Bioimaging - Core Centers - Delaware Health Sciences Alliance

Core Center Resources:

Resources for Bioimaging

Bioimaging – James Keen, Ph.D. Kimmel Cancer Center (KCC), TJU

The goal of the Kimmel Cancer Center (KCC) Bioimaging Shared Resource is to provide detailed light microscopic image analysis capabilities to KCC investigators in order to promote the basic and medical science research programs. The Bioimaging Shared Resource provides multicolor analysis of fixed and living specimens, Z series, image processing and production of publication-ready images for the investigator. The Bioimaging Shared Resource operates with a well-trained technician and a faculty supervisor, Dr. James Keen. Through the operation of this Shared Resource, individual KCC investigators are assured of a reliable shared resource operated with a high degree of technical expertise, and are relieved of the obligation for substantial capital investment for equipment.

Equipment

Confocal Laser Microscopy: The Bioimaging Shared Resource is equipped with a state-of-the-art Zeiss LSM 510 META Confocal Laser Scanning Microscope System interfaced to a Zeiss Axiovert 200M inverted microscope equipped with 5 objectives ranging from 10x to 63x. The confocal uses multiple lasers with excitation lines at 405nm, 458nm, 477nm, 488nm, 514nm, 543nm, and 633nm. The system’s three PMT detectors provide the ability to simultaneously image up to eight fluorophores with emission between 417nm and 730nm. The META detector also permits resolution of very closely emitting dyes. Images are analyzed with Zeiss’s AIM Image Examiner software. The system is built on an isolation table to dampen vibration and support multiposition, Z-stack, and time lapse functions. A Zeiss temperature-controlled chamber is available for high and low temperature imaging. A programmable pump and perfusion chamber is also available for use with this microscope system to allow media exchange and reagent addition during continuous sample monitoring.
Widefield microscopy: This resource is equipped with a widefield epifluorescence imaging system specifically designed for high quality acquisition of living cell images. This “Live Cell System” is built around a Zeiss Axiovert 200M inverted microscope equipped with 5 objectives ranging from 10x to 100x that can be used for both epifluorescence and brightfield microscopy. The system uses a broad spectrum Xenon excitation lamp with Lambda-64 quick filter changer and built-in epifluorescence reflectors produced by Chroma Technologies to provide flexible emission filtering and rapid imaging of multiply-stained samples. A Roper Scientific CoolSnap HQ CCD camera is used for image capture with Metamorph Image Acquisition and Analysis software v7.0 (Universal Imaging), as well as with Image J and Adobe Photoshop. The instrument is enclosed in a temperature, CO2, and humidity controlled chamber for long-term time-lapse imaging of live cells. The system is built on an isolation table to dampen vibration and support multi-position, Z-stack, and time-lapse functions. A programmable pump and perfusion chamber is also available for use with this microscope system to allow media exchange and reagent addition during continuous sample monitoring.
Microinjection: This system is set up on a Nikon Eclipse TE-2000-S inverted microscope with an attached Eppendorf Micromanipulator 5171 and Microinjector 5242. An independent stand allows adjustment of injection angle suitable for injection of cultured cells or C. elegans.

Bioimaging – Joseph Rabinowitz, Ph.D., Center for Translational Medicine, TJU

Within the Center for Translational Medicine (CTM) at TJU, Dr. Rabinowitz has technology available through his laboratory to image light emission and fluorescence in live animals, critical to stem cell and gene therapy research being done in the CTM. The in vivo bioluminescent system is the Xenogen Series (now Caliper) IVIS 100 Imaging System. It is sensitive and has a large field of view. The focusing stage is adjustable from 10-25 cm resulting in a field of view large enough for two rats or five mice or small enough for only one mouse. The system features a 25 mm (1.0 inch) square back-thinned, back-illuminated CCD, which is cryogenically cooled to greater than –100°C via a closed cycle refrigeration system to minimize electronic background and maximize sensitivity. The systems animal handling features include gas anesthesia within the chamber and in a specialized holding compartment. The chamber has a heated shelf to maintain the comfort of the animals during imaging. The Living Image software is easy to use and to transfer files for presentations.

Small Animal Imaging – Matthew Thakur, Ph.D., KCC, TJU

The Small Animal Imaging Shared Resource provides state-of-the-art in vivo molecular imaging capabilities to the investigators of the Kimmel Cancer Center. It was established under the direction of Dr. Matthew Thakur in April 2004 supported, to date, by the Thomas Jefferson University and the Department of Radiology. This resource provides non-invasive small animal imaging, with PET, SPECT, CT and ultrasound. These modalities offer innovative basic, as well as pre-clinical translational, research capabilities for studying patho-physiology and genesis of cancer. In addition to providing comprehensive imaging services the expertise exists to

Custom-synthesize probes specific for targeted bio-markers;
Develop new image generation and analysis techniques;
Train cancer investigators to harness the strengths of molecular imaging.

