Confocal microscopy provides the ability to focus on a single focal plane within the specimen. Consequently, out-of-focus light coming from above and below the plane being imaged is rejected, resulting in improved signal-to-noise resolution. Optical sectioning of the sample in sequential z planes yields multiple scans that can be combined to generate 3D reconstructions of the sample. Localization and co-localization of multiple fluorescent probes and their spatial inter-relationship in live and fixed specimen can be best studied with confocal microscopy.
Confocal images can be obtained by single-point, linear-slit, or multi-point scanning of the samples.   Laser-based confocal microscopes are mostly single-point/slit scanners while multi-point scanning is achieved by using spinning disks with multiple pinholes. The latter can be lamp-based or laser-based systems. Advantages of the spinning disk-based confocal microscopes using lamps as light sources include greater speed and reduced phototoxicity compared to laser-based, point-scanning confocal systems. Thus, live cell confocal time lapse imaging is preferentially carried out with such systems.

The Center offers a BioRad MRC-1024ES point-scanning laser confocal system attached to an inverted Nikon Diaphot microscope. The gas Argon Ion 100mW laser (major lines at 488 and 514 nm) is operated by the LaserSharp controller and analysis software. A high pressure mercury lamp is also included. Three-color detection is provided by highly sensitive PMTs.  A wide selection of objectives is available. The system sits on an anti-vibration air table.

The Center houses a CARV multi-point confocal system (BD Bioscience, Rockville, MD) configured on a Zeiss Axioskop FS up-right microscope and equipped with a full-spectrum mercury lamp, a cooled CCD camera, and CARVer software for image acquisition and 3D reconstruction.  High speed, multi-point confocal scanning is achieved by a Nipkow spinning disk.  A highly reproducible Z focus system enables accurate Z steps of 28 nm.

The microscope and software are quite simple to use, making it possible for a new user to obtain good confocal images with little training.  An added feature is the ability to directly view the confocal image through the binocular eyepieces so that the specimen can be easily surveyed, just like one would observe a fluorescent specimen under wide field fluorescent microscopy.  The microscope can be operated in standard wide field mode and, by simply sliding a lever, the spinning disk is engaged into the light path, creating confocal images.



For High Content Screening (HCS), the Center offers an automated spinning-disk confocal fluorescence microscopy station (BD™ Pathway Bioimager, BD Bioscience, Rockville, MD) equipped with a confocal scanning module identical to the CARV system described above.  The station includes a stationary sample stage for placement of slides, culture dishes, or 96 or 384 well plates and an on-board fluidic dispenser for unattended automated imaging and compound addition for live cell imaging. In a less automated mode, the system can be used for higher resolution confocal imaging with high magnification objectives such as a PlanApo 63X, na 1.4 oil immersion objective.  Full spectrum arc lamp illumination and 16 bandpass interference filters enable any wavelength of excitation to be selected. The image is detected with a 12 bit Hamamatsu Orca ER cooled CCD camera with any of the 8 emission wavelengths selected by filters. For more details on the Pathway Bioimager, see Drug Discovery, under Services.