- FILM-Users - imperial.ac.uk

[FILM-Users 00831] FLIM-FRET seminar - Sean Warren- on Monday 12th November from 1-2pm
by Bruckbauer, Andreas 09 Nov '18

09 Nov '18

Sean Warren, who is a former member of the Photonics group in Physics and who wrote the FLIMfit software package for analysing fluorescence lifetime image data, will be giving a seminar on Monday 12th November from 1-2pm in Blackett room B636: Dr. Sean Warren Garvan Institute of Medical Research In vivo quantification of FRET biosensors using multispectral FLIM-FRET Intravital imaging of genetically expressed Förster resonance energy transfer (FRET) biosensors can provide unique insights into the function of biological processes in a native context. However, quantitative analysis of intravital FRET data is often compromised by motion, tissue or drug autofluorescence and scattering. We demonstrate tools to overcome these challenges and so reliably obtain accurate FRET quantification in vivo. We first demonstrate a new open source software tool, Galene, for image-based correction of sample motion blurring in both time resolved and conventional laser scanning fluorescence microscopy data in two and three dimensions. We show that we can resolve intravital FRET images of intra-abdominal organs in a murine model. We then introduce a multispectral fluorescence lifetime imaging (FLIM)-FRET analysis approach which allows reliable quantification of FRET biosensors in a variety of intravital contexts. We demonstrate that we can measure FRET activity in the presence of high levels of autofluorescence from both tissue and administered small molecule drugs. Finally, we demonstrate that this multispectral approach can be used to image two highly overlapping FRET biosensors using a genetically engineered mouse model expressing FRET biosensors for the prototypical RhoGTPases RhoA and Rac1. This approach allows us to directly visualise the mutually inhibitory response of RhoA and Rac1 on a subcellular level to treatment with small molecule inhibitors and activators. Best wishes Andreas ---------------------------------------------------- Dr Andreas Bruckbauer FILM facility manager Sir Alexander Fleming Building South Kensington Campus Imperial College London Exhibition Road London, SW7 2AZ SAF 4th floor, open space desk 401 +44 (0)20 7594 2023 a.bruckbauer(a)imperial.ac.uk<mailto:a.bruckbauer@imperial.ac.uk> http://www.imperial.ac.uk/medicine/facility-for-imaging-by-light-microscopy/ http://www.imperial.ac.uk/people/a.bruckbauer ----------------------------------------------------

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[FILM-Users 00829] Reminder for data saving
by Gaboriau, David C A 06 Nov '18

06 Nov '18

Dear all, Just a quick reminder to save your microscopy data on the local hard drive of the microscope you are using, and then copy all the data to your folder on the new network drive Scratch2 (Scratch has been discontinued). You can map Scratch2 at the following address: \\film-nas2.med.ic.ac.uk\scratch2<file:///\\film-nas2.med.ic.ac.uk\scratch2> All the best and Happy Imaging! David David Gaboriau, PhD FILM - Facility for Imaging by Light Microscopy High-Content Microscopy Specialist Sir Alexander Fleming Building, desk 408 Imperial College London / South Kensington Exhibition Road London SW7 2AZ, UK Phone: +44 (0)2075 949 793 Email: d.gaboriau(a)imperial.ac.uk<mailto:d.gaboriau@imperial.ac.uk> Website: http://imperial.ac.uk/imagingfacility Page: http://www.imperial.ac.uk/people/d.gaboriau

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[FILM-Users 00828] TODAY: Seminar at Imperial , SAF G120, 12.30
by Gaboriau, David C A 23 Oct '18

