Andor Technology Ltd is a developer and manufacturer of high performance light measuring solutions . It became a subsidiary of Oxford Instruments after it was purchased for £176m in December 2013.Andor Technology was set up by its founders, Dr. Hugh Cormican, Dr. Donal Denvir and Mr. Mike Pringle in the mid-1980s. While studying at Queen's University Belfast, they "used their physics know-how to build a highly sensitive digital camera...as a tool for their laser research." They subsequently set up Andor Technology to develop it into a commercial product for use in scientific research.Andor Technology Ltd was established in 1989, as a spin out from Queen's University, Belfast. In December 2004 the company became a PLC when it listed on the Alternative Investment Market of the London Stock Exchange.The company is based in Belfast, Northern Ireland, and it designs and manufactures and sells scientific imaging equipment including charge-coupled device , electron-multiplying CCD and Intensified charge-coupled device camera systems, spectroscopy instrumentation, and microscopy systems. The cameras can be used for low light imaging, spectroscopy, X-ray, time resolved, and microscopy studies and have a wide range of users including physicists, biologists, life scientists, geneticists and nano-technologists all around the world.Andor introduced its first EMCCD camera, the DV 465 in 2001 and the company was awarded The Photonics Circle of Excellence Awards from Laurin Publishing, which recognizes the 25 Most Technically Innovative New Products of the Year.EMCCD is based on a CCD chip that incorporates electron multiplication, or EMCCD technology. It is used in fields such as drug discovery, where scientists need to watch vats of chemicals in real time, astrophysics, and oceanography.In June 2010, Andor Technology announced a profits rise by 87%. Wikipedia.
All in vitro experiments used C57BL/6 mice younger than 5 weeks old, except for adult DRG temperature threshold experiments (for example, Fig. 1c, d), in which 3-month-old adult mice were used (these mice were from the same group as was used for two-plate thermal preference tests experiments, see Fig. 3). Trpm2−/− mice were gifts from Y. Mori, and were generated as reported previously27, 28. Mice were maintained on a 12 h day/12 h night cycle. All mice used in two-plate thermal preference tests had been backcrossed onto the parental C57Bl6/6J strain for 7 generations, and wild-type and Trpm2−/− mice were littermates from breeding pairs of Trpm2+/− heterozygote mice. PPG, SCG and paravertebral chain ganglia were extracted from 3 or more mice and DRG from a single mouse. Ganglia were incubated in papain (2 mg ml−1 in Ca2+-free and Mg2+-free HBSS) for 30 min at 30 °C, followed by incubation in collagenase (2.5 mg ml−1 in Ca2+-free and Mg2+-free HBSS) for 30 min at 37 °C. Ganglia were re-suspended and mechanically dissociated in Neurobasal-A/B27 growing medium, which was prepared with Neurobasal-A Medium supplemented with 0.25% (v/v) l-glutamine 200 mM (Invitrogen), 2% (v/v) B-27 supplement (Invitrogen), 1% (v/v) penicillin-streptomycin (Invitrogen), and nerve growth factor (NGF) (Sigma-Aldrich) at 50 ng ml−1. Dissociated neurons were centrifuged and plated onto coverslips pre-coated with poly- l-lysine (10 μg ml−1) and laminin (40 μg ml−1). Neurons were kept in a 37 °C incubator with a 95% air / 5% CO atmosphere for at least 3 h before use, and all neurons were used within 24 h. The growth medium used for PC12 cell culture was RPMI-1640 (Sigma-Aldrich), supplemented with: 1% (v/v) penicillin-streptomycin (Invitrogen), 1% (v/v) L-glutamine 200 mM (Invitrogen), 10% (v/v) horse serum (Invitrogen) and 5% (v/v) fetal bovine serum (FBS, Invitrogen). The differentiation medium for PC12 cells was RPMI-1640 supplemented with: 1% (v/v) penicillin-streptomycin (Invitrogen), 1% (v/v) l-glutamine-200 mM (Invitrogen), 1% (v/v) horse serum (Invitrogen), and NGF (Sigma-Aldrich) with final concentration at 100 ng ml−1. PC12 cells were incubated and maintained in a 37 °C incubator with a 95% air / 5% CO atmosphere. Medium was changed every 2 days, and cells were split every 3–4 days when grown to 90% confluency. The PC12 cells were seeded for imaging on coverslips pre-coated with poly-l-lysine (1 mg ml−1; Sigma-Aldrich) and collagen IV (1 mg ml−1; Sigma-Aldrich). PC12 cell lines were not authenticated and were not tested for mycoplasma contamination. MAH cells were kind gifts from A. Tolkovsky and S. Birren18. The growth medium used for MAH cell culture was L-15 medium (Sigma-Aldrich) supplemented with: 1% (v/v) penicillin-streptomycin (Invitrogen), 1% (v/v) l-glutamine 200 mM (Invitrogen), 10% (v/v) fetal bovine serum (FBS, Invitrogen), 17% (v/v) NaHCO (150 mM), and dexamethasone (Sigma-Aldrich) at 5 μM. The differentiation medium for MAH cells was the same as the growth medium except dexamethasone was replaced