- FILM-Users - imperial.ac.uk

[FILM-Users 00219] server update
by Martin Spitaler 29 May '12

29 May '12

Dear microscopists, we've now had a service session with Netgear, the manufacturer of our server, they think they have sorted out the problems behind our frequent troubles recently. Now, the server is up and running again, hopefully that's all sorted then. Sorry for all the troubles, ICT have worked hard behind the scenes to sort them out, it just was something very unusual. Good imaging, Martin -- ------------------------------------------------------------------------ *Martin Spitaler, PhD* *FILM - Facility for Imaging by Light Microscopy* - Facility Manager - Sir Alexander Fleming Building, desk 401 Imperial College London / South Kensington Exhibition Road London SW7 2AZ UK Tel. +44-(0)20-759-42023 E-mail m.spitaler(a)imperial.ac.uk <mailto:m.spitaler@imperial.ac.uk> Website: http://imperial.ac.uk/imagingfacility

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[FILM-Users 00218] Scratch Server Maintenance
by Rothery, Stephen M 28 May '12

28 May '12

Dear All The Film server will undergo maintenance /testing this afternoon in an attempt to sort out our recent problems Unfortunately during this work the server will not be available We apologise for any inconvenience caused Best wishes Steve FILM - Facility for Imaging by Light Microscopy ************************************************************** Steve Rothery FILM Technician Sir Alexander Fleming Building, desk 403 Imperial College London / South Kensington Exhibition Road London SW7 2AZ Tel. +44-(0)207 594 9793 E-mail s.rothery(a)imperial.ac.uk<mailto:s.rothery@imperial.ac.uk> Website: http://imperial.ac.uk/imagingfacility **************************************************************

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[FILM-Users 00216] TODAY: FILM Club Friday 25 April 2012: Charanjit Singh: Quantitative imaging of plasmid DNA in live human airway epithelial cells
by Martin Spitaler 25 May '12

25 May '12

Dear microscopists, TODAY is our MAY FILM CLUB, again with a very interesting presentation: Charanjit Singh (Eric Alton lab) will tell us about "QUANTITATIVE BIOLOGICAL IMAGING OF PLASMID DNA IN LIVE HUMAN AIRWAY EPITHELIAL CELLS FOLLOWING NON-VIRAL GENE TRANSFER" FRIDAY 25 MAY 2012, 1PM, SAFB-120, SOUTH KENSINGTON CAMPUS The UK CF Gene Therapy Consortium is interested in non-viral gene therapy for cystic fibrosis (CF). It is widely accepted that in addition to extracellular barriers responsible for inefficient uptake, there are key intracellular obstacles to the nuclear delivery of the therapeutic plasmid DNA (pDNA). Thus, we are investigating the intracellular fate of pDNA following transfection, using the clinically relevant cationic Genzyme Lipid (GL) 67A formulation, using transmission electron microscopy (TEM) combined with three-dimensional Spinning-Disk confocal microscopy (SDCM), to track, quantitate and provide high resolution ‘snapshots’ of pDNA at the single molecule level in transfected primary human airway epithelial cells (AECs) grown at the air-liquid interface (ALI). The pDNA was tagged with 1.4 nm gold nanoparticles (NG- pDNA) or fluorescent quantum dots (QDot-pDNA) for use in TEM or fluorescence studies, respectively. We were able to detect significantly (p<0.05) more electron-dense gold spots in gold-enhanced TEM sections from NG-pDNA-transfected cells compared with those transfected with unconjugated pDNA. Approximately 50% and 10% of the total internalised pDNA localised to nuclei within 1 hour transfection in SDCM (123 AECs, 8 independent experiments) and TEM (48 AECs, 6 independent experiments) studies respectively. In experiments to track the intracellular trafficking of the NG-pDNA, we found that the nuclear delivery of NG-pDNA increase with increasing transfection time from 15 to 60 min but there was no any change after 360 min transfection. Parallel confocal experiments following the intracellular fate of Qdot-pDNA over 120 min in living AECs confirm the TEM experiments. See you there, good imaging, Martin -- Martin Spitaler, PhD FILM - Facility for Imaging by Light Microscopy - Facility Manager - Sir Alexander Fleming Building, desk 401 Imperial College London / South Kensington Exhibition Road London SW7 2AZ UK Tel. +44-(0)20-759-42023 E-mail m.spitaler(a)imperial.ac.uk Website: http://imperial.ac.uk/imagingfacility