23 Oct '18

Dear all, There is a FILM Seminar today in SAF, G120 at 12.30 by Dr Valentina Caorsi, from Abbelight: See details below. All the best and hope to see you there! David From: Gaboriau, David C A Sent: 12 October 2018 12:05 To: film-users <film-users(a)imperial.ac.uk> Subject: Seminar at Imperial and workshop at King's College Dear all, Dr Valentina Caorsi, from abbelight, will give a talk on single molecule localization microscopy at FILM, as described below: Date: Tuesday 23rd October Time: 12.30-13.30 Room: G120 in SAF Building Into the nanoworld Breaking the resolution limit of conventional microscopy opened the way to investigation of cellular structures at the nanoscale, from individual proteins to entire organelles. Different approaches have been proposed from structured illumination microscopy (SIM) to stimulated emission depletion (STED) and single molecule localization microscopy approaches (SMLM). SMLMs, such as ﬂuorescence photoactivated localization microscopy ((F)PALM) and direct stochastic optical reconstruction microscopy (dSTORM), can easily provide lateral localization precision down to 5–10 nm. However, 3D multicolour nanoscopy is still a challenge and a lot of effort has been made by the nano-community to develop quantitative and reproducible 3D super-localization methods. In this panorama abbelight developed a new nanoscope allowing precise isotropic 3D localization precision (15X15X15nm) by decoupling the lateral and axial detection. For the latter, two different sources of axial information are retained: the supercritical angle fluorescence (SAF) that provides an absolute z-reference (0-600nm) and a strong astigmatism-based PSF measurement allowing an extended axial depth (up to 5•m). Thus, nanoscopy can be accessible, to gain more details in 3D and extract quantitative information (numbers, size, distributions, spatial organization of individual molecules, clusters or organelles). Additionally, abbelight is organising a 3-days workshop on SMLM at King’s College London (see attached poster), and there are spaces for Imperial College researchers to attend and test abbelight’s DAISY module with their own samples. If interested, please contact Valentina: vcaorsi(a)abbelight.com<mailto:vcaorsi@abbelight.com> All the best, David

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[FILM-Users 00827] Reminder: FLOW/FILM user meeting today 14:00 - 16:00
by Bruckbauer, Andreas 17 Oct '18

17 Oct '18

Dear all, It's today! The FILM and Flow facilities will be having a combined user meeting on Wednesday the 17th of October from 14:00 - 16:00 (free cookies!!!). This will be on the 6th floor SAF in the glass walled meeting area. FLOW will be hosting the first 60 minutes and FILM the second, you can choose whether you come to one or both and both sessions will be in the following format: * 15 minute presentation by the facility. * A chance for users to discuss problems or helpful hints from their own data. * Feedback for the facility. If you would like a chance to present your work in the presentation slot, please contact me! Best wishes Andreas ---------------------------------------------------- Dr Andreas Bruckbauer FILM facility manager Sir Alexander Fleming Building South Kensington Campus Imperial College London Exhibition Road London, SW7 2AZ SAF 4th floor, open space desk 401 +44 (0)20 7594 2023 a.bruckbauer(a)imperial.ac.uk<mailto:a.bruckbauer@imperial.ac.uk> http://www.imperial.ac.uk/medicine/facility-for-imaging-by-light-microscopy/ http://www.imperial.ac.uk/people/a.bruckbauer ----------------------------------------------------

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[FILM-Users 00826] Seminar at Imperial and workshop at King's College
by Gaboriau, David C A 12 Oct '18

12 Oct '18

Dear all, Dr Valentina Caorsi, from abbelight, will give a talk on single molecule localization microscopy at FILM, as described below: Date: Tuesday 23rd October Time: 12.30-13.30 Room: G120 in SAF Building Into the nanoworld Breaking the resolution limit of conventional microscopy opened the way to investigation of cellular structures at the nanoscale, from individual proteins to entire organelles. Different approaches have been proposed from structured illumination microscopy (SIM) to stimulated emission depletion (STED) and single molecule localization microscopy approaches (SMLM). SMLMs, such as ﬂuorescence photoactivated localization microscopy ((F)PALM) and direct stochastic optical reconstruction microscopy (dSTORM), can easily provide lateral localization precision down to 5–10 nm. However, 3D multicolour nanoscopy is still a challenge and a lot of effort has been made by the nano-community to develop quantitative and reproducible 3D super-localization methods. In this panorama abbelight developed a new nanoscope allowing precise isotropic 3D localization precision (15X15X15nm) by decoupling the lateral and axial detection. For the latter, two different sources of axial information are retained: the supercritical angle fluorescence (SAF) that provides an absolute z-reference (0-600nm) and a strong astigmatism-based PSF measurement allowing an extended axial depth (up to 5•m). Thus, nanoscopy can be accessible, to gain more details in 3D and extract quantitative information (numbers, size, distributions, spatial organization of individual molecules, clusters or organelles). Additionally, abbelight is organising a 3-days workshop on SMLM at King’s College London (see attached poster), and there are spaces for Imperial College researchers to attend and test abbelight’s DAISY module with their own samples. If interested, please contact Valentina: vcaorsi(a)abbelight.com<mailto:vcaorsi@abbelight.com> All the best, David

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[FILM-Users 00825] Reminder: FLOW/FILM user meeting 17/10 14:00 - 16:00
by Bruckbauer, Andreas 11 Oct '18