with a cocktail of neurotrophic factors: CNTF (10 ng ml−1; Peprotech), bFGF (10 ng ml−1; Peprotech), and NGF (50 ng ml−1; Sigma-Aldrich). Medium was changed every 2 days and MAH cells were split every 4 days when grown to 90% confluency and incubated and maintained in a 37 °C incubator with a 95% air / 5% CO atmosphere. MAH cells used for imaging were seeded on coverslips pre-coated with poly-l-lysine (1 mg ml−1; Sigma-Aldrich) and laminin (40 μg ml−1; BD Science). MAH cell lines were not authenticated and were not tested for mycoplasma contamination. Unless otherwise specified, all experiments were carried out with extracellular solution containing 140 mM NaCl, 4 mM KCl, 1.8 mM CaCl , 1 mM MgCl , 10 mM HEPES and 5 mM glucose; pH was adjusted to 7.4 with NaOH and osmolarity was between 295–305 mOsm. Sodium-free extracellular solution is prepared with the formulation above except for replacing sodium chloride with equimolar choline chloride. Calcium-free extracellular solution is prepared with the formulation above except for removal of calcium chloride. An 8-line manifold gravity-driven system controlled by an automated solution changer with a common outlet was used to apply solution to the cells. The temperature in three lines was heated or cooled with a Peltier device regulated by a proportional gain feedback controller designed by V. Vellani (CV Scientific). The temperature in each experimental protocol was recorded by a miniature thermocouple immediately before the solution entered the bath or (in separate control experiments) at the cell location at the tip of the solution outlet. All compounds applied were prepared as stock solutions first and then diluted to the concentration needed before experiments. Capsaicin was dissolved in ethanol to make 5 mM stock solutions. Pregnenolone sulphate was dissolved in DMSO to make 500 mM stock solutions. 2-APB was dissolved in DMSO to make 500 mM stock solutions. The TRPV4 agonist, PF-4674114, was dissolved in DMSO to make 5 mM stock solutions. Nifedipine was dissolved in DMSO to make 100 mM stock solutions. TTX was dissolved in pH 4.8 citrate buffer to make 100 mM stock solutions. Verapamil, ruthenium red, and H O were dissolved in extracellular solution on the day of experiments. Cells were loaded with 5 μM fura-2 AM (Invitrogen) with 0.02% (v/v) pluronic acid (Invitrogen) for 30 min. After loading, coverslips were put in an imaging chamber and transferred to a Nikon Eclipse Ti-E inverted microscope. Cells were continuously perfused with extracellular solution and were illuminated with a monochromator alternating between 340 and 380 nm (OptoScan; Cairn Research), controlled by WinFluor 3.2 software (J. Dempster, University of Strathclyde, UK). Emission was collected at 510 nm and the resulting pairs of images were acquired every two seconds with a 100 ms exposure time using an iXon 897 EM-CCD camera (Andor Technology, Belfast, UK). Image time series were converted to TIFF files and processed with ImageJ software. Images of the background fluorescence intensity were obtained for both wavelengths and subtracted from the respective image stack before calculating the F ratio images. A minority of neurons (<10%) exhibited an unstable F baseline in the absence of any applied stimulus, usually caused by poor dye loading but in some cases apparently due to low-frequency repetitive firing even in the absence of any treatment, and were removed from analysis. In experiments to identify neurons responding to known TRP channel agonists (Fig. 1a), we found that PS caused a very slow increase in F ratio in some neurons, clearly distinguishable from the rapid elevation in [Ca] seen in TRPM3-expressing DRG neurons. This slow response can probably be attributed to an off-target effect of PS as it was also seen in autonomic neurons which do not appear to express TRPM3 (Fig. 2). A positive response to all agonists was therefore defined from the rate of increase of [Ca] following agonist application, as an increase of F ratio, between two consecutive time points following application of agonist, which exceeds the mean + 3.09 s.d. (cumulative probability value of 99.9%) of all such differences in the absence of any agonist. A heat-sensitive neuron is defined as a neuron with a peak increase in F during a heat stimulus larger than the mean + 3.09 s.d. of the peak increase in F of the glial cells in the same experiment (see Extended Data Fig. 1b). The thermal threshold of a heat-responsive neuron (see Fig. 1d, e) was defined as the temperature when the increase in F ratio between two consecutive time points is larger than the mean + 3.09 s.d. of the increase in F of the glial cells between two consecutive time points in the same experiment. For MAH and PC12 cell cultures, where no glial cells were present, we used the value of mean + 3.09 s.d. obtained from glial cells in similar experiments on neuronal