National Cerebral and Cardiovascular Center,
Department of Cell Biology
Background
The team at Department of Cell biology at the National Cerebral and Cardiovascular Center is focused on exploring the molecular mechanism by which blood vessel formation and cardiogenesis are regulated during development. To meet their research goals, the team must determine how cardiovascular development is precisely regulated.
Understanding of the development of cardiovascular system contributes to developing new strategy for cardiovascular regeneration. Currently the team is using zebrafish to investigate cardiovascular development. Zebrafish provide great advantages with quick development, extra-embryonic development, translucency and easy gene manipulation. The embryos expressing fluorescent proteins under the cardiovascular-specific promoters can be visualized as they are developing.
Figure 1 The heart of zebrafish embryo expressing green fluorescence and red fluorescence was imaged on an Olympus IX81 equipped with the Evolve 512 camera and double-view. Video rate image could be obtained by transfer speed of the image with the Evolve camera.
Challenge
The only way to obtain the high speed needed (video rate image of fluorescence), is through the combination of CCD and spinning disk confocal microscopy. The imaging solution used must support the ability for high speed image acquisition of live zebrafish embryos expressing green fluorescence and red fluorescence.
The quality and speed of the Evolve 512 EMCCD camera has enabled us to capture zebrafish embryo developments under challenging conditions.
Solution
Having used the CoolSNAP HQ2 CCD from Photometrics in the past, the decision was made to pursue a more advanced solution from the same company. The team selected the Evolve® 512 EMCCD camera (new series now available). “The quality and speed of the Evolve 512 EMCCD camera has enabled us to capture zebrafish embryo development under challenging conditions,” states Mr. Mochizuki.
Spinning Disk Confocal In Vivo ImagingCustomer Stories
Dr. Scot Kuo
Microscope Facility Director and Associate Professor in Biomedical Engineering and Cell Biology
Johns Hopkins University School of Medicine
Background
The Johns Hopkins University School of Medicine Microscope Facility has an expansive selection of research microscopes, including multiple laser scanning confocal microscopes, a multiphoton microscope, an atomic force microscope, a TIRF microscope and multiple spinning disk confocal microscopes. The wide variety of microscopy options allows the university team to work with researchers to develop techniques for specialized research projects.
Figure 1In vivo imaging of CD8+ T cell-mediated elimination of malaria liver stages. Cockburn IA, Amino R, Kelemen RK, Kuo SC, Tse SW, Radtke A, Mac-Daniel L, Ganusov VV, Zavala F, Ménard R. PNAS 2013 110 (22) 9090-9095 P. yoelii – GFP: Parasites PyTCR : Activated CD8+ T-cells OT-I: Control CD8+ T- cells
Challenge
To understand the cellular immune response protecting against malaria infection in its early liver stage, a colleague from the Johns Hopkins Malaria Research Institute approached the team about conducting in vivo imaging of infected mice. Malaria parasites and specifically activated T-cells (CD8+) are both capable of extremely fast motility in liver tissue. Using a non-fluorescent mouse host, their colleagues wanted to monitor the motility of injected parasites and T-cells, each expressing different fluorescent proteins. To address specificity, non-activated control T-cells labeled with a third fluorescent protein are often included in the same mice. In addition to choosing an appropriate microscope system, additional challenges of this experimental system includes the photosensitivity of these labeled cells and the high autofluorescence of liver tissue. Fast motility requires fast imaging, but the phototoxicity limits excitation intensity.
Due to phototoxicity and speed of imaging, the Evolve 512 EMCCD camera is essentially running at single-molecule sensitivities in our experiments. We conducted side-by-side camera comparisons… Only the Evolve was suitable for this research.
Solution
On an Intelligent Imaging Innovations (3i) system based on the Yokogawa spinning disk confocal microscope, the team selected the Photometrics Evolve® 512 EMCCD camera (new series now available) as the best camera solution. Although the spinning disk confocal microscope limits the effects of the tissue’s autofluorescent ‘haze’, the exceptional signal-to-noise of the Evolve camera provided the additional discrimination needed to see labeled cells. Given the 3D motility of T-cells, z-stacks are required, so the system is running continuously to provide the spatiotemporal resolution needed. It cannot run any slower for fear of missing abrupt changes in motility as T-cells ‘discover’ and attack malaria parasites.
