Inverted Microscope Imaging System
Microscope
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 for a range of contrast-enhancing techniques including differential interference contrast (DIC) and phase contrast. 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 4x, 10x, 20x, 40x, 63x (water), 63x (oil), and 100x (oil) provide an ideal range of magnification options with excellent optical performance. A 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. Details regarding appropriate matching of objectives, optivar settings, and camera resolution for optimal sampling are also available.
Accessories
Epi-Fluorescent Filter Sets. Filter cubes purchased from Semrock, Chroma Technology Corp. and Carl Zeiss, Inc. for UV, FITC, GFP, YFP, Cy3, Rhodamine, and Texas Red provide a range of opportunities for single or multi-label fluorescent samples. Filter cube specifics are available in PDF format. Semrock documentation of fluorophores best for their FITC, DAPI-contrast, DAPI-brightness, Texas Red, and TRITC filter cubes are also available. An EXFO X-cite 120 XL light source provides the fluorescent illumination. This metal halide light source has a spectrum similar to mercury arc lamps but has a much longer lamp life and requires no bulb alignment.
Motorized External Controls. This inverted microscope has been equipped with a Mac 5000 Controller System from Ludl Electronic Products Ltd. to provide motorized, automated control of the Z-axis/Focus. 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 repeatibility. 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. The Mac 5000 Controller brochure is available in PDF format. A table indicating ideal z-step sizes is also available