cultures. Supplementary Information Video 1 shows an example series of calcium images. The intracellular solution for concurrent calcium imaging and patch clamp (see Fig. 2b) contained 140 mM KCl, 1.6 mM MgCl , 2.5 mM MgATP, 0.5 mM NaGTP, 10 mM HEPES and 167 μM fura-2; pH was adjusted to 7.3 with KOH. The intracellular solution for current-voltage relationship determination, in which Ca2+ and K+ currents were blocked (see Fig. 2c), contained 130 mM CsCl, 2.5 mM MgATP, 0.5 mM NaGTP, 10 mM HEPES, 10 mM TEA, and 5 mM 4-AP; pH was adjusted to 7.3 with CsOH. The osmolarity of both of the intracellular solutions was between 295–305 mOsm. The extracellular solution for current–voltage relationship determination contained 125 mM NaCl, 2 mM CaCl , 10 mM HEPES and 5 mM glucose, 10 mM TEA, 5 mM 4-AP, 2 μM tetrodotoxin, and 100 μM CdCl . All patch clamp experiments were carried out with an Axopatch 200B patch-clamp amplifier (Axon Instrument, USA). Patch pipettes (Blaubrand 100 μl borosilicate glass, Scientific Laboratory Supplies, Germany) were pulled using a Flaming/Brown P-97 horizontal micropipette puller (Sutter Instruments, USA) and had a resistance between 3 and 5.5 MΩ. A giga-ohm seal was formed between the patch pipette and the cell membrane and the pipette capacitance transients were cancelled before achieving the whole cell configuration. All experiments were begun in voltage clamp mode with holding potential at −60 mV at the time of entering whole cell mode. After entering the whole-cell mode, series resistance was adjusted to be lower than 20 mega-ohm. Resting membrane potential was tested and only neurons with membrane potentials more negative than −50 mV were used for recording. Data were acquired and analysed with pClamp10 software (Axon Instruments, USA) and whole cell currents and voltages were filtered at 1 kHz and sampled at 10 kHz. MAH cells were trypsinized and collected as a cell pellet before lysis. RNA extraction was performed with the miRNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. Two samples from MAH cells grown in growth medium and 2 samples from MAH cells grown in differentiation medium were sent to Oxford Gene Technology to complete the rest of the steps for RNA-sequencing. Sequencing libraries were prepared with the Illumina TruSeq RNA Sample Prep Kit v2. A total of 4 samples (two cold-sensitive MAH cells and two cold-insensitive MAH cells) were sequenced on 2 lanes on the Illumina HiSeq2000 platform using TruSeq v3 chemistry. All sequences were paired-end and sequencing was performed over 100 cycles. Read files (Fastq) were generated from the sequencing platform via the manufacturer’s proprietary software. Reads were processed through the Tuxedo suite. Reads were mapped to their location to the appropriate Illumina iGenomes build using Bowtie version 2.02. Splice junctions were identified using TopHat, version v2.0.9. Cufflinks version 2.1.1 was used to perform transcript assembly, abundance estimation and differential expression and regulation for the samples. Visualization of differential expression results were performed with CummeRbund. RNA-seq alignment metrics were generated with Picard. Coverslips were marked on the periphery with a diamond knife to assist localization of the imaged region and were then calcium-imaged as above to identify novel heat-sensitive neurons. Following calcium imaging, coverslips were rinsed with phosphate-buffered saline (PBS) then fixed in 4% paraformaldehyde (PFA) at 4 °C for 20 min. TRPM2 mRNA was detected with digoxigenin-labelled antisense probes against mouse Trpm2 (NM_138301.2). We are very grateful to Y. Mori of Kyoto University for providing the mouse Trpm2 gene cloned into the pCI-neo plasmid (Promega). For the Trpm2 antisense probe the plasmid was linearized with EcoRI and transcribed with T3 RNA polymerase and for the Trpm2 sense probe, which was used as a negative control, the plasmid was linearized with SalI and transcribed with T7 RNA polymerase. Fixed coverslips were rinsed in PBS with 0.1% Triton, and were then incubated in in situ hybridization solution without probe at 47 °C for 30 min as pre-hybridization step. After pre-hybridization, coverslips were transferred into in situ hybridization solution with antisense or sense probes for hybridization at 47 °C overnight29. Following hybridization coverslips were washed in 2× SSC and then 0.2× SSC at 47 °C for 30 min for each solution. Coverslips were then washed twice with KTBT solution at room temperature for 5 min for each washing. 