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

2. Laser Scanning Confocals Leica - SP2 aobs UV 351, 364 nm blue 458, 488 nm green514, 561 nm red 633 nm Reflection Transmitted DIC X-Z scanning scanning FRAP, FRET UltraView blue 488 nm green 568 nm red 633 nm transmitted Biosensors 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 CY5, DRAQ 5, Alexa 635 (far red, invisible) CFP/YFP, dsRed, etc., fluorescent proteins many others… Heated chambers, 5% CO 2 Tissues, small animals possible with setup

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

4. Resources: – Digital Light Microscopy Instrumentation Microscopes: 6 x Compound, 2x Dissecting, 3x Confocals, Laser Micro-Dissection, Atomic Force, Flat bed scanner (standard reflected and transparency) Imaging Processing, Analysis, morphometry 2D, 3D, 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 Michael Hooker Microscopy Facility

5. Wide Field Microscopes Leica DMIRB inverted Transmitted light Nomarski (DIC) Fluorescence B/W camera (OrcaER) Color camera Nikon upright Transmitted light Hi resolution color camera Nikon TE2000 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 Dissecting Scopes Fluorescent & motorized Simple transmitted light

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

7. 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)

8. Michael Hooker Microscopy Facility 6129 Thurston Bowles Location:

9. 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

10. Resources: Image Analysis & Processing Image Processing Work Stations (4x) \\Nomarski – general purpose – full Leica confocal off line \\Snell – 3D Volocity, full Leica & Zeiss confocal off line \\Zernike – Windows 64 bit RAM 8GByte OpenGL 512MByte Video \\Young – video tape capture – scanner with transparency Analysis Co-localization, fluorograms, FRAP, FRET Measurements – area – intensity – tracking - etc. Enhancements – min/max/ave projections – local contrast – etc. Etc., etc., etc….. Software (examples understated!) General:- C-Imaging 6, Metamorph 7.1, ImageJ 3D Rendering:- Volocity, Slicer (T3D), Voxblast Volocity licence server http://ai.med.unc.edu:15003 2D:- Photoshop, Illustrator, ThumbsPlus 7 Video:- Quicktime Pro 7, VidEdit 1.1, Premiere 1.5,

11. Resources: –Space 850 sq.ft –Microscopes 10x –Workstations 4x –Software 3x high packages –Web pages & booking calendars http://microscopy.unc.edu –File servers / AD-DC / Terabyte Raid Windows files/directory sharing –Personnel 2x 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 5day with discount to those who give salary support/ Users group meetings $free –Support Cystic Fibrosis Center, Alcohol Studies Center, Gene Therapy Center, SukWon Jin lab. (R01) –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: P60 0.1 »Jin: RO1 - 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: –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 Summary:

12. Other Resources \\Marvinfile server 4.6 x 10 12 bytes Active Directory Domain Controller \\Minskyfile server 1.3 x 10 12 bytes Domain Controller \\AIfile server 9.4 x 10 12 bytes Backup server Shared network resources, e.g: \\MINSKY\USERS2 public (any MHMF users) \\MINSKY\USERS1public & AppleTalk \\MINSKY\PI-lab share: lab. group \\MINSKY\yourname share: private \\MINSKY\Phaser8550-MHMFshare: dye sub color printer \\computername\shareddirectory\filename UNC ( Universal Name Convention) Incubator 37 o C, 5% CO 2, 95% humidity Refrigerator Heated Stages

13. Using: The Resources Using the resources: Training/orientation for each microscope User are issued with computer account on mhmicroscopy domain Users may request after hours access Wet sample restrictions 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)

14. http://microscopy.unc.edu ftp://152.19.58.173

15. Michael Hooker Microscopy Facility http://microscopy.unc.edu