Confocal Microscope Imaging System
Microscope
A Zeiss LSM 710 laser scanning confocal microscope provides advanced imaging capabilities to faculty and students at Wake Forest University. This confocal microscope, newly released in the summer of 2008, possesses many significant new features and capabilities. Among these are full spectral imaging with no emission filters and down to 3nm step sizes; increases in signal to noise due to reduced laser reflection, new algorithms, almost no dark noise, and spectral recycling; and user control of collimation and alignment for calibration and diagnostics. Additionally, the design of the new laser module will allow easy replacement and upgrade of lasers in the future.
The 710 confocal scan head is attached to an AxioObserver Z.1 inverted microscope. The inverted microscope body provides versatility for live and dead, slide and dish imaging applications. The Z.1 is a fully motorized microscope, allowing software control of objectives; fluorescent cubes; transmitted, epi-fluorescent, and laser illumination; Z-position, and X/Y on the stage. This microscope complements an existing AxioObserver D.1 in the main Core Facility, promoting greater user familiarity as well as accessory compatibility.
The purchase of this laser scanning confocal microscope was made possible by a NSF Major Research Instrumentation award in 2007. This award was the result of an interdisciplinary effort led by the PI, Dr. Anita McCauley, and faculty from Biology (Drs. Conner, Fahrbach, Johnson, and Muday) and Physics (Drs. Guthold and Williams). The university has renovated Winston Hall Room 009 in order to create a dedicated room for confocal microscopy and related sample preparation and teaching.
Accessories
The AxioObserver Z.1 is equipped with two fluorescent filter cubes, Zeiss set 10 and Zeiss set 43, which allow conventional fluorescent imaging of green and red emitting samples. Full details about Filter set 10 and Filter set 43, as well as a list of fluorophores visible with each cube, are available. The microscope is outfitted with a range of dry and immersion objectives, all with high numerical apertures and specifically designed for confocal imaging. These include EC Plan-Neofluar 10x/0.30, Plan-Apochromat 20x/0.80, and Plan-Apochromat 40x/0.95 dry objectives, Plan-Apochromat 40x/1.3 and Plan-Apochromat 63x/1.4 oil-immersion objectives, and a Plan-Apochromat 63x/1.2 water-immersion objective.
Image acquisition and analysis for the LSM 710 is controlled using the recently released Zen software. In addition to the basic image acquisition and analysis modules, we have four add-on modules for advanced imaging functions. Modules for FRET, FRAP, and Physiology allow the acquisition and analysis of these dynamic microscopic imaging techniques. A module called Multi-time allows for advanced control of multi-dimensional image capture sequences.
Applications
The LSM 710 is being used for a wide range of research and educational programs. Research uses include imaging of fluorescent protein expression in plant roots and stems, fluorescent protein and immunohistochemical label in Drosophila nervous system, morphological analysis of neuronal structure in both culture and brain sections, and FRAP and colocalization studies of DNA mismatch repair proteins in living cells. The microscope has also been utilized in an undergraduate laboratory course, Optics -- taught by grant co-PI Richard Williams, and a graduate course, Microscopy in the Biological Sciences -- taught by grant PI Anita McCauley.
These images, of Molecular Probes prepared slides, illustrate the quality of images that can be acquired with this microscope.