To minimize phototoxicity, the 3i system has the excitation lasers electronically slaved to the Evolve 512’s ExposeOut synchronization signal. The lasers are only active when the camera is collecting photons, thus limiting exposure times to the absolute minimum. Even so, the lasers are running at very low power levels to preserve cell viability. Dr. Kuo explains, “Due to phototoxicity and speed of imaging, the Evolve is essentially running at single-molecule sensitivities in our experiments. We conducted side-by-side camera comparisons on the same microscope system, including two different sCMOS cameras and a less deeply cooled EMCCD camera. The results clearly showed that these other cameras were inappropriate. Cells were lost in the noise even though exposures were ~250ms. Only the Due to phototoxicity and speed of imaging, the Evolve is essentially running at single-molecule sensitivities in our experiments. We conducted side-by-side camera comparisons… Only the Evolve was suitable for this research.” The team’s malaria experiments continue and have expanded their research to include other tissues (skin) and other stages of malaria infection (transfer into blood vessels).
More about advanced imaging technologies for cell and subcellular studies is available at: www.jhu.edu/cmml/
Single Molecule and TIRFCustomer Stories
Dr. Steven Magennis
The University of Glasgow, School of Chemistry
Background
The School of Chemistry has a long history of excellence in both research and teaching. It maintains a superb research environment with world leading research groups and facilities. The School supports a wide variety of research topics, from all aspects of Chemistry, as well as interfaces with biology, materials science and physics.
Dr. Magennis and his team focus on single-molecule techniques, looking for ways to reveal details about molecular systems that are otherwise obscured by conventional ensemble methods. They use powerful tools for the study of systems that cannot be synchronized, display static or dynamic heterogeneity, have transient intermediates or undergo rare events. Underlying themes in their research are the use of single molecule spectroscopy and imaging to probe the structure, dynamics and reactions of biomolecules, and the development of new tools using ultrafast lasers.
Figure 1: Multiphoton TIRF microscopy of immobilised CdSe/ZnS quantum dots (see Lane et al. Optics Express, 2012, Vol. 20, p 25948-25959)
Challenge
Due to the sophisticated nature of their research, the team needed to ensure reliable detection of single molecules in their imaging process. Intrinsically weak signals and interference from background sources of scattering and fluorescence make imaging a challenge and difficult to achieve. Of highest importance was the need for good signal to noise capability.
The quality and speed of the Evolve 512 EMCCD camera has enabled us to capture zebrafish embryo developments under challenging conditions.
Solution
The team successfully used the Evolve® 512 EMCCD camera (new series now available) for a number of years for prior research. They were happy to continue using the Evolve as their camera of choice. They also implemented the Photometrics DV2 multichannel system. The DV2 is an emission splitting system enabling a user to acquire two spatially identical but spectrally distinct images simultaneously.
Dr. Magennis shares “In combination with total internal reflection fluorescence (TIRF) microscopy, the Evolve 512 has allowed us to routinely detect and analyze the fluorescence from single molecules, providing long-time dynamic information on immobilized molecules and particles.”
Research at Sussex University focuses on the development and application of ultrasensitive optical techniques for the detection and manipulation of single molecules. Representing the ultimate level of sensitivity in the analysis and control of matter, single molecule techniques have many advantages over conventional ensemble methods, namely the measurement of static and dynamic heterogeneity in molecular systems.
Dipole images of a TMRbiocytin ligand oriented within a streptavidin protein nanoenvironment.
Intensity images of Quantum Dots undergoing fluorescence intermittency.
Challenge
The lab uses TIRF imaging on customized laser microscopy platforms to investigate a diverse range of molecular systems, from protein-ligand interactions by dipole imaging and protein-protein interactions in yeast using PALM, to quantum dot (QD) activation and blinking. They set out to pursue a way to improve the quality of defocused dipole images, reduce uncertainties in the location of molecules in PALM and achieve reliable measurements of QD quantum yields.
The quality and speed of the Evolve 512 EMCCD camera has enabled us to capture zebrafish embryo developments under challenging conditions.
Solution
The research team selected the Evolve® 512 EMCCD camera (new series now available) and are now using it for single photon counting and to support their diverse set of challenging imaging requirements. “The camera has performed exceedingly well under challenging and photon poor conditions,” states Osborne. The team is also using Micro-Manager microscopy software for image acquisition and processing.
Super Resolution Microscopy
(SptPALM/STORM)Customer Stories
Dr. Deepak Nair
Indian Institute of Science, Centre for Neuroscience
Background
Research at the Nanoorganization Lab, Centre for Neuroscience, Indian Institute of Science in Bangalore India borders on the interface of single molecule spectroscopy and molecular and cellular neuroscience. Here, team members develop and adapt state of the art paradigms in ultra-high and super resolution microscopy to image molecules at the synapses of living neurons.
