The Zeiss AxioObserver is a high-performance research instrument delivering multi-modal imaging capabilities. This inverted light microscope stand is equipped for transmitted and epi-fluorescent illumination and is specialized for phase contrast microscopy. A motorized filter cube turret, motorized shutters for transmitted and reflected light, and encoded objectives enhance the image acquisition process. Plan Neofluar and Plan Apochromatic objectives at 5X/0.15NA, 10x/ 0.25NA, 20x /0.5NA, 40x /0.65NA, long working 40x /0.6NA, and 100x/ 1.3NA (oil) provide an ideal range of magnification options with excellent optical performance. The Plan Neofluar 40x with long working-distance is ideal for cell culture applications. An optivar with 1.25x and 1.6x settings allow additional control of image magnification.
Epi-Fluorescent Filter Sets. Filter cubes purchased from Semrock, Chroma Technology Corp. and Carl Zeiss, Inc. for UV, CFP, FITC, GFP, YFP, Cy3, Rhodamine, mCherry, Texas Red, and sets for ET-FURA2 and CFP/YFP FRET provide a range of opportunities for single or multi-label fluorescent samples. An EXFO X-cite 120 XL light source provides the fluorescent illumination.
Motorized External Controls. This inverted microscope has been equipped with a Mac 6000 Controller System from Ludl Electronic Products Ltd. to provide motorized, automated control of the Z-axis/Focus as well as rapid switching of excitation wavelengths. The Z-axis/Focus controller with the microstepping motor, linear encoder, and digipot allows precise focus positioning with 0.05 micron step sizes and 0.1 micron repeatability. This equipment enables the acquisition of optical sections through a sample with minimal photobleaching, creating 3-dimensional optical stacks that can be used for image restoration, 3-D reconstruction, and volume analysis.
Live-Cell Perfusion System. A heated stage and perfusion system from Warner Instruments enables live cell imaging and imaging under changing physiological conditions. A dual channel heater and solution heater ensure that the sample and solutions are heated, while a chamber insert allows cells in 35mm culture dishes to be heated and perfused.
Digital Image Acquisition. A Hamamatsu Orca C4742-80-12AG digital camera provides high-performance 12-bit monochromatic digital image capture. This camera has a 1.37 megapixel progressive-scan interline CCD chip that delivers excellent spatial resolution and sensitivity in low-light imaging situations.
High performance color image capture is available through a Hamamatsu Orca-3CCD cooled digital camera. The Orca C7780-20 digital camera has three separate 1.37 megapixel progressive-scan interline chips to provide high spatial and temporal resolution color images.
Imaging Software. Hamamatsu HCImage software is used for acquisition of standard, 3D or time-lapse images. Automated acquisition protocols controlling filter, camera, microscope, and shutter settings can be saved for individual users.
Funding Support. The acquisition of these instruments and software for 3 and 4-D epi-fluorescent imaging has been made possible by support from the National Science Foundation (Susan Fahrbach; AnitaMcCauley), the U.S. Department of Agriculture (Gloria Muday), the Cannon Foundation of North Carolina (Anita McCauley), and the WFU Center for Molecular Signaling, and Wake Forest University.
Leaf Stomata Opening Assay –