The 710 confocal scan head is coupled to the AxioObserver Z1 inverted microscope. This inverted microscope body provides versatility for live and fixed, slide, well-plate, and dish imaging applications. The Z1 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 D1 in the main Core Facility, promoting greater user familiarity as well as accessory compatibility. Laser lines available on on this microscope include 405nm (25mW diode), 440nm (25 mW diode), 458nm, 488nm, 514nm (35mW Argon laser), 561nm (25mW diode), 594nm (2mWHeNe laser), and 633nm (5mW HeNe laser). Advanced features include full spectral imaging with no emission filters with 3-9nm emission channels
The AxioObserver Z1 is equipped with four fluorescent filter cubes, Zeiss sets 49 (blue), 38 (green), 49 (red), and 76 HE (far red) which allows conventional fluorescent imaging of multi-channel emitting samples. Full details about these Zeiss filter sets, as well as a list of fluorophores visible with each cube, are available here
. 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/0.45, 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/1.3 water-immersion objective.
A full live-cell incubation system is also available. This includes a temperable stage (temperable insert P S1), capable of heating or cooling slides or dishes, modules to control temperature, carbon dioxide and humidity (Temp Module S1, CO2 module S1, O2 module S1, and heating device humidity S1),an enclosure to maintain the environment (Incubator PM S1) and a temperable objective ring. Additionally, a Definite Focus module is available to maintain precise Z-position, a feature particularly useful during longer duration time-lapse imaging experiments.
Image acquisition and analysis for the LSM 710 is controlled using 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.
The purchase of this laser scanning confocal microscope and live cell imaging accessories was made possible by two NSF Major Research Instrumentation awards, in 2007 and 2010. These awards were the result of interdisciplinary efforts led by the PI, Dr. Anita McCauley, and faculty from Biology (Drs. Conner, Fahrbach, Johnson, and Muday), Chemistry (Dr. King), Physics (Drs. Bonin, Guthold and Williams), and faculty from WFUHS (Drs. Poole and Scarpinato). The university has renovated Winston Hall Room 009 in order to create a dedicated room for confocal microscopy and related sample preparation and teaching.
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 undergraduate laboratory courses, Optics — taught by grant co-PI Richard Williams, Biology 214: Cell Biology, and Biology 369: The Cell Biological Basis of Disease, and a graduate course, Biology 775: Microscopy in the Biological Sciences. The confocal microscope has been a centerpiece in recent regional meetings, including regional microscopy workshops, the department’s annual Perspectives in Biology symposium, and the SYNAPSE 2010 meeting hosted at WFU.