11 Oct '18

Dear all, Only one week to go! Title of my presentation: Types of detectors, Signal to Noise and optimum settings in confocal microscopy. The FILM and Flow facilities will be having a combined user meeting on Wednesday the 17th of October from 14:00 - 16:00 (free cookies!!!). This will be on the 6th floor SAF in the glass walled meeting area. FLOW will be hosting the first 60 minutes and FILM the second, you can choose whether you come to one or both and both sessions will be in the following format: * 15 minute presentation by the facility. * A chance for users to discuss problems or helpful hints from their own data. * Feedback for the facility. If you would like a chance to present your work in the presentation slot, please contact me! Best wishes Andreas ---------------------------------------------------- Dr Andreas Bruckbauer FILM facility manager Sir Alexander Fleming Building South Kensington Campus Imperial College London Exhibition Road London, SW7 2AZ SAF 4th floor, open space desk 401 +44 (0)20 7594 2023 a.bruckbauer(a)imperial.ac.uk<mailto:a.bruckbauer@imperial.ac.uk> http://www.imperial.ac.uk/medicine/facility-for-imaging-by-light-microscopy/ ----------------------------------------------------

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[FILM-Users 00824] Start-up company for bioimage analysis using artificial intelligence
by Bruckbauer, Andreas 08 Oct '18

08 Oct '18

Please see email below if you are looking for a collaboration on image segmentation using artificial intelligence, please contact them directly. Andreas ---------------------------------------- From: Edward Dowling [mailto:edward.dowling@cellari.io] Sent: 25 September 2018 11:35 To: Bruckbauer, Andreas <a.bruckbauer(a)imperial.ac.uk> Subject: Quick question Dear Dr. Bruckbauer, I was referred to you by Daniel Elson as someone who could help me with some medical imaging related questions. I work for Cellari, which is an AI startup helping researchers, and non-specialists to take full advantage of recent developments in artificial intelligence. Cellari provides a very precise "active learning model" for bioimage segmentation, combined with an intuitive and easy-to-use user interface for teaching artificial intelligence to carry out repetitive, manual bioimaging tasks, such as counting and analysing cells. An automated statistical testing service is being implemented to ensure statistical validity of clinical and research related results along with unparalleled review and methodology transparency functionalities. Cellari is free for non-profits and individuals and aims to make image science robust and reproducible, while saving a lot of time. See the short demonstration video on https://cellari.io<https://cellari.io/>. Question: 1) I am looking for collaborators with interesting image-related problems to solve. Do you know any? 2) I am interested in finding out which pieces of machinery produce the data in which most time is wasted doing reproducible image related tasks. Do you have any insights? Also if you have any images you'd like to test out feel free to give it a go yourself for free here<http://cellari.io/>! Thanks in advance and hope to hear from you soon. Best, Edward Dowling [https://www.linkedin.com/in/edward-dowling/] <https://www.linkedin.com/in/edward-dowling/> BSc (Physics) Head of Technological Applications at Cellari Mobile (UK): +44 7766566158 | Skype: Edward Dowling Email: edward.dowling(a)cellari.io<mailto:Edward.Dowling@Cellari.io>

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[FILM-Users 00823] lone working - please read
by Bruckbauer, Andreas 04 Oct '18

04 Oct '18

Dear FILM users, This is to clarify my email from Tuesday regarding access to the FILM rooms: FILM is part of the National Heart and Lung Institute, Faculty of Medicine. The Faculty of Medicine classifies all work in labs (which includes our microscopy rooms) on weekdays from 7 pm - midnight as well as weekends 7 am - midnight as 'lone working' and this is to be avoided. If this is not possible you need apply for lone working (see below). Furthermore, Imperial College has an 'Access to Buildings Out of Hours Policy' which does not allow access from midnight to 7 am, Monday - Sunday and at any time during College closure days. Written permission must be sought from the Head of Department if access is required. The Faculty of Medicine page for lone working can be found here: https://www.imperial.ac.uk/medicine/fom-staff/support-and-services/health-a… It has a link 'how to apply' https://www.imperial.ac.uk/medicine/fom-staff/support-and-services/health-a… There is also a link regarding the risk assessment with an example and template https://www.imperial.ac.uk/medicine/fom-staff/support-and-services/health-a… To apply for lone working, use the electronic system, the link is on the bottom of this page and directs to 'My Imperial' : http://www.imperial.ac.uk/safety/safety-by-topic/lone-working/ Please choose 'National Heart & Lung Institute' as 'Department I want to lone work in', the end time is one year maximum, afterwards you need to reapply. Do a risk assessment (see templates in the link above), I suggest to write under lasers: Contained in microscopes and secured by interlock, no risks during routine operation. (The use of the laser tweezers on the CF2 in room 527 is not permitted out of hours). Security number for SAF is 4444 from a College phone or 020 7589 1000 from a mobile. Select the room(s) you will be working in (See our webpage for the location of the microscopes) As approvers, select your supervisor, the FOM Faculty Safety Advisor Helga Koch and myself for South Kensington or Sukwinder Singh and Stephen Rothery for Hammersmith. This will typically take a few days to process. You can find your application on 'My Imperial' https://my.imperial.ac.uk/ Please work safely! Andreas ---------------------------------------------------- Dr Andreas Bruckbauer FILM facility manager Sir Alexander Fleming Building South Kensington Campus Imperial College London Exhibition Road London, SW7 2AZ SAF 4th floor, open space desk 401 +44 (0)20 7594 2023 a.bruckbauer(a)imperial.ac.uk<mailto:a.bruckbauer@imperial.ac.uk> http://www.imperial.ac.uk/medicine/facility-for-imaging-by-light-microscopy/ ----------------------------------------------------