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[FILM-Users 00215] TOMORROW: FILM Club Friday 25 April 2012: Charanjit Singh: Quantitative imaging of plasmid DNA in live human airway epithelial cells
by Martin Spitaler 24 May '12

24 May '12

Just a reminder about tomorrow's FILM Club, see you all there, Martin > > *From:*film-users-bounces@imperial.ac.uk > [mailto:film-users-bounces@imperial.ac.uk] *On Behalf Of *Martin Spitaler > *Sent:* 18 May 2012 19:14 > *To:* film-users > *Subject:* [FILM-Users 00211] FILM Club Friday 25 April 2012: > Charanjit Singh: Quantitative imaging of plasmid DNA in live human > airway epithelial cells > > Dear microscopists, > > our next FILM CLUB is coming up, again with a very interesting > presentation: > > Charanjit Singh (Eric Alton lab) will tell us about "QUANTITATIVE > BIOLOGICAL IMAGING OF PLASMID DNA IN LIVE HUMAN AIRWAY EPITHELIAL > CELLS FOLLOWING NON-VIRAL GENE TRANSFER" > > FRIDAY 25 MAY 2012, 1PM, SAFB-120, SOUTH KENSINGTON CAMPUS > > The UK CF Gene Therapy Consortium is interested in non-viral gene > therapy for cystic fibrosis (CF). It is widely accepted that in > addition to extracellular barriers responsible for inefficient uptake, > there are key intracellular obstacles to the nuclear delivery of the > therapeutic plasmid DNA (pDNA). Thus, we are investigating the > intracellular fate of pDNA following transfection, using the > clinically relevant cationic Genzyme Lipid (GL) 67A formulation, using > transmission electron microscopy (TEM) combined with three-dimensional > Spinning-Disk confocal microscopy (SDCM), to track, quantitate and > provide high resolution 'snapshots' of pDNA at the single molecule > level in transfected primary human airway epithelial cells > (AECs) grown at the air-liquid interface (ALI). The pDNA was tagged > with 1.4 nm gold nanoparticles (NG- pDNA) or fluorescent quantum dots > (QDot-pDNA) for use in TEM or fluorescence studies, respectively. We > were able to detect significantly (p<0.05) more electron-dense gold > spots in gold-enhanced TEM sections from NG-pDNA-transfected cells > compared with those transfected with unconjugated pDNA. Approximately > 50% and 10% of the total internalised pDNA localised to nuclei within > 1 hour transfection in SDCM (123 AECs, 8 independent experiments) and > TEM (48 AECs, 6 independent experiments) studies respectively. In > experiments to track the intracellular trafficking of the NG-pDNA, we > found that the nuclear delivery of NG-pDNA increase with increasing > transfection time from 15 to 60 min but there was no any change after > 360 min transfection. Parallel confocal experiments following the > intracellular fate of Qdot-pDNA over 120 min in living AECs confirm > the TEM experiments. > > See you there, good imaging, > > Martin > > -- > > ------------------------------------------------------------------------ > > *Martin Spitaler, PhD* > > *FILM **- Facility for Imaging by Light Microscopy* > - Facility Manager - > Sir Alexander Fleming Building, desk 401 > Imperial College London / South Kensington > Exhibition Road > London SW7 2AZ > UK > > Tel. +44-(0)20-759-42023 > E-mail m.spitaler(a)imperial.ac.uk <mailto:m.spitaler@imperial.ac.uk> > Website: http://imperial.ac.uk/imagingfacility >

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[FILM-Users 00214] FILM microscopes in L-Block on Hammersmith campus
by Martin Spitaler 22 May '12