The facility is supported by three AALAC accredited animal houses and two veterinarian staff members. Thus far this shared resource has supported fifteen federally funded investigations related to oncology and enhanced productivity as depicted by several publications in peer-reviewed journals.

Equipment

The imaging facility is housed in 1018 Bluemle Life Science Building. It is strategically located adjacent to the animal housing where a majority of the Cancer Center laboratory animals reside and in the building where a majority of the investigators perform cancer research. The 250 square feet imaging facility currently houses the Philips Mosaic PET scanner, the Imtek microCAT II CT scanner and a host of support equipment.

The Mosaic small animal PET scanner (Philips Medical Systems) uses 2x2x10 mm3 GSO (gadolinium oxyorthosilicate) crystals coupled to 19 mm diameter photomultiplier tubes (PMT) via a continuous slotted light guide. The detectors are arranged to produce a transaxial field of view (FOV) of 128 mm and an axial FOV of 120 mm. The absolute coincidence sensitivity is 1.3% for a point source and the transverse resolution is 2.2 mm at full-width half- maximum (FWHM). This scanner is operated exclusively in the 3D volume imaging mode, leading to about 55% coverage of the total solid angle (in singles) for a point source at the center of the scanner field-of-view. The Anger-logic detector achieves high spatial resolution due to the good crystal discrimination (2 mm crystals), and reduced scanner dead-time by restricting the scintillation light spread within a 7-PMT cluster (central PMT plus 6 adjacent PMTs). The energy resolution averaged over a large array of crystals is 17% at 511 keV. Good energy resolution is needed so that a high-energy discriminator (380 keV) can be used to reject scatter. The integral software and the in-house programming capability provide excellent data analysis.
The ImTek microCAT II small animal CT scanner has two main roles: as an ultra-high resolution system for anatomical imaging down to 50 μm, and to generate anatomical reference images for correlation with the functional PET and SPECT data. Radionuclide images typically lack sufficient anatomical detail to permit delineation of organs, thus requiring a co-registered reference image from a modality such as CT. In addition, the high-resolution CT images will enable accurate and reproducible measurements of lesion size and structure, and may be able to locate metastatic sites. Changes in the size and shape of tumors on in vivo microCT images will be an important marker for cancer proliferation and response to treatment. Supporting equipment in the imaging facility includes laminar flow hood, injection stages, infusion pump, micro centrifuge, dose calibrator, scintillation counter, survey meter, shielded assay area, small animal bio-monitoring system, image processing and analysis computers and software.
Complimentary to the imaging facility is the 2000 square feet radiochemistry facility run by Dr. Thakur. This lab includes a large array of equipment for the development of novel radiotracers, and for the analysis of blood and tissue samples from experimental animals. The equipment includes: HPLC, TLC, gamma counters, fraction collectors, lyophilizer, PAGE system, gel filtration system, gel electrophoresis system, microscopes, a GE StarCam gamma camera, fume hoods, and a biosafety cabinet.
A variety of portable ultrasound scanners dedicated to research are available. These include an Aplio scanner (Toshiba America Medical Systems, Tustin, CA), a Voluson 730 and a Logiq 9 system (both from GE Healthcare, Milwaukee, WI) as well as two Sonoline Elegra scanners (Siemens Medical Solutions, Issaquah, WA). However, the highest imaging frequency available with these clinical ultrasound systems is 17 MHz and this limits the achievable resolution to around 200 mm. There are plans to invest in a dedicated small animal imaging scanner: the Vevo 770 from Visualsonics Systems (Toronto, Canada). This scanner operates at imaging frequencies up to 82.5 MHz, which enables resolution on the order of 30 mm. Moreover, it has software packages specifically designed for molecular imaging and contrast specific modes such as high frequency pulse inversion nonlinear imaging. Due to the Vevo 770’s open architecture it is also possible to implement novel contrast imaging modes developed by our group on this unit.

Bioimaging – Director: Kirk Czymmek, Ph.D., DBI, UD

The Bioimaging Center is a multi-user facility, which provides state-of-the-art microscopic imaging instrumentation and technology. The center is open to university researchers and collaborators on a fee-for-service basis; outside industrial users are accommodated when scheduling permits. Access to the center is available through a web-based reservation system. The staff is available for project consultations and provides regular user training. The center is professionally staffed by a Director, Dr. Kirk Czymmek, Dr. Liz Adams, an expert in Scanning Probe Microscopy, Deborah Powell to provide SEM support, Shannon Modla, to provide TEM support and David Scheiblin and Bekki Helton as laboratory assistants.