Figure 1: Upper Panel: The epifluorescence image of a COS7 cell stained with tubulin antibody (A) which is then revealed by a secondary antibody coupled to the fluorophore Alexa 647. and Corresponding super resolution image (B).
Lower Panel: Gallery of epifluorescence images of different regions (C, E, G, I) from the cell and their corresponding super resolution images (D, F, H, J).
Challenge
The research team is interested in the molecular and cellular mechanisms that mediate basal synaptic transmission and plasticity. The routine imaging techniques used include FRAP, Photoactivation, Single Molecule Tracking and Stochastic Super Resolution imaging.
The team was interested in a widefield detection system with a high dynamic range that could handle both ensemble and single molecule fluorescence. Single molecule tracking in live cells is challenging due to heterogeneity in the signals and auto fluorescence from cells which make the contrast difficult to achieve. Most importantly the data needs to have good signal to noise ratio.
The quality and speed of the Evolve 512 EMCCD camera has enabled us to capture zebrafish embryo developments under challenging conditions.
Solution
The Evolve® 512 Delta (new series now available) was recommended and having used a prior version of the Evolve camera, the team was happy to integrate a newer model into their system. They had also previously used TwinCam (Cairn Research) to implement a multichannel imaging system to acquire spatially similar but spectrally different images at the same time.
“We were interested in the Evolve Delta for its low read out noise, sensitivity and ability to image very high frame rates per second,” Dr. Nair shares. By coupling the dual camera detector unit with a total internal reflection microscope (Azimuthal TIRF, Roper France), they were able to meet expected demands.
“The Evolve cameras also gave us the ability to interface with third party software to drive the acquisition, which made it optimal for a wide range of applications,” Dr. Nair continues. The microscopy configuration also enabled the ability to collect routine images in the widefield and single molecule regime. The camera’s sensitivity and dynamic range extended the microscopic capabilities from micrometers to tens of nanometers.
“The Evolve 512 Delta is great for stochastic super resolution microscopy and meets our expectations in both ensemble and single molecule microscopy,” Dr. Nair concludes.
Additional information about the research lab at the Centre for Neuroscience at the Indian Institute of Science in Bangalore is available at: http://www.cns.iisc.ernet.in/deepak/
Optics and NanomaterialsCustomer Stories
Darin Peev, PhD Student
University of Nebraska, Walter Scott Engineer Center
Background
The Walter Scott Engineer Center maintains extensive research facilities that include active and passive remote sensing facilities, an optical polarimetric scatterometer, an atomic force/scanning tunneling microscope facility and a microwave anechoic chamber facility.
In the optics and nanomaterials lab, ellipsometry is the technique that is used. This technique involves probing materials with light that is sent to the sample, causing the signal to bounce, which is then detected by a simple detector. When the light interacts with the sample, it changes its polarization state. The team was specifically interested in optimizing the detection of the sample’s attachment.
Challenge
Slow detection is the greatest challenge in studies involving ellipsometry. Important in this research are the speed, accuracy and high spatial resolution properties of the polarimeter, which can also be used as a polarization microscope. Because detection is very slow, it increases the potential to miss important interactions. The research team needed a solution that would increase speed while improving spatial resolution, and also enable them to capture reflection dynamics of sample interactions. They also wanted the ability to study how samples changed over time.
The quality and speed of the Evolve 512 EMCCD camera has enabled us to capture zebrafish embryo developments under challenging conditions.
Solution
Ellipsometry studies don’t usually use a camera as the detection device, however the team decided it was the best solution to help them achieve their goals. They selected the Evolve® 512 Delta EMCCD camera (new series now available) as their detection device and are now obtaining results much more quickly. “It used to take eight seconds to image and now we can acquire data in 20 milliseconds. We now have more time to collect more data and get results faster,” states Peev. The low read noise of the camera also gives us a better signal to noise ratio.
The team now has much more flexibility in their research. The instrument can be used in different regimes such as dark-field microscopy or even as a Mueller matrix ellipsometric system. Using the low noise characteristics of the Evolve 512 Delta camera, they have the ability to normalize their measurements by using the imaged area as a reference, and using captured intensities for normalization, thus greatly suppressing the source fluctuations.