22 May '12

Dear microscopists, as you may already know, FILM is about to expand to Hammersmith Campus, setting up two brand-new microscopes in the new L-Block building. With generous funding from a BHF fund to NHLI, two top-class microscopes have been purchased, a Zeiss LSM-780 confocal with GaAsP metadetector and an AxioObserver with LED illumination for fast multi-channel acquisition, a Hamamatsu Flash 4.0 high-resolution / high-sensitivity sCMOS camera and a Vivatome for fast 3D acquisition. Details of the microscopes will soon be available on our website and in our WIKI, I'll keep you posted. Installation starts this week, then the microscopes will be available to users in NHLI as well as other Imperial College users. The microscopes will be part of FILM, so you can expect the same training and support as so far on the South Kensington campus. The new FILM branch will be started with two OPENING AND TRAINING DAYS on THURSDAY+FRIDAY 14+15 JUNE 2012 THURSDAY 14 JUNE MORNING SESSION (Wolfson Building LT2): Presentations 10.00-10.10 (Prof Tony Magee, head of FILM): Welcome and opening of the L-Block branch of FILM 10.10-10.20 (Martin Spitaler, FILM): Overview and specifications of the new microscopes 10.20-11.20 (David Crook, Zeiss): Functions and applications of the widefield microscope setup (AxioObserver with Vivatome, Colibri LED illumination and Hamamatsu Flash 4.0 camera) # hardware specifications # software # high-speed acquisition (fast LED switching, fast camera options) # automated tiled, multi-position and multi-well acquisition # 3D acquisition with the Vivatome 11.20-11.30 coffee break 11.30-13.00 (Graham Brown, Zeiss): Functions and applications of the confocal microscope setup (LSM-780 Meta) # hardware specifications # confocal-specific software functions # setup of fluorophor-specific light paths, PMT versus Meta-GaAsp detectors # spectral acquisition with the Meta detector and spectral unmixing 13.00-14.00 lunch break AFTERNOON SESSION (Microscope Room L-Block 314): 14.00-17.00: hands-on session for users with their own sample (advance booking) FRIDAY: Further hands-on session for users with their own sample, according to demand To get your personal introduction with your own samples, please book your personal 2-hour slot by email to Stephen M Rothery <s.rothery(a)imperial.ac.uk> (this introduction will NOT replace your own personal training session, which will still be provided later) Presentations on Thursday morning will be open to everybody. Good imaging, Martin -- ------------------------------------------------------------------------ *Martin Spitaler, PhD* *FILM - Facility for Imaging by Light Microscopy* - Facility Manager - Sir Alexander Fleming Building, desk 401 Imperial College London / South Kensington Exhibition Road London SW7 2AZ UK Tel. +44-(0)20-759-42023 E-mail m.spitaler(a)imperial.ac.uk <mailto:m.spitaler@imperial.ac.uk> Website: http://imperial.ac.uk/imagingfacility

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[FILM-Users 00210] server service
by Martin Spitaler 21 May '12

21 May '12

Dear microscopists, because of the frequent problems with the FILM server recently, ICT will do a service, i.e. reinstall the operating system. During that time, the server will be unavailable on MONDAY 21 MAY, MORNING UNTIL NOON (approximately). This should not affect the data storage, the data should all stay untouched, but as always, we don't guarantee for the data on this scratch disk. Good imaging, Martin -- ------------------------------------------------------------------------ *Martin Spitaler, PhD* *FILM - Facility for Imaging by Light Microscopy* - Facility Manager - Sir Alexander Fleming Building, desk 401 Imperial College London / South Kensington Exhibition Road London SW7 2AZ UK Tel. +44-(0)20-759-42023 E-mail m.spitaler(a)imperial.ac.uk <mailto:m.spitaler@imperial.ac.uk> Website: http://imperial.ac.uk/imagingfacility

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[FILM-Users 00212] FILM Club Friday 25 MAY(!) 2012: Charanjit Singh: Quantitative imaging of plasmid DNA in live human airway epithelial cells
by Martin Spitaler 19 May '12

