About the facility: Robarts Research Institute at the Western University London, Ontario, Canada The London Regional Cell and In Vitro Molecular Imaging Facility is located within the Robarts Research Institute at the Western University. The facility operates as a multi-user resource and is available to all researchers at university as well as the entire research community within the region. The facility was established in 1999 by Dr. Stephen Ferguson, former Facility Director, and has gone through significant expansion over the years under his direction. It has grown from a single confocal microscope within a 100 sq ft room to a multi-site facility with 9 fluorescence imaging systems, a digital imaging laboratory and an on-site cell culture facility. Members of User Committee: Dr. Jane Ryllet (current facility s Director), Dr. Bryan Heit, Dr. Steve Kerfoot, Dr. Marco Prado, Dr. Stephen Pasternak, Dr. Sean Cregan, Dr. Greg Dekaban, Dr. Michael Poulter. Hours of Operation: The imaging facility is unlocked 9:00am-5:00pm Monday to Friday, but is available to all users 24 hrs a day, 7 days a week. Each user can apply for after-hours card access to the building and the facility. NOTE: Technical assistance is available 9:00am-5:00pm, Monday-Friday Workstations for Image Analysis: The facility has 5 workstation computers that are available to all users. These workstations are equipped with a variety of image processing software including the full Zeiss LSM 510, Zen 2009, Olympus FV10 Leica LAS software, Imaris, Image J, Fiji and Photoshop, etc. There are no fees and no time limit for use of the workstation computers and are available on a first-come basis. Cell Culture Facility: The facility is equipped with a fully functional cell culture room that is available to all users. The hood is located at room 3260C and the incubator is located at room 3281. NOTE: Each user must provide their supplies (i.e. pipettes, tips, dishes, etc) 7
Access to the facility: The imaging facility is available to all researchers at the Robarts, Western University and within the London regional research community. The following requirements must be met by each laboratory for access to the facility: 1. Please fill the form for New Lab or New User. The forms can be found in our website page, under the Access window (http://www.robarts.ca/confocal/access.html) 2. Please provide a brief summary of the research and need for the facility with an emphasis on the relevant microscopy experiments. This summary will be evaluated with respect to laser-dye compatibility, specimen type and applicability of the various imaging techniques. This process is not to discourage use but to ensure that the investigator is able to achieve the desired results from the facility before any financial and time commitments have been made. 3. Not knowingly contributed to the damage of the microscope 4. Are in good financial standing with the facility About the equipment: The main site of the facility is located in Room 3260 within the J. Allyn Taylor Centre for Cell Biology at Robarts and contains the image analysis workstations, a cell culture facility and 3 Zeiss LSM 510 METAs, one equipped with a ConfoCor2 for FCS and another with a femtosecond laser for multiphoton microscopy. We have expanded to include a Leica TCS SP5 II, Leica AM TIRF MC, and Olympus FV1000. Those equipments are located in the second site of our facility: room 3281 at Robarts. We have recently incorporated a Zeiss Apotome microscope that is located at room 3250. Through a generous donation from the Kelman family in 2013, we have acquired a Ground State Depletion (GSD) super resolution Microscope from Leica Microsystems. This system is one of the latest technology in the optical imaging field and provides resolution up to 20 nm (10 times improvement comparing to Confocal Microscope). This equipment is been available for general use since October 2013. With the 9 imaging systems and their various hardware configurations, the facility provides researchers with diversity and flexibility with respect to experimental approaches and techniques for molecular imaging. 7
A brief summary of our equipment and imaging techniques is outlined below: Microscope Zeiss Meta 510 Zeiss Meta 510 NLO Zeiss Meta 510 FCS Zeiss Apotome Leica TCS SP5 II Leica AM TIRF MC Leica SR GSD Olympus FV1000 PTI Delta RamV 405 458 477 488 Laser Lines (nm) 514 543 561 633 642 720-920 Epi Confocal TIRF Applications Multiphoton Fura-2 imaging FCS Structure 8
Illumination Accessories Super Resolution FRET/FRAP wizard Spectral imaging Image Tiling and Stitching Motorized stage CO2 controller Temperature controller Note: the lasers on the Leica AM TIRF MC are all solid state lasers (405, 488, 561 and 635nm) The lasers on the Leica GSD are fiber lasers (405, 488, 532 and 642nm) The Apotome has a LED X-Cite lamp and the PTI has a Xenon Lamp 9