Electrophysiology and
Calcium ImagingCustomer Stories
Nick Spencer, PhD, Associate Professor
Flinders University, Department of Human Physiology, School of Medicine
Background
The Department of Human Physiology was established in 1974 as the first department in the School of Medicine at Flinders University. The department has a research focus in neurosciencein three major areas; sensory and autonomic neurobiology, roles of neurotrophic factors and neurodegenerative diseases. The team is interested in how pain is detected from internal organs, such as the gastrointestinal tract.
Traditionally, all recordings from nerves that detect pain have been made outside the organ of interest, as these nerves project toward the spinal cord. However, these recording sites are not where sensory transduction takes place. The team wanted to identify and record directly from the nerve endings that detect pain from internal organs, from the site where sensory transduction occurs. This is a challenging task and one that has not been performed previously
Figure 1: Panel A, shows a superimposed image of calcium fluorescence (green) and CGRP fluorescence (red) in live mouse colon enteric ganglia. The axons and nerve endings in red indicate neural tissue that expresses the CGRP neuropeptide. Calcium imaging is performed on these immunoreactive nerve endings. B, shows a recording using the Evolve 512 Delta EMCCD camera. Each yellow circle represents the mean dynamic calcium fluorescence from discrete CGRP positive nerve endings.
Figure 2: Upper panel image shows CGRP expressing nerve endings (red) and calcium fluorescence (green). The numbers 1-4 represent regions of interest in CGRP expressing nerve endings and the numbers 5-10 represent the regions of interest of dynamic calcium fluorescence in non-CGRP expressing nerve axons. During peristalsis, enteric ganglia (regions 5-10) show synchronised activation that is independent of the synchronised activation of CGRP expressing spinal afferent nerve endings. These nerve endings in red are the likely nerve endings that encode nociception from the gastrointestinal tract.
Challenge
The team has succeeded in using calcium imaging to record dynamic changes in excitability from multiple sites along single axons that underline pain perception. However, they encountered many technical challenges.
One of the greatest was recording from nerve endings with electrophysiology. They found that using this approach, they could not record from multiple sites simultaneously. Another major obstacle was making sure that the nerve endings that they recorded from were in fact spinal afferent neurons that underlie pain perception.
The quality and speed of the Evolve 512 EMCCD camera has enabled us to capture zebrafish embryo developments under challenging conditions.
Solution
The team’s challenges were overcome with the Evolve® 512 Delta (new series now available). Using the camera for calcium imaging enabled them to record from multiple sites simultaneously along single axons of interest. They were also able to ensure that nerve endings were recorded accurately by stimulating dorsal nerve roots and demonstrating that those calcium transients were evoked in the same nerve endings that were activated by stretch or acid. This work has recently been published.
Of significance, due to the sensitivity of the Photometrics range of EMCCD cameras, the team has been able to perform the first and only recordings from nerve endings that detect pain from internal organs.
They are now answering many new and exciting questions using this technique. “We have been stunned by the incredible sensitivity of the EMCCD cameras produced by Photometrics,” states Dr. Spencer. Also, the high frame rate acquisition of the Evolve Delta camera at full resolution is just what was needed to record dynamic physiological activation of fine nerve endings.
Center for Quantitative Biology, Peking University
Background
The Tang Lab at the Center for Quantitative Biology at the Peking University is interested in quantitative studies of biological systems. They apply, develop and integrate theoretical, computational and experimental methods to address key biological questions. They believe that an interdisciplinary approach focusing on quantitative questions at a systems level will uncover new biological principles and help them better understand complex disease and design new therapeutic strategies. Current research areas include cell cycle regulation, cellular decision-making, the relationship between function and topology in biological networks, developmental landscapes, information processing in biological systems and network-based complex disease mechanisms. To achieve their research goals, the team must acquire stable, high resolution images for their quantitative studies.
HeLa cells. CFP was over-expressed and localized across the whole cell. 40X air objective.
Fission yeast, Schizosaccharomyces pombe.
Histone HTA2 was tagged with mCherry, which was localized in the nucleus. 60X oil objective.
Challenge
The quantitative studies of biological systems require the ability to capture high resolution images. Using standard CCD cameras, the team found they could not conduct quantitative analysis because they were unable to observe weak fluorescence signals. The research was dependent on a more advanced camera technology that could provide higher sensitivity and resolution for capturing accurate and reproducible data.
The quality and speed of the Evolve 512 EMCCD camera has enabled us to capture zebrafish embryo developments under challenging conditions.
Solution
Previously, Chao Tang, professor of the Tang Lab, worked at the University of California in San Francisco. There, he used both Photometrics and QImaging cameras, and was very satisfied with the cameras’ performance.