19 May '12

...sorry, meant 25 MAY of course (as below), this coming week, Martin Dear microscopists, our next FILM CLUB is coming up, again with a very interesting presentation: Charanjit Singh (Eric Alton lab) will tell us about "QUANTITATIVE BIOLOGICAL IMAGING OF PLASMID DNA IN LIVE HUMAN AIRWAY EPITHELIAL CELLS FOLLOWING NON-VIRAL GENE TRANSFER" FRIDAY 25 MAY 2012, 1PM, SAFB-120, SOUTH KENSINGTON CAMPUS The UK CF Gene Therapy Consortium is interested in non-viral gene therapy for cystic fibrosis (CF). It is widely accepted that in addition to extracellular barriers responsible for inefficient uptake, there are key intracellular obstacles to the nuclear delivery of the therapeutic plasmid DNA (pDNA). Thus, we are investigating the intracellular fate of pDNA following transfection, using the clinically relevant cationic Genzyme Lipid (GL) 67A formulation, using transmission electron microscopy (TEM) combined with three-dimensional Spinning-Disk confocal microscopy (SDCM), to track, quantitate and provide high resolution 'snapshots' of pDNA at the single molecule level in transfected primary human airway epithelial cells (AECs) grown at the air-liquid interface (ALI). The pDNA was tagged with 1.4 nm gold nanoparticles (NG- pDNA) or fluorescent quantum dots (QDot-pDNA) for use in TEM or fluorescence studies, respectively. We were able to detect significantly (p<0.05) more electron-dense gold spots in gold-enhanced TEM sections from NG-pDNA-transfected cells compared with those transfected with unconjugated pDNA. Approximately 50% and 10% of the total internalised pDNA localised to nuclei within 1 hour transfection in SDCM (123 AECs, 8 independent experiments) and TEM (48 AECs, 6 independent experiments) studies respectively. In experiments to track the intracellular trafficking of the NG-pDNA, we found that the nuclear delivery of NG-pDNA increase with increasing transfection time from 15 to 60 min but there was no any change after 360 min transfection. Parallel confocal experiments following the intracellular fate of Qdot-pDNA over 120 min in living AECs confirm the TEM experiments. See you there, good imaging, Martin -- ------------------------------------------------------------------------ *Martin Spitaler, PhD* *FILM - Facility for Imaging by Light Microscopy* - Facility Manager - Sir Alexander Fleming Building, desk 401 Imperial College London / South Kensington Exhibition Road London SW7 2AZ UK Tel. +44-(0)20-759-42023 E-mail m.spitaler(a)imperial.ac.uk <mailto:m.spitaler@imperial.ac.uk> Website: http://imperial.ac.uk/imagingfacility

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[FILM-Users 00211] FILM Club Friday 25 April 2012: Charanjit Singh: Quantitative imaging of plasmid DNA in live human airway epithelial cells
by Martin Spitaler 18 May '12

18 May '12

Dear microscopists, our next FILM CLUB is coming up, again with a very interesting presentation: Charanjit Singh (Eric Alton lab) will tell us about "QUANTITATIVE BIOLOGICAL IMAGING OF PLASMID DNA IN LIVE HUMAN AIRWAY EPITHELIAL CELLS FOLLOWING NON-VIRAL GENE TRANSFER" FRIDAY 25 MAY 2012, 1PM, SAFB-120, SOUTH KENSINGTON CAMPUS The UK CF Gene Therapy Consortium is interested in non-viral gene therapy for cystic fibrosis (CF). It is widely accepted that in addition to extracellular barriers responsible for inefficient uptake, there are key intracellular obstacles to the nuclear delivery of the therapeutic plasmid DNA (pDNA). Thus, we are investigating the intracellular fate of pDNA following transfection, using the clinically relevant cationic Genzyme Lipid (GL) 67A formulation, using transmission electron microscopy (TEM) combined with three-dimensional Spinning-Disk confocal microscopy (SDCM), to track, quantitate and provide high resolution 'snapshots' of pDNA at the single molecule level in transfected primary human airway epithelial cells (AECs) grown at the air-liquid interface (ALI). The pDNA was tagged with 1.4 nm gold nanoparticles (NG- pDNA) or fluorescent quantum dots (QDot-pDNA) for use in TEM or fluorescence studies, respectively. We were able to detect significantly (p<0.05) more electron-dense gold spots in gold-enhanced TEM sections from NG-pDNA-transfected cells compared with those transfected with unconjugated pDNA. Approximately 50% and 10% of the total internalised pDNA localised to nuclei within 1 hour transfection in SDCM (123 AECs, 8 independent experiments) and TEM (48 AECs, 6 independent experiments) studies respectively. In experiments to track the intracellular trafficking of the NG-pDNA, we found that the nuclear delivery of NG-pDNA increase with increasing transfection time from 15 to 60 min but there was no any change after 360 min transfection. Parallel confocal experiments following the intracellular fate of Qdot-pDNA over 120 min in living AECs confirm the TEM experiments. See you there, good imaging, Martin -- ------------------------------------------------------------------------ *Martin Spitaler, PhD* *FILM - Facility for Imaging by Light Microscopy* - Facility Manager - Sir Alexander Fleming Building, desk 401 Imperial College London / South Kensington Exhibition Road London SW7 2AZ UK Tel. +44-(0)20-759-42023 E-mail m.spitaler(a)imperial.ac.uk <mailto:m.spitaler@imperial.ac.uk> Website: http://imperial.ac.uk/imagingfacility

