showing the progression of a Ca2+wave as it progresses though a rabbit urethral muscle cell.Kinetic plots derived from offset ROIs within the cell show temporal progression of the Ca2+flux. Using the same field of view and no binning, frame rates were then pushed to 120fps for better temporal resolution of events. Courtesy of Dr Mark Hollywood, Smooth Muscle Research Centre, Dundalk Institute of Technology.
In many tissue types, ion flux changes occur rapidly, very often in the low millisecond time domain. As such, ion-binding fluorescence dyes tend to have rapid responses, enabling changes in concentrations of free ions to be imaged with high temporal resolution, requiring a means of fast quantitative imaging of these fluorescence intensity changes. Ultimately however, we are practically limited by the binding constants of these dyes – the rate of imaging does not need to not exceed the time taken for dyes to diffuse, bind and respond to free ions, irrespective of how rapidly the ion concentration changes. One is better determining this binding constant of the dye, and using an exposure time suited to this. Using too short an exposure time adversely affects signal to shot noise ratio, the ultimate limiting detection phenomenon (which can make your image look grainy and unresolved), as would be the case even in the presence of a "perfect" photon detector (100% Quantum Efficiency (QE) + zero noise floor).Another factor that must be taken into consideration is the concentration of ion dye used. Ideally, lowest possible concentrations of dye are used as indicators of free ion concentrations, such that the dye binds to negligible amounts of ions as compared to the total quantity of the ion species in question, and so it does not significantly alter the statistical equilibrium of bound and free ions. The remedy is to lower the concentration as far as possible whilst still maintaining an adequate signal to noise ratio for the exposure time (and therefore frame rate) required.
Andor's EMCCD technology, whether as a key component in our Revolution confocal live cell imaging system, or as a "stand-alone" EMCCD + iQ imaging software solution, is the proven solution for ultra-sensitive rapid imaging of ion concentrations. The Signal to Noise (S/N) achievable at rapid frame rates is ideally suited to intracellular visualization and measurement of common ion-binding dyes such as the calcium-specific Fluo-4. EMCCD technology enables readout noise to be completely negated, even at very fast readout rates, ideally suited to temporal requirements of calcium transient processes, such as calcium sparks or waves. The iXonEM+ DU860 back-illuminated for example is capable of > 500 frames/sec @ 128 x 128 pixel format. Through use of EMCCDs, calcium dye concentrations have also been reduced, enabling propagation of calcium waves along the full length of elongated smooth muscle cells for example. High sensitivity also enables excitation power to be attenuated. Through minimization of excitation powers, whether laser or lamp based, photobleaching rates of the dyes can be dramatically reduced. In living cell environments, these "milder" excitation conditions also reduce the phototoxic effects that would otherwise kill the living cells, enabling them to be studied over longer periods of time.
The iXonEM+ and LucaEM imaging EMCCD platforms each displays single photon sensitivity combined with high QE at multi-MHz rapid readout speeds.
Andor's pioneering iXonEM+ is a revolutionary range of CCD cameras that provides single 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 LucaEM is the latest EMCCD innovation, a highly cost-effective option making EMCCD available to every laboratory carrying out ion signalling using widefield fluorescence microscopy. Operate "gain off" for conventional CCD operation under brighter conditions: turn on the EM gain when the photons become scarce!LucaEM DL-8658M provides single photon detection sensitivity and ~52% QE at 30 full frames/sec, in a cooled USB 2.0 platform.
Quantum Efficiency and Fluorescent Dyes relevant to Ion Signalling Microscopy
Widefield and confocal fluorescence microscopy are common techniques for quantitative imaging of intracellular ion flux. Confocal fluorescence microscopy delivers inherently low fluorescence background, and as such 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.
Andor iXonEM DU897-BV and iQ software is used to image calcium with Fura2. Dr Fayuk studies neuron receptors in brain sections and cultures to elucidate processes of addiction. In this example a cultured neuron was patch-clamped and an iontophoretic electrode used to generate a 50ms Choline injection (200nA) at the spot shown. Synchronization to electrophysiology apparatus is achieved with Andor’s iQ software and Precision Controller Box (PCB). Courtesy Drs Dmitriy Fayuk and Jerry Yakel’s Neurobiology Laboratory, NIEHS, NC.
For imaging techniques that are fundamentally restricted by out-of-focus fluorescence, such as widefield fluorescence microscopy, 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:
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Ratio Imaging - A triplet of HEK cells showheterogenous response to Ach stimulation. Courtesy Dr Paul Thomas, UEA, Norwich, UK.
Revolution XD system
Andor's Revolution ultra-sensitive, rapid live cell confocal systems enable ion fluxes to be studied with minimal photo-bleaching and shorter exposures/faster frame rates, through multiple dimensions. Access the capability to do searingly fast high-resolution confocal imaging or even fast z-sectioning through calcium sparks.ntrations. Full rendering and analysis of ion flux changes is accessible through Andor iQ software.
Consecutive images from a 1500 frame kinetic series, recorded with Revolution 488nm @ 60 frames/sec with 256x256 pixel sub-array of iXonEM+ DU-897E, showing the initiation and spread of a single calcium spark from a smooth muscle cell loaded with Fluo-4 dye. Laser power was at minimum possible setting to reduce photo-bleaching and phototoxic effects.
Revolution live cell confocal microscopy solutions are based around ultrasensitive EMCCD technology, the systems offering perfect synchronization of camera with other key hardware components such as the CSU laser confocal spinning disk or fast z-stage, each integrated seamlessly by Andor iQ multi-dimensional imaging software.This effective and unique implementation of EMCCD means that many of the traditional challenges of live cell microscopy are overcome, enabling reduced phototoxicity and photo-bleaching, faster frame rates and lower fluorophore concentrations. Full rendering and analysis of ion flux changes is accessible through Andor iQ software
Revolution’s confocal dual spinning disk technology provides an ideal platform for high speed, high SNR imaging, with low bleach rate and low photoxicity
Revolution 488 is Andor's single-line 488nm confocal solution for fluorescence imaging of the Fluo family of calcium binding dyes, with the unparalleled sensitivity, speed and resolution.
Revolution is also readily adapted for fast confocal and fast ratiometric capability, combined on the one system. Widefield imaging of the ratiometric calcium dye Fura, is achieved with either Till Polychrome V or Cairn Optoscan rapid excitation wavelength switchers, synchronized through Andor iQ to the iXonEM+ 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.
The extreme sensitivity of the Revolution 488 system, facilitates not only lower illumination levels and shorter exposures, but also lower dye concentrations. In this case Fluo-4 concentrations have been reduced significantly, allowing propagation of the calcium wave to the extremities of the cell. Prior to the use of the iXon EM+ DU897-BV, the concentration required gave inadequate signal to noise and also buffered the calcium wave propagation. Courtesy of Dr Mark Hollywood, Smooth MuscleResearch Centre, Dundalk Institute of Technology.
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