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The MCC is a campus-wide resource for light microscopy, immunocytochemistry and in situ hybridization techniques.
We are part of the Biomolecular Research Facilities program and are located in room 120 of the Life Sciences Center.

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LASER SCANNING CONFOCAL MICROSCOPY

The MCC has a Zeiss Meta NLO 2 photon laser scanning confocal microscope system (click here to view the contributors), which has the ability to simultaneously image up to 8 fluorochromes in a sample, effectively subtract the autofluorescence from the original signal using linear unmixing, spectrally separate two or three closely related fluorophores, perform 3D tile scans covering large sample areas in multiple channels and view specimens with increased penetration (depth along Z axis) along with uniform intensity in samples such as brain slices and whole mounts using the 2-photon Chameleon laser. The system visible laser lines for fluorophore excitation at 458 nm, 477 nm, 488 nm, 514 nm, 543 nm, and 633 nm. The Chameleon 2-photon laser system is a pulsed laser tuneable to any wavelength between 730 nm and 930 nm; this laser can excite most fluorophores in the visible spectrum, but is most efficient with UV and near-UV excitable dyes.

The MCC also has a BioRad Radiance 2000 confocal system coupled to an Olympus IX70 inverted microscope which has the ability to optically section thick sections free of out-of focus fluorescence. This system has krypton-argon and red diode lasers with excitation lines of 488, 568, and 637 nm and can simultaneously image all 3 fluorescent channels as well as a high quality transmitted light Nomarski-type image. This system cannot collect images of UV excitable dyes.

WIDE-FIELD MICROSCOPY

An inverted Olympus IX70 eq uipped with bright field, phase, DIC, and fluorescence optics and digital camera is available. IX70 system has computer controlled filter wheels with Chroma HiQ fluorescence filters and a z-motor drive for computer controlled acquisition of fluorescence images at multiple focal planes and wavelengths. Click here for a list of objectives and fluorescence filters.

An inverted Leica DMI4000 B with integrated Eppendorf Microinjection system and a 1.4 megapixel, 12-bit, cooled CCD camera from Q-Imaging (QICAM) .

The upright Nikon Optiphot 2 is equipped with bright field, phase, DIC, dark field, and fluorescence optics.

A Leica stereoscope equipped with epi-fluorescence excitation and Chroma HiQ fluorescence filters is also available.

LONG WORKING DISTANCE OBJECTIVES FOR OLYMPUS IX-70:
Long working distance objectives are available for viewing samples in Petri dishes, culture plates or on other thick substrates. For the best image, ask MCC staff member to place the correct cap on the end of each of these objectives to compensate for the thickness of the substrate.

OIL & WATER IMMERSION OBJECTIVES:
Slides to be used with immersion objectives must have clean surfaces with no mounting medium of any type leaking out or present on the surface. Core staff may require certain samples be sealed with nail polish or mounted in alternative acceptable formats. All of our immersion objectives are meant to be used for viewing samples adjacent to a #1.5 glass coverslip. They will not work with samples on thick substrates such as plastic dishes. Depending on the particular objective, they must be used with either oil or water between the objective and the coverslip.

PHASE CONTRAST OBJECTIVES
Phase contrast is an appropriate method for getting a transmitted light view of unstained tissues. It is similar to Nomarski optics but works with specimens on thicker substrates. In phase, one can get a bright halo around dark objects or a dark halo around bright objects. This can be helpful in viewing the specimen but can interfere with measurements. The effect is especially prominent at boundaries between regions. The following phase contrast objectives are available for the BioRad Radiance 2000 confocal system and the Olympus IX-70 widefield microscope:

10x UPlan Fluor NA 0.3 Ph1
20x LWD LCPlan Fluor, NA 0.40 Ph2
40x Plan Fluor, NA 0.6 Ph2
60x Plan Fluor, NA 0.7 Ph2

PREPARATIVE EQUIPMENT
Includes everything required for fixation, embedding and sectioning tissues for immunocytochemistry or in situ hybridization including paraffin microtomes, cryostat, ultramicrotome for semi-thin and ultrathin plastic or frozen sections and an LKB knife maker. Fluorescent labels for immunohistochemistry are available on-site through our Invitrogen Supply Center.

PRESENTATION GRAPHICS
Several high-end computers with extensive software for image processing, analysis, and graphic presentation are available for client use or fee-based service work. Adobe Photoshop software is available for staff-produced final output for publications or for training of clients by Core staff.

IMAGE PROCESSING WITH 2D & 3D DECONVOLUTION & 3D AND 4D VOLUME RENDERING
The core now has Autodeblur from Autoquant with 2D and 3D blind deconvolution algorithms for widefield, confocal and 2-photon raw data sets/images. Deconvolution is a mathematical approach of increasing image resolution. Even optimal data sets acquired under the best possible microscopic settings can be improved with this technique (examples).

For image analysis and processing, we have the Imaris Image Processing Suite, Metamorph, and ImagePro software packages. Image analysis allows standard morphometric measurements (e.g, lengths, surface area, or automated counting of features of interest). Raw or deconvolved data sets can be uploaded for orthogonal plane analysis or characterization of co-localization. 3D, 4D, and isosurface projections can be made and recorded as animated movies.

If you have any other questions, please contact us at 882-4895