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[FILM-Users 00209] Fwd: Electron Microscopy and Tomography Position - University of Exeter
by Martin Spitaler 16 May '12

16 May '12

...forwarded, for your information, Martin ================================================= -------- Original Message -------- Subject: Electron Microscopy and Tomography Position - University of Exeter Date: Tue, 15 May 2012 12:22:07 +0100 From: Paszkiewicz, Konrad <K.H.Paszkiewicz(a)EXETER.AC.UK> Reply-To: Paszkiewicz, Konrad <K.H.Paszkiewicz(a)EXETER.AC.UK> To: <UK-EUROBIOIMAGING-PROJECT(a)JISCMAIL.AC.UK> Dear all, Could you please circulate this to any interested colleagues? This full time post is primarily to support the Exeter Bio-Imaging Centre, as part of a new Biomedical Informatics Hub, which is funded by a recent Wellcome Trust Institutional Strategic Support Fund award. This Biomedical Informatics Hub will provide informatics support to a range of emerging technologies and support multiple research areas. The post holder will carry out research activities and support grant and income generation related to Wellcome Trust projects. The post holder will provide support to cell biologists studying fundamental aspects of cellular organisation, membrane trafficking, the cytoskeleton, and microbial pathogenesis. We are particularly interested in applications from candidates with expertise in advanced electron microscopy and related computational techniques (including tomography and 3D reconstruction of ultra-structural data from biological specimens; reconstruction of single molecule images). Candidates should have expertise in all aspects of electron microscopy sample preparation, immunogold labelling, usage and running of an electron microscopes, and computational and image analysis experience. The post holder will personally undertake sample preparation, perform imaging and analysis on behalf of researchers and as part of collaborative research projects, and will be expected to contribute to research papers and grant proposals as required. For job particulars please see http://www.admin.ex.ac.uk/personnel/jobs/R11159.pdf and apply online at https://jobs.exeter.ac.uk For further information contact Professor Gero Steinberg, e-mail G.Steinberg(a)exeter.ac.uk or telephone (01392) 723476. Dr Konrad Paszkiewicz Head, Exeter Sequencing Service, Biosciences, Stocker Road, University of Exeter, Exeter EX4 4QD, UK. http://biosciences.exeter.ac.uk/facilities/sequencing/ http://biosciences.exeter.ac.uk/facilities/exbiss/ -- UK-EUROBIOIMAGING Visit the website http://www.bioimaginguk.org To e-mail the group, send messages to UK-EUROBIOIMAGING-PROJECT(a)JISCMAIL.AC.UK To subscribe visit https://www.jiscmail.ac.uk/cgi-bin/webadmin?A0=UK-EUROBIOIMAGING-PROJECT --

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[FILM-Users 00208] Fwd: [neuroscience-technology-network] Connectomics Seminar - Professor George Augustine
by Martin Spitaler 16 May '12

16 May '12

...forwarded, for your information, Martin ====================================================================================== -------- Original Message -------- Subject: [neuroscience-technology-network] Connectomics Seminar - Professor George Augustine Date: Mon, 14 May 2012 15:19:14 +0000 From: Fenton, Rob J <r.fenton(a)imperial.ac.uk> To: neuroscience-technology-network <neuroscience-technology-network(a)imperial.ac.uk> Dear Colleague, Please note below the forthcoming lecture that may be of interest to you. Professor George Augustine 'Optogenetic mapping of brain circuitry' 4pm in RSM 2.28 on Monday 21st May Biography: Prof. Augustine is a distinguished neurobiologist and a Faculty Professor at Duke-NUS Medical School in Singapore. He is also Director of the new Center for Functional Connectomics at the Korea Institute of Science and Technology. He has broad interests in cellular and circuit neuroscience, and will talk about his recent work using novel optogenetic tools to map the fine organisation of nervous system I am in process of drawing up a schedule for individual meetings; please let me know if you'd like to meet with Prof. Augustine Thanks, Paul Chadderton ____________________________ Paul Chadderton Department of Bioengineering Imperial College London Prince Consort Road London SW7 2BW T: +44-20-7594-0770 E: p.chadderton(a)imperial.ac.uk <mailto:p.chadderton@imperial.ac.uk>

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