Cell motility is required for many important physiological processes during development, such as cell migration during gastrulation, axon guidance, tissue regeneration and embryological development. Unregulated cell migration can be the cause for progression of cancer, e.g during metastasis. The challenge to perform rapid, multi-dimensional imaging of motile cells is fundamental to our understanding of above mentioned processes.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. Historically the microscopy of motile cells has, from the instrument standpoint, been marred by the need for greater speed and sensitivity at high resolution. For example, it can be desirable to image rapid protrusion of lamellipodia and filopodia.It can also be fundamental to visualize the cytoskeletal dynamics and membrane morphology of moving cells with high resolution and sensitivity, such that the underlying mechanisms of protrusion and retraction can be understood in the context of the interactions and growth of actin (e.g. stress fibers), mictrotubule and intermediate filament cytoskeletons.Underlying all direct imaging studies of living cells or organisms, is the desire to preserve the living subject for as long as possible, through minimization of both phototoxic cell/tissue damage and photobleaching of the incorporated fluorophores.
The iXonEM+ EMCCD has provided the solution to the challenges described above, enabling high resolution, high signal-to-noise (S/N) movies to be acquired of cell systems and their chemotactic response, without sacrificing any of the critical imaging parameters. Furthermore, through reducing the excitation power, phototoxic effects are minimized, enabling cells to be followed for much longer periods.
iXonEM+ and LucaEM imaging EMCCD platforms each display single photon sensitivity combined with high Quantum Efficiency (QE) at multi-MHz rapid readout speeds.
iXonEM+
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.
Courtesy of Dr Richard Berry, Molecular Motors Research Group, Dept. of Physics, Oxford University, UK.
Description of Dr Berry's images:Steps in the rapid rotation of ATP-driven flagellar motor have been resolved by single molecule fluorescence microscopy, using a widefield epifluorescence configuration. 200nm diameter fluorescent beads were attached to the sticky flagellar filaments of E.Coli, prior to imaging with the back- illuminated iXonEM DV860 at a frame rate of 2.4 kHz. Shown here are: (a) selected frames at 21ms intervals during one rotation. Red and yellow dots mark the calculated bead centre in the current and previous images respectively. Each pixel is 80nm square. (b) Stepping rotation of flagellar motors with a range of average speeds. Bead positions are shown in the insets (scales in nanometers); bead angles are plotted against time in the main figure.
Andor's LucaEM is the latest EMCCD innovation, a highly cost-effective option making EMCCD available to every laboratory carrying out Cell Motility studies 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 DL658M provides single photon detection sensitivity and high QE at 30 full frames/sec, in a TE cooled, USB 2.0 camera platform.
LucaEM EMCCD Camera
For further information on the LucaEM range, please refer to the Products-Cameras section.
Quantum Efficiency and Fluorescent Dyes relevant to Cell Motility
Widefield (+ deconvolution or structured illumination) and confocal fluorescence microscopy are both common techniques for Cell Motility of living cells. For techniques such as spinning disk 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 Cell Motility techniques that are fundamentally restricted by out-of-focus fluorescence (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 inherent 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:
iQ is a state-of-the-art multi-dimensional imaging software package, ideal for even the most complex imaging protocols.Andor has developed a modular approach for the Andor iQ software. So depending on your application we have bespoke solution to match your needs, making it a cost effective yet high-performance solution.
Still image from a time-lapse movie of a Rat2 fibroblast (left) with kymograph lines marked in red (1-6). Corresponding kymographs or time-space plots (right). Kymographs give detailed information about lamellipodial dynamics. Courtesy of Dr James E Bear, Lineberger Comprehensive Cancer Centre, NC.
Revolution XD-system
A powerful and flexible system solution for cell motility studies is Revolution 488 or Revolution XD (depending on fluorophore requirements), incorporating the Piezo Z100 stage for rapid volumetric monitoring of the motile cell environment and cytoskeleton, and adapted also for standard widefield fluorescence microscopy. It can be important to have a capability to monitor beyond a single narrow confocal plane of excitation, since movement of cells 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 core 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.
Figure 4 - Courtesy Dr Stefan Diez, Max Planck Institute, Dresden, Germany.
Description of figure 4:Quantum dots are immuno-linked to dynein molecules, which carry their cargo along extra-cellular microtubules (not visible here). Using Andor iQ and iXon DV887 back-illuminated, 4000 frames were acquired with 30 ms exposure in frame transfer mode and simultaneously displayed and streamed to hard disk at 30 fps using iQs ImageDisk functionality. The data has been processed by maximum intensity time encoding, in which each pixel is replaced by the time value when it was brightest and then pseudo-colored. Red pixels are the latest in time, while blue are the earliest, giving a map of the rate and direction of transport.
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