Now at the University of Peking, Tang and his team use two Evolve® 512 cameras (new series now available) from Teledyne Photometrics.
“The Evolve EMCCD cameras are very sensitive, so we can detect weak fluorescence signal and get high resolution images. The advanced camera features enable us to acquire accurate and reproducible data,” states Tang.
Tang adds, “The Evolve cameras provide high quality images for quantitative analysis, and have fast image acquisition speed. The cameras are very stable, fast and efficient.”
Live Cell Imaging, Membrane DynamicsCustomer Stories
Dr. Raghu Padinjat
National Centre for Biological Sciences (NCBS)
Background
The ability to perceive external stimuli and respond accordingly is a fundamental characteristic of biological systems. The stimuli are then converted to signals that can be read by cells via the process of signal transduction, of which there are many different kinds. The G-protein coupled receptor (GPCR) pathway is one such kind of pathway, one particular class of which is driven by the cleavage of lipid molecules known as phosphoinositides, specifically, phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2]. This action further propagates the signal and results in a physiological response. The maintenance of PI(4,5)P2 levels is vital for a sustained response to a continuous signal, requiring the recycling of PI(4,5)P2 to be tightly regulated. PI(4,5)P2 resynthesis is a multi-step pathway, distributed over different membranes and involving several lipid intermediates and lipid-modifying proteins. The visual transduction of the fruit fly, Drosophila melanogaster has proven to be an ideal model system in which to study the regulation of GPCR signaling.
Figure A: Diagrammatic representation of the experimental protocol used to study PI(4,5)P2 dynamics in the intact eye. The blue symbols indicate the time of image acquisition. The color of the bar indicates the light condition at which the fly was kept during the experiment. Black color indicates total dark and red indicates in red light illumination. The time points are labeled above the bar in minute. The detailed experimental procedure is discussed in material and methods section.
Figure B: Fluorescent deep pseudopupil (DPP) imaging to study PI(4,5)P2 dynamics using flies expressing the PI(4,5)P2 biosensor. The time scale of the imaging is indicated on the top of each panel. Arrows indicate the timing of a 90 ms flash of blue light used for imaging the dpp. Images were acquired from control, dPIP5K18 and norpAP24. The genotypes used for the image acquisition are labeled at the left of the image panel. norpAP24, which is a protein null mutant of PLCβ, is used to show the dependence of DPP dynamics on PLCβ activity.
Figure C: Quantitative representation of PI(4,5)P2 dynamics. X-axis represents time in minutes between the depleting flash of blue light and the next image acquired. During this period eyes were illuminated in red light. Y-axis represents the level of fluorescence represented as a % of the value in the initial image. Error bars represents mean +/− S.D from five flies. p values were calculated using an unpaired t-test. The stars represent level of significance (***p< 0.001; **p< 0.01; *p< 0.05)
Challenge
In order to actually determine the specifics of PI(4,5)P2 turnover during signaling, the movement of the lipid must be monitored. Since fluorescently labelling the lipids in vivo is not a feasible prospect, only alternative methods that enable the ability to follow movement of the lipids involved in GPCR signaling can be used.
Live imaging in whole flies is achieved by expressing fluorescently-tagged, lipid-binding probes in the fly eye, which can be used as a readout of the kinetics of the lipid itself. Drosophila eyes contain various optically active elements, often contributing a significant amount of background noise to the preparation.
Researchers have been seeking imaging-based methods to interrogate cellular lipid behavior, with minimal success. The Evolve® 512 EMCCD camera (new series now available) was selected to support the challenging imaging requirements of this research because it offers a singular advantage — the noise is almost completely eliminated. The camera gives the team the ability to reliably track and analyze the movement of the fluorescent probe alone.
The quality and speed of the Evolve 512 EMCCD camera has enabled us to capture zebrafish embryo developments under challenging conditions.