Fees: For all the microscopes (except the Apotome) the fees are: $25/hr for the first 100 hrs $12, 50/hr for all subsequent hrs For the Apotome system the fees are: $6/hr for regular Wide field microscope $10/hr for the use of the Apotome slider For the Chameleon laser the fee is: $12, 00/hr The fiscal year for this Facility is counted as from May01 st to April 30 Th of the next year. The Invoices are sent four times a year (every three months) as follow: Charges regarding period of: May 01 st to July 31 st August 01 st to October 31 st November 01 st to January 31st February 01 st to April 30 th Invoices will be sent on: August November February May Training: The imaging facility at Robarts provides in-depth training for all users of the facility. The applicable introductory training session(s) are mandatory for all new users and must be completed before access to the facility is permitted. Users can only access equipments they have been trained on. Users are NOT permitted to train other new users. The introductory training sessions include an instructional portion as well as a hands-on practical portion. All of the introductory training sessions cover: The confocal facility s rules of operation Fundamentals in Fluorescence Microscopy Start-up procedures Working with Fluorophores spectra Setting the configurations Acquiring an image Z-stack and time lapse Saving and exporting Shut-down procedures 10
Below is a brief summary of the training sessions available. Please note any of these training sessions can be tailored to suit your needs. 1. Introduction to Confocal Microscopy This introductory training session is required for access to any of the confocal systems: LSM 510 META, Leica SP5 II or Olympus FV1000 Prerequisite: An understanding of the basic concepts of fluorescence Fee: $240 Time: 1 hr instruction + 2 hrs practical 2. Introduction to Wide Field Microscopy This introductory training session is mandatory for access to the Zeiss Apotome system. Prerequisite: An understanding of the basic concepts of fluorescence and confocal microscopy Fee: $120 Time: 2 hr of instruction + practical 3. Multiphoton Microscopy This training session is intended for users that have previously completed the introductory training session and is mandatory for all users before the Chameleon laser on the LSM 510 META-NLO system can be used. Prerequisite: Introduction to LSM 510 META and a basic understanding of multiphoton microscopy Fee: $120 Time: 1.5 hrs instruction 4. Combined Introduction to LSM 510 META and Multiphoton Microscopy: This training session is intended for new users that require both the introductory and multiphoton microscopy training. Prerequisite: A basic understanding of fluorescence, confocal microscopy and multiphoton microscopy Fee: $300 Time: 2 hrs instruction + 2 hrs practical 11
5. Introduction to TIRF Microscopy This introductory training session is required for access to the Leica AM TIRF MC system. Prerequisite: A basic understanding of fluorescence and TIRF microscopy Fee: $420 Time: 2 hrs instruction + 4 hrs practical 6. Spectral Unmixing (Emission Fingerprinting) This training session covers the steps involved in separating the emission of fluorochromes with significantly overlapping emission spectra (Emission Fingerprinting). Prerequisite: the introductory training session on the applicable system (Zeiss LSM 510 META or Leica TCS SP5 II) Fee: $240 Time: 1 hr instruction + 2 hrs practical 7. Ratiometric Calcium Imaging with Fura-2 This training session is done at the TIRF microscope, using the wide field mode. It specifically focuses on imaging Fura-2. Prerequisite: An understanding of the basic concepts of fluorescence and Fura-2 imaging Fee: $120 Time: 1 hr instruction + 1 hr practical 8. Refresher The refresher training sessions are intended for users that have previously been trained but would like some additional training. These sessions involve an overview of topics previously covered during the initial training. Prerequisite: the applicable introductory training session Fee: $80/hr 9. Introduction to Super resolution imaging (GSD Training) This training session covers the concepts of resolution, principles of ground state depletion super resolution microscopy, comparison with other techniques offered at our facility and sample preparation. 12
Prerequisite: - A thorough understanding of the concepts of the main Fluorescence Microscopy Techniques (Wide Field, Confocal and TIRF) - Introductory training on Confocal Microscopy In order to book the training, the supervisor must submit a letter justifying the use of GSD. This process is not to discourage use but to ensure that this technique is suitable to achieve the results desired. Fee: $420 Time: 1 hr instruction + 3 hrs practical 10. FRET Training This training session covers the basic concepts on FRET microscopy, focusing on Sensitized Emission and Acceptor Photobleaching approaches. It also covers the analysis part, using pfret plugin on Image J. Prerequisite: the introductory training session on the applicable system (Zeiss LSM 510 META or Leica TCS SP5 II) Fee: $420 Time: 3 hrs practical + 1 hr analysis 11. Additional Training The introductory training sessions only cover the very basic topics. All of our systems can be used for a variety of specialized applications. Since many of the applications are not relevant to many users, they are not covered in the introductory training. Additional training sessions are available and can be tailored to the individual user's needs. Prerequisite: the applicable introductory training session Fee: $80/hr Facility s Policies After prime-hours access to the facility and building access: The facility is open Monday to Friday from 9am to 5pm. Users who need access to the facility after-prime hours and/or over the weekend should contact the manager. A requisition will then be sent by the manager to the security in order to grant access. After the requisition has been sent, the user needs to go to security (2 nd floor of Robarts building) in order to have his card activated. The user will be notified by the manager when the requisition has been sent. 13
