Michael Hooker Microscopy Facility

Dr. Michael Chua microscopy@unc.edu 6007 Thurston Bowles843-3268

http://microscopy.unc.edu

Dr. Neal Kramarcy neal_kramarcy@med.unc.edu 6033a Thurston Bowles966-7051

User training hardware & softwareRun workshops / user meetings / Answer questions / consultationsTrouble shoot hardware & applicationsRepairs / upgrades / maintenance

Maintain / update softwareMaintain servers & raid storageProcess usage data for invoicingGrant applicationsUser account updates

Laser Scanning ConfocalsLeica - 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% CO2

Tissues, small animals possible with setup

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

Resources: – Digital Light Microscopy•Instrumentation Microscopes: 6x 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

AdviceTrainingHelpSeminars/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

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

Michael Hooker Microscopy FacilityMission:

• 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

6129 Thurston BowlesLocation:

Laser Micro Dissection

Leica AS-LMDSection on penfoil slide (polyethylene coated)Outline area on monitor imageCut with pulsed UV laserCollect cut out in Eppendorf tube capProtein mass spec. / DNA / RNA analysisAblation of Zebra fish vasculature

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,

• 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 26oC)– 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:

Other Resources \\Marvin file server 4.6 x 1012 bytes Active Directory Domain Controller

\\Minsky file server 1.3 x 1012 bytes Domain Controller

\\AI file server 9.4 x 1012 bytes Backup server

Shared network resources, e.g:\\MINSKY\USERS2 public (any MHMF users)\\MINSKY\USERS1 public & AppleTalk \\MINSKY\PI-lab share: lab. group\\MINSKY\yourname share: private\\MINSKY\Phaser8550-MHMF share: dye sub color printer\\computername\shareddirectory\filename

UNC (Universal Name Convention)

Incubator 37oC, 5% CO2, 95% humidity

Refrigerator

Heated Stages

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 accessWet 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)

http://microscopy.unc.eduftp://152.19.58.173