Solution
Considering the challenges, lipid-binding protein domains can be harnessed as probes, using their movement as a proxy for kinetics of the lipid itself. Invertebrate eyes, being compound eyes, exhibit a phenomenon known as the deep pseudopupil (DPP). In this scenario, the images of several ommatidia (individual units of the eye) are superimposed to form one large, virtual image of a single ommatidium formed on a plane below the surface of the eye. The DPP can be observed using a microscope and fluorescent probes expressed in the photoreceptors can be clearly seen using appropriate fluorescence optics. The PH domain of PLCδ, fused to GFP, has long been used a reliable probe for PI(4,5)P2 and, when expressed in fly photoreceptors, forms a fluorescent DPP. The team’s assay focuses on determining the kinetics of PI(4,5)P2 in different genetic backgrounds, where protein components of the signaling pathway have been perturbed. A live fly prep is used for DPP imaging, where a flash of blue light (ƛmax488nm) is used to both stimulate phototransduction and excite GFP. The blue light used to excite GFP is also the stimulus to rapidly convert the majority of rhodopsin to metarhodopsin thus activating the phototransduction cascade and triggering depletion of PI(4,5)P2. Between the blue light stimulations, photoreceptors are exposed to long wavelength (red – ƛmax660nm) light for incremental time periods, which reconverts metarhodopsin to rhodopsin. The recovery in DPP fluorescence intensity with time indicates translocation of the probe from cytoplasm to cell membrane upon PI(4,5)P2 re-synthesis, giving insight into the timescale of the process.
When asked how the Evolve 512 performed for image acquisition in these experiments, Dr. Raghu Padinjat, principal investigator, shared, “The Evolve’s high sensitivity and dynamic range were integral to the acquisition of high-quality images for analysis.” He adds, “The camera’s high signal-to-noise ratio and EM gain feature allowed the acquisition of the extremely low intensity images required to carry out studies of membrane function using fluorescently tagged lipid-binding proteins.”
In Drosophila, two enzymes exist that synthesize PI(4,5)P2 – dPIP5K and SKTL – from its major precursor PI4P. In order to determine which enzyme is responsible for PI(4,5)P2 synthesis during phototransduction, individual mutants for both proteins were assayed for defects in photoresponses. The DPP imaging assay was successfully used to monitor PI(4,5)P2 kinetics in both wild-type (WT) flies and in flies mutant for one of the enzymes (dPIP5K) that synthesizes PI(4,5)P2 in photoreceptors. While WT flies show a characteristic fluorescence recovery curve for the PLCδPH-GFP probe, mutants for dPIP5K show delayed recovery kinetics when compared to that of WT flies. Mutants for SKTL show no notable defects in phototransduction, pointing to dPIP5K as the enzyme that synthesises PI(4,5)P2 required for the photoresponse.
Team members in the lab hope to incorporate dual-color imaging in the near future. This will allow them to monitor more than one phosphoinositide species at any given time, enabling them to better understand the regulatory mechanisms involved in lipid homeostasis during GPCR signaling. For this, they believe the DV2 two-channel simultaneous imaging system from Photometrics would be an ideal addition to their imaging apparatus.
Calcium ion (Ca2+) is a second messenger involved in various physiological phenomena. In contrast to the high Ca2+ concentration (2 mM) in the extracellular fluid, cells maintain the cytosolic Ca2+at nM concentration. However, in the presence of external stimuli, the cytosolic Ca2+concentration increases approximately 100 times through the activation of calcium channels on the plasma membrane, and triggers a diverse array of physiological responses. In order to delineate the physiological significance of these calcium channels, the Laboratory of molecular Biology at Kyoto University studies voltage dependent calcium channels (VDCCs) and transient receptor potential (TRP) channels at various levels of an organism using molecular, cellular and physiological techniques.
Smooth muscle derived A7r5 cells were illuminated by fluorophore-fused PH domain proteins
Custom written software was built in MATLAB to the dual-view based FRET measurement.
Challenge
The primary challenge in this research is the ability to capture simultaneous measurements of the ion channel activity by patch clamp and the FRET dynamics of lipid biosensor. A sophisticated imaging solution is essential.
The quality and speed of the Evolve 512 EMCCD camera has enabled us to capture zebrafish embryo developments under challenging conditions.
Solution
The Photometrics Evolve® 512 EMCCD camera (new series now available) and the DualView multichannel system were both demonstrated. After discussions with a knowledgeable imaging expert, the team selected these cameras as the imaging solutions that would support their research. Since implementing the cameras, they have found that their research has greatly improved with the ability to capture images with such high speed and low noise.
The team simultaneously and quantitatively measured the lipid dynamics by fluorescence changes and ionic flow from ion channels by combining with the patch-clamp. The fluorescence detection was completely dependent on the Evolve 512. “We have chosen this camera because of its high sensitivity, high temporal resolution, and low electrical noise,” states Dr. Mori. As the result, the membrane lipid component (PIP2) was clearly synchronized to the Ca2+ and Na+ flow to activate the cells. Dr. Mori concludes, “The scientific imaging solutions from Photometrics has made evolutional progress on our electrophysiology and FRET research applications.”