Confocal microscopy: The center houses four confocal microscopes. Two of the systems are a Zeiss LSM 510 VIS attached to an Axiovert 100M microscope with one system having laser lines at 458nm, 488nm, 543nm and 633nm and the other system having 488nm, 568nm and 633nm. The multiphoton system is a Zeiss LSM 510 NLO with a PECON Environmental Incubation System with laser lines 458nm, 477nm, 488nm, 514nm, 543nm, 633nm and 730nm. The newest system is a Zeiss LSM5 DUO, with META and Live Scanhead with laser lines of 405nm, 458nm, 477nm, 488nm, 489nm, 514nm, 560nm, 561nm, 633nm, and 635nm. The four systems have over 35 objectives lens ranging from 1.25x to100x.
Light Microscopy: The Zeiss Axioskop2 is an upright microscope and the Zeiss M2BIO is a dissecting microscope. Each can be equipped with either of the centers two digital cameras, a Zeiss AxioCamHR or a Hamamatsu Orca-ER.
Electron Microscopy: The Field Emission Scanning Electron Microscope (FE-SEM) is a Hitachi S4700 and has a Gatan Alto 2500 Cryotransfer System and an Oxford INCA Energy (EDS) System. The system has collaborative capabilities via the Internet. The Transmission Electron Microscope (TEM) is a Zeiss Libra 120 with EELS capabilities.
Scanning Probe Microscopy: The Veeco Bioscope II has a Nanoscope V controller; 8 data channels including phase, inphase, quadrature, z sensor etc, at higher resolution. Also Force volume, Q control and HarmoniX applications. Tapping and contact in both air and fluid, as well as temperature control and flow chambers. It can be used with a wider range of samples, from petri dishes, to glass slides. Objective lenses include a 10x, 20x and 40x. The Veeco Multimode has a Nanoscope IIIA controller with Quadrex Electronincs (Phase capabilities); Scanners include an A, J and PF, TUNA/conductivity module, PicoForce controller and PicoAngler. Tapping and contact in both air and fluid, as well as force modulation and lateral force modes.
Laser Capture Microdisection: The Laser Capture Microdissection Microscope (LCM) is a PALM Combisystem with optical tweezers. It has a Pulsed nitrogen laser of 337nm and an IR laser of 1064nm. It is capable of transmitted and epifluoresence and has 5x, 10x, 20x, 40x, 63x and 100x objective lenses.
Microtomy: Includes two Reichert-Jung Ultracut E microtomes, a LKB 7800 Knifemaker and two Leica CM3050 S Cryostats, one of which has an Instrumedics CryoJane Tape-Transfer System.
Specimen Preparation equipment: Includes a Leica EM PACT for high-pressure freezing, Leica EM AFS for automated freeze substitution, an Autosamdri-815B, Series A critical point dryer, a Leica EM IGL for immunogold labeling and a Denton Bench Top Turbo III for Carbon or Gold/Palladium coating.
Image Analysis and Enhancement workstations: There are two computer workstations; one dell and one Alienware computer are available which have an array of image processing and analysis software (Improvision’s Volocity, Mercury’s AMIRA and Zeiss AIM 4.2 for 3-D rendering and analysis and Zeiss AIM 4.1 and SVI Huygens Pro for Deconvolution). A Fujifilm Pictography 4000 Printer with publication quality print capability, a Nikon Coolpix 5700 digital camera, an Agfa Duoscan T2500 scanner and an Umax Powerlook 3000 scanner are additional tools available to center users. In addition, the center has a facility for specimen preparation and a darkroom with a Kodak X-Omat.

Bioimaging – Bruce Boman, M.D., Ph.D., Christiana Care

The Center for Translational Cancer Research (CTCR) currently has a Zeiss motorized AxioObserver Z1 equipped for brightfield, phase contrast, varel contrast, and epi-fluorescence, with both color and monochrome digital cameras, X -Y scanning stage, anti-vibration platform, 10x, 20x, 40x and 100x objectives, and Apotome confocal imaging device with a high-performance PC, a flat-panel monitor, complete Axiovision software solution, and an Axiocam MRm digital camera. The system is configured to be upgraded to a to a complete Zeiss LSM 710 laser scanning confocal microscope, which is planned for opening of the new CTCR in 2009.

The Bioimaging facility supports research involving immunofluorescence that requires a variety of excitation and emission filters to cover a broad spectrum of fluorophores. The LSM 710 Confocal Microscope uses a series of lasers to excite sample fluorophore(s) at very specific wavelengths and a wide array of emission filters that allows selection of several possible collection wavelengths. Because the Confocal uses separate detectors to collect different channels, multiple color samples can be employed simultaneously.

Imaging – Jeffery Twiss, M.D., Ph.D., Nemours

The Imaging Shared Resource Laboratory provides access to the LEICA DMIRE2 Confocal Microscope System. Training and troubleshooting support is available for microscopy and image processing software.