For users outside Robarts who need access to this building, the process is the same. Please note that it takes about one week to complete this process, so the users should plan their experiments accordingly. Bookings: Each laboratory is entitled to book 4 hrs per microscope/per day advanced booking (maximum one week in advance), during prime-time hours (8:00am-6:00pm). Please note that the 4 hrs per microscope/per day is the total of hours that all the users of the same lab can book in each microscope of the facility. Unlimited bookings during non-primetime hours (before 8:00am, after 6:00pm, and weekends) are available to all users. Cancellations with <24hrs notice as well as any unused time will be charged for at the regular rate. Each lab pays for the booked and used hours. Reports from the online calendar and the computers of each microscope are generated in the end of each month. Those reports are used for invoicing. Please note that if users do not show up for time they have booked and they do not cancel a booking within 24 hours of the start time they will be charged for the time booked. Memory sticks and hard drives: Memory sticks and any sort of hard drives are not allowed in any of our microscope computers. The only exceptions to this rule are: to copy tile image files from Olympus, NLO or Confocor computer and to copy data from the GSD computer. Your drive MUST BE SCANNED for virus before using in our computers. Misuse: Misuse of our machines must be reported and avoided. Misuse is defined as careless use of any equipment in our facility. Here are some examples of misuse: Leaving the microscope on overnight or all weekend Leaving fluorescent lamp on overnight or all weekend Leaving laser, remote control or any other component of the scope on overnight or all weekend Not cleaning or not cleaning properly the objectives Use of wrong immersion on objectives Liquid spills on objectives and microscope Infecting computers with virus Inappropriate use of internet Not following the shutdown procedure rules Leaving the laser(s) or fluorescent lamp on overnight or all weekend implies in charges during the time the components have been left on. Users with repeated history of misuse will have their privilege of using the facility taken away. Problems with the equipments: Users will NOT be charged if the equipment is not functioning. Please inform the facility manager immediately if there is a problem with the equipment. 14
Saving files on server: All the data generated in the microscopes should be saved in our server (gonad). It is the user s responsibility to back up and remove the files from the server using one of the workstations that are not connected to any microscope. The use of memory sticks or hard drives are not allowed on the microscope s computers. Please refer to the Memory sticks and hard drive session for the exceptions to this rule. Shutdown procedure rules: The fluorescent lamp and lasers require a proper cool down time before they can be used again. Failure in letting those components to cool down can result in damage and possible explosions. Here are our rules for shutting the systems down or leaving on standby: 1- Always check the online calendar before turn the system off or leave in stand by 2- If the next user is coming within 1 hour, put the argon laser on standby, log off your account and leave everything on for him/her 3- If the next user is coming with more than 1 hour after you finished, cool down the lasers properly and turn all the components off The current user is responsible for: - Checking the online calendar and making sure to follow the correct procedure (shutting down or leaving on standby). The following user is responsible for: - Checking the signing sheet and make sure the system has been properly cooled down before starting use it - In case of canceling his booking, make sure to contact the facility manager or the previous user to guarantee that the system won t be left on overnight Use of workstations for imaging analysis: Our facility has five workstation dedicated to imaging analysis. Besides the microscope software (LSM Browser, Leica LAS AF, Zen and Olympus FV10) we also have CorelDraw, Adobe Photoshop, ImageJ, Fiji and other basic software installed. Users are not allowed to install software in these computers unless authorized by the manager. Memory sticks and hard drives must always be scanned for virus before used in the workstations. There is no charge to use those computers as long as the work done is related to the facility. 15
Use of safety glasses: According to the Western s Biosafety Policies and Procedures, you MUST carry your safety goggles with you when using this facility. You are not required to use it when looking at your sample under the microscope but you are required to use it when manipulating biological samples (preparing and mounting or working with live cells). For more Information, please visit our website at http://www.robarts.ca/confocal/ or contact our manager: Fabiana Caetano Crowley, PhD Technical Specialist and Research Associate London Regional Cell and In Vitro Molecular Imaging Facility Robarts Research Institute, London, Ontario 519 663 5777ext 24151; fcaetano@robarts.ca 16