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[FILM-Users 00822] Out of hours access
by Bruckbauer, Andreas 02 Oct '18

02 Oct '18

All film-users, There is strictly no access to the FILM labs without explicit out of hours access for our rooms, this access has to be approved by your supervisor and myself and NHLI health and safety advisors via the electronic system! This applies on weekdays after 7 pm, see below! You all agreed to the following rules on PPMS when registering: When using the FILM facility outside the normal hours (07:00 - 19:00 from Monday to Friday) the College's Out of Hours and Lone Working Policies apply and it is the responsibility of the user to get prior authorisation from his or her supervisor and complete a risk assessment. Copies of both, authorisation and risk assessment, have to be shown to FILM staff prior to commencement of the lone working. See: http://www.imperial.ac.uk/safety/safety-by-topic/lone-working/ And also the faculty of Medicine Lone Working policy https://www.imperial.ac.uk/medicine/fom-staff/support-and-services/health-a… best wishes Andreas ---------------------------------------------------- Dr Andreas Bruckbauer FILM facility manager Sir Alexander Fleming Building South Kensington Campus Imperial College London Exhibition Road London, SW7 2AZ SAF 4th floor, open space desk 401 +44 (0)20 7594 2023 a.bruckbauer(a)imperial.ac.uk<mailto:a.bruckbauer@imperial.ac.uk> http://www.imperial.ac.uk/medicine/facility-for-imaging-by-light-microscopy/ ----------------------------------------------------

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[FILM-Users 00821] BBSRC grant to fund an automated slide scanner
by Rothery, Stephen M 25 Sep '18

25 Sep '18

Please do not reply to this e-mail but contact Andrew Scott directly if interested – email link below Dear FILM-users, I am investigating the possibility of applying for a BBSRC grant to fund an automated slide scanner (a Nanozoomer S360, S60 or similar) for our work on cardiac microstructure. The BBSRC 18ALERT: Mid-Range Equipment grants fund instrumentation for multi-user/multi-project use and I am wondering whether any FILM users might also have projects within the BBSRC remit that would benefit from access to a slide scanner? Details of the scheme are here: https://bbsrc.ukri.org/funding/filter/2018-alert-mid-range-equipment-initia… There is an internal college deadline of 5th October for outline applications, so please get it touch if you are interested. Kind regards, Andy Andrew D Scott PhD a.scott(a)rbht.nhs.uk<mailto:a.scott@rbht.nhs.uk> a.scott07(a)imperial.ac.uk<mailto:a.scott07@imperial.ac.uk> -- Senior Physicist Cardiovascular Magnetic Resonance Unit The Royal Brompton Hospital -- Honorary Senior Research Fellow National Heart and Lung Institute, Imperial College London — Academic Fellow Data Science Institute Imperial College London On 25 Sep 2018, at 09:28, Rothery, Stephen M <s.rothery(a)imperial.ac.uk<mailto:s.rothery@imperial.ac.uk>> wrote: Hi Andy Yes sure this should be fine Do you want to draft a message and we will send it out Bw Steve -----Original Message----- From: Scott, Andrew D Sent: 24 September 2018 21:34 To: Rothery, Stephen M Subject: FILM users email Hi Steve, I’m looking into whether some colleagues and I can put together a BBSRC grant application for a whole slide scanner (a Nanozoomer S360 or similar). We would need some collaborators from other groups/projects and I wondered whether it would be OK if I post to the FILM-users list to ask if anyone is interested? All the best, Andy

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