May 2012 - FILM-Users - imperial.ac.uk

[FILM-Users 00221] Sharepoint upgrade complete
by Martin Spitaler 30 May '12

30 May '12

Dear microscopists, the upgrade of our Sharepoint booking system is now complete, the booking is now available at its new site: <https://share.imperial.ac.uk/fom/NHLI/Facilities/FILM> _ _ Rotimi from ICT and her colleagues have done an excellent job, everything has been transferred and checked and should be exactly as before, including all bookings and your message alerts, and links will be redirected automatically for a while, but please let us know if you discover anything unusual. IMPORTANT NOTES: # CHECK YOUR BOOKINGS! And note: bookings made today until now (when you shouldn't have done any) will probably not have migrated! # CHECK YOUR ALERTS: Click on your name in the top-right corner of the page, go to MY SETTINGS and from there to MY ALERTS; you should have one 'MESSAGES' alert for every microscope / software / offline computer you are using # to MAKE NEW BOOKINGS: click on the calendar to make the menu bar appear on the top; under LIST TOOLS / LIST you will find the familiar NEW ITEM icon # UPDATE YOUR BOOKMARKS, the re-direct is only temporary and will expire after a while # to change the view from WEEK to DAY or MONTH, go to CALENDAR TOOLS # to change the view from the default CALENDAR VIEW to e.g. the list view ALL ITEMS, go to LIST TOOLS / LIST / CURRENT VIEW and select one from the pull-down menu For more information, see Rotimi's email below. Good imaging, Martin _ _ MESSAGE FROM ROTIMI NELSON, ICT: Please note that the .... SharePoint site has now been migrated to SharePoint 2010 as part of an on-going upgrade project being carried out by ICT. You will notice some cosmetic changes to your sites, as the new version of SharePoint has adopted the look and feel of other more recent Microsoft Office applications - the biggest difference being the change to using a Ribbon Style toolbar. If you have any bookmarks set then please can you update them to refer to the new URL which is: <https://share.imperial.ac.uk/fom/NHLI/Facilities/FILM> ICT have put in an automatic redirect on the original SharePoint site, so if you forget, you will be automatically redirected to the new area. We have done our best to check through and ensure that everything works as it did on the original site, but if you do spot any differences, please first check the 'SharePoint FAQ' list on the SharePoint help pages before raising any queries. Please raise your issues by completing the dedicated form which would be sent on to the relevant support team with ICT. Sharepoint help: <https://share.imperial.ac.uk/help/SitePages/Home.aspx> Sharepoint problem report form: <https://www.imperial.ac.uk/ict/servicedesk/mfd/login.aspx?ReturnUrl=%2fict%…> Kind Regards Rotimi -- ------------------------------------------------------------------------ *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 00217] Sharepoint upgrade
by Martin Spitaler 29 May '12

29 May '12

Dear microscopists, due to the upgrade of Sharepoint to the new version across the College, Wednesday next week (30 May) our booking system will be unavailable for half a day. All bookings and alerts should be migrated to the new version, but during the transfer you can't make or change bookings or send messages. The planned schedule will be: Wednesday 30 May: until 8.00: Sharepoint available as usual 8.00-14.00 (approximately): no FILM bookings or messages after 14.00 (to be confirmed on the day, I will send you an email): Sharepoint available on the new site, all links should be redirected (so you can update your bookmarks later). IMPORTANT: during the migration (8.00-~14.00), it might be technically possible to access the old Sharepoint system, but any changes might not appear in the new system or interfere with migration of the data - so PLEASE DON'T MAKE ANY CHANGES (you can view it though). Just wait until the move is completed. Please let me know if you have any questions or problems. 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 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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