Michael Hooker Microscopy Facility http microscopy unc edu

Michael Hooker Microscopy Facility http: //microscopy. unc. edu User training hardware & software Run workshops / user meetings / Answer questions / consultations Trouble shoot hardware & applications Repairs / upgrades / maintenance Dr. Michael Chua microscopy@unc. edu 6007 Thurston Bowles 843 -3268 Maintain / update software Maintain servers & raid storage Process usage data for invoicing Grant applications User account updates Dr. Neal Kramarcy neal_kramarcy@med. unc. edu 6033 a Thurston Bowles 966 -7051

Laser Scanning Confocals Leica - SP 2 aobs UV 351, 364 nm blue 458, 488 nm green 514, 561 nm red 633 nm Reflection Transmitted DIC X-Z scanning l scanning FRAP, FRET Zeiss - 510 meta blue 458, 490 green 514, 543 red 633 nm Reflection Transmitted DIC Spectral unmixing FRAP, FRET Fluorophores: DAPI, Hoechst, Alexa 350, etc FITC, GFP, Alexa 488, etc. Rhodamine, Texas Red, Alexa 543, 568 & 594 CY 5, DRAQ 5, Alexa 635 (far red, invisible) Ultra. View blue 488 nm green 568 nm red 633 nm transmitted Biosensors CFP/YFP, ds. Red, etc. , fluorescent proteins many others… Heated chambers, 5% CO 2 Tissues, small animals possible with setup

USERS in 2009: 205 users - 104 labs, ~60 Departments/Centers

Michael Hooker Microscopy Facility Resources: – Digital Light Microscopy • Instrumentation Microscopes: 6 x Compound, 2 x Dissecting, 3 x Confocals, Laser Micro-Dissection, Atomic Force, Flat bed scanner (standard reflected and transparency) • Imaging Processing, Analysis, morphometry 2 D, 3 D, time lapse, FRAP, FRET, Fura-2, tracking Storage, color printing (Xerox dye sub) File storage (raid arrays, Ethernet accessible) • Instruction Advice Training Help Seminars/courses/workshops on microscopy • Collaborations • Fee for use – recharge facility • Credit for block support e. g. PPG, SCOR cores • Users from UNC-CH, EPA, Pharmas, NCCU

Wide Field Microscopes Dissecting Scopes Fluorescent & motorized Simple transmitted light Leica DMIRB inverted Transmitted light Nomarski (DIC) Fluorescence B/W camera (Orca. ER) Color camera Nikon upright Transmitted light Hi resolution color camera Nikon TE 2000 inverted Transmitted light Nomarski (DIC) Phase contrast Fluorescence Time lapse Ratio imaging e. g. Fura-2 [Ca], FRET Multimode B/W cameras Intensified camera Motorized filters changers

Michael Hooker Microscopy Facility Mission: • Research light microscopy facility • Standard and advanced digital light microscopy image acquisition • Image processing and analysis resources • Provide instrumentation and instruction • Fee for use, training, assistance • Users scan and analyze images from samples that they have prepared • Occasional users receive scanning assistance (no training)

Michael Hooker Microscopy Facility Location: 6129 Thurston Bowles

Laser Micro Dissection Leica AS-LMD Section on penfoil slide (polyethylene coated) Outline area on monitor image Cut with pulsed UV laser Collect cut out in Eppendorf tube cap Protein mass spec. / DNA / RNA analysis Ablation of Zebra fish vasculature

Resources: Image Analysis & Processing Image Processing Work Stations (4 x) \Nomarski – general purpose – full Leica confocal off line \Snell – 3 D Volocity, full Leica & Zeiss confocal off line \Zernike – Windows 64 bit RAM 8 GByte Open. GL 512 MByte Video \Young – video tape capture – scanner with transparency Software (examples understated!) General: - C-Imaging 6, Metamorph 7. 1, Image. J 3 D Rendering: - Volocity, Slicer (T 3 D), Voxblast Volocity licence server http: //ai. med. unc. edu: 15003 2 D: - Photoshop, Illustrator, Thumbs. Plus 7 Video: - Quicktime Pro 7, Vid. Edit 1. 1, Premiere 1. 5, Analysis Co-localization, fluorograms, FRAP, FRET Measurements – area – intensity – tracking - etc. Enhancements – min/max/ave projections – local contrast – etc. Etc. , etc…. .

• Resources: – – – – • Space 850 sq. ft Microscopes 10 x Workstations 4 x Software 3 x high packages Web pages & booking calendars http: //microscopy. unc. edu File servers / AD-DC / Terabyte Raid Windows files/directory sharing Personnel 2 x What we do: – – – – User Training, on demand – New users $140 confocals, $70 widefields, Consultations (in person, on demand, e-mail, phone) $9. 50 / 15 minutes Collaborations User problems $9. 50 / 15 mins, but mostly not logged Image analysis and data processing development $9. 50 / 15 mins, somwhat logged Annual Workshops $350 to $700 5 day with discount to those who give salary support/ Users group meetings $free Support Cystic Fibrosis Center, Alcohol Studies Center, Gene Therapy Center, Suk. Won Jin lab. (R 01) – – • Director salary » CF: Scor 0. 25 ended Aug 2009 » CF: Core-D 0. 20, » CF-GT: MTCC 0. 10 » GT: PPG 0. 1 end Oct 2009 » Alc: P 60 0. 1 » Jin: RO 1 - 0. 05 » Recharge: q. v. Resource maintenance/repairs/trouble shooting/system development, including computer IT Book keeping/grants/l. o. s. Olympus Center out reach (No details of activities yet) What we do not do: – – • Summary: Tissue preparation (but advice given) Scan for users (some exceptions $9. 50 /hr + machine cost) Short comings: – – – No wet bench space No sterile hood (bad for biosensor experiments) Server room (With 3 servers overnight office temperature is 26 o. C) Older equipment approaching end of service life Have to compete with UCRF supported facilities – i. e. PIs do not want to contribute to shared instrumentation grants. Those cores have lower prices. Need more core grants with UNC groups

Other Resources \Marvin \Minsky \AI file server 4. 6 x 1012 bytes Active Directory Domain Controller 1. 3 x 1012 bytes Domain Controller 9. 4 x 1012 bytes Backup server Shared network resources, e. g: \MINSKYUSERS 2 public (any MHMF users) \MINSKYUSERS 1 public & Apple. Talk \MINSKYPI-lab share: lab. group \MINSKYyourname share: private \MINSKYPhaser 8550 -MHMF share: dye sub color printer \computernameshareddirectoryfilename UNC (Universal Name Convention) Incubator 37 o. C, 5% CO 2, 95% humidity Refrigerator Heated Stages

Using: The Resources Using the resources: Training/orientation for each microscope User are issued with computer account Users may request after hours access Wet sample restrictions on mhmicroscopy domain Booking: On line http: //microscopy. unc. edu/booking/ Walk up if machine is not booked Assistance with experiments, consultations, collaborations: Schedule an appointment Ad Hoc if less than 15 minutes (Please do not assume we are available without an appointment)

http: //microscopy. unc. edu ftp: //152. 19. 58. 173