25% normal goat serum was then used for blocking cells for 1 h at room temperature. Coverslips were then incubated in 25% normal goat serum containing pre-absorbed anti-digoxygenin antibody coupled to alkaline phosphatase for 2 h at room temperature, followed by washing 3× in KTBT for 15 min each wash, and then twice in alkaline phosphatase buffer at room temperature for 10 min each wash. Coverslips were then developed in alkaline phosphatase buffer containing 337.5 μg ml−1 NBT and 175 μg ml−1 BCIP in the dark for 8 h before being washed in KTBT, fixed in 4% PFA for 10 min, washed in PBS, and then mounted in SlowFade Gold Antifade Mountant with DAPI29. DIC transmitted-light images were acquired through a Plan Fluor 10× Ph1 DLL objective with a DS-Qi2 monochrome camera on a Nikon Eclipse Ti-E inverted microscope. A GFPHQ filter was used to enhance the dark purple colour. The images were rotated, cropped, and resized with ImageJ to be aligned with the images obtained in calcium imaging. DRG neurons on marked coverslips were calcium imaged as above then rinsed with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde (PFA) at 4 °C for 20 min. After fixation, coverslips were washed twice in PBS with 0.1% Triton then incubated in solution containing 10 μg ml−1 IB4 bound to Alexa Fluor 594, 10% normal goat serum, 2% bovine serum albumin, 0.1% Triton, and 10 mM sodium azide for 1 h at room temperature followed by washing with PBS three times. DIC transmitted-light images were acquired through a Plan Fluor 10× Ph1 DLL objective with a DS-Qi2 monochrome camera on a Nikon Eclipse Ti-E inverted microscope. A Texas Red HYQ filter was used to capture the Alexa 594 signal. The images obtained were rotated, cropped, and resized with ImageJ to be aligned with the images obtained in calcium imaging. To eliminate as far as possible any extraneous genetic influences the Trpm2−/− mice were backcrossed onto the parental C57Bl6/6J line for 7 generations27, 28. To minimize environmental effects, wild-type and Trpm2−/− littermates from heterozygous matings were compared in behavioural experiments. Sample size to achieve significance was determined from trial experiments but no power analysis was performed. All mice were tested at all temperatures so no randomization of experimental groups was necessary (see Fig. 3 legend). We used a two-plate thermal preference test (BioSeb, France) with one plate maintained at a temperature of 33 °C, which other studies have shown is the preferred temperature11, and the other at a variable temperature. The temperatures of test and control plates were reversed after 30 min to control for any influence of environmental cues. Other studies have observed sex differences in mouse thermal behaviour12 so we followed other authors5, 11 in using only adult males (10–16 weeks old) in behavioural experiments. Two hot/cold-plate machines (Bioseb, France), placed back to back, formed the two-plate thermal apparatus. Plates were enclosed in a plexiglass chamber divided into two lanes, with an opaque compartment between them, and two mice were tested simultaneously in adjacent lanes (see Supplementary Information Video 2). The temperature of each plate was controlled by T2CT software (Bioseb, France). Plate temperatures were tested with an infrared thermometer (Bioseb) and were found to be accurately controlled to within 0.2 °C of the command temperature over the entire plate area. One plate was maintained at the preferred temperature of 33 °C, and mice were initially placed onto the plate with starting temperature other than 33 °C (‘plate A’, see Fig. 3) before initiating recording. The movements of the mice between the two plates were recorded for 3,495 s without human presence and the mouse position was determined with an automated video tracking system (Bioseb), so operator blinding was not necessary. The temperatures of the two plates were exchanged 1,800 s after initiation of recording; plate temperature settled to within 0.2 °C of the new temperature within 180 s of the change. Experiments were performed between 8 a.m. and 10 p.m., with room temperature at 20 °C. For experiments testing thermal preference between the two mildest temperatures, 28 °C versus 33 °C and 33 °C versus 38 °C, mice were tested again, with the starting temperatures of the two plates exchanged, 3–5 h after the first recording. For other temperatures recordings were made only once on a particular mouse. Mice were tested with the protocols, in order, of 28 °C versus 33 °C, 33 °C versus 38 °C, 23 °C versus 33 °C and 33 °C versus 43 °C. Sample size was based on pilot experiments. When making statistical comparisons variances were checked to ensure that it was similar between groups being compared. All animal experiments were approved by the Animal Welfare and Ethical Review Body (AWERB), King’s College London. Supplementary Information Video 2 shows an example of a thermal-choice behavioural experiment. All data are expressed as means ± s.e.m. Analyses were performed with GraphPad Prism version 6.01 or SigmaPlot 11.0. The particular statistical test used is stated either in the text or figure legends. Biological replicates are stated in the legends for each figure. Given the nature of these experiments, technical replicates were not possible.
Oreopoulos J.,Spectral Applied Research |
Berman R.,Spectral Applied Research |
Browne M.,Andor Technology
Methods in Cell Biology | Year: 2014
Live-cell imaging requires not only high temporal resolution but also illumination powers low enough to minimize photodamage. Traditional single-point laser scanning confocal microscopy (LSCM) is generally limited by both the relatively slow speed at which it can acquire optical sections by serial raster scanning (a few Hz) and the higher potential for phototoxicity. These limitations have driven the development of rapid, parallel forms of confocal microscopy, the most popular of which is the spinning-disk confocal microscope (SDCM). Here, we briefly introduce the SDCM technique, discuss its strengths and weaknesses against LSCM, and update the reader on some recent developments in SDCM technology that improve its performance and expand its utility for life science research now and in the future. © 2014 Elsevier Inc. Source
Andor Technology | Date: 2013-03-25
Scientific equipment and apparatus for use in spectroscopy and scientific imaging comprising multichannel detector systems, scientific charge coupled devices, intensified charge coupled devices, detector head controllers and microcontrollers, spectrograph apparatus, fiber optics and fiber optic accessories for collecting, measuring, transporting and projecting light; computer software for use in spectroscopy and scientific imaging. Engineering services and design services in relation to scientific equipment and apparatus for spectroscopy and scientific imaging; computer software development and design and computer programming.
Agency: Cordis | Branch: FP7 | Program: CP-FP | Phase: HEALTH-2009-1.2-1 | Award Amount: 2.34M | Year: 2010
Since the histopathological diagnosis of tumours is based on microscopy, it would be highly desirable to possess a microscopical technique allowing morphological investigation with a wide zoom range, high resolution and the implementation of multiplex staining. Thus, the goal of the REMEDI project is to explore the possibilites of novel light microscopy techniques, which overcome the diffraction limit of visible light and extend the range of observation to the molecular level of proteins in tumour cells and analyze their interaction and spatial distribution. This proposal aims at the use of single-molecule-microscopy of cancer tissue samples with ultra-high sensitivity, simplified handling and increased speed of analysis. This shall be accomplished by the development of a novel resolution-enhanced microscopy platform with an integrated novel CMOS camera and an adequate image acquistion and analysis software. The REMEDI platform will be validated with two relevant applications: diagnostic/experimental pathology of breast cancer and alterations of plasma membrane components of lymphoma cells.
Agency: Cordis | Branch: H2020 | Program: MSCA-ITN-ETN | Phase: MSCA-ITN-2014-ETN | Award Amount: 3.88M | Year: 2015
Cell migration (cell motility) is a fundamental biological process that is pivotal in (i) tissue formation and repair (health) and (ii) tissue invasion during carcinogenesis (disease). Understanding and controlling cell migration will have major clinical impact. Clarifying mechanisms driving cell motility has been challenging due to the complex underlying cellular mechanisms; these involve multiple components coordinated by structural, chemical and physical signals in terms of time and space. To accomplish breakthroughs in this field, researchers are needed who (i) master cutting-edge experimental techniques for monitoring the different cellular processes at high resolution and (ii) have competencies in theoretical science for integrating the resulting data sets into mechanistic mathematical models for predicting motile cell behaviour. The Research Training Network on Integrated Component Cycling in Epithelial Cell Motility (InCeM) aims to endow up-and-coming researchers with exactly these competencies. They will be able to develop and apply innovative devices for microscopic recording, image processing techniques, data analysis tools and modelling procedures for mechanistic understanding of cell migration. InCeM will focus on epithelial cells, since inducing motility in this cell type is clinically relevant for wound healing and cancer invasion. The ultimate goal is to control and manipulate cell migration for clinical applications. A dedicated multidisciplinary team of 11 beneficiaries from universities (4), research institutions (4) and industry (3), based in 5 European countries and Israel, together with 17 associated partners from the public and private sector, will train 15 Early Stage Researchers (ESRs) to use the relevant technologies and sciences and will offer business training to prepare them for successful careers in both academic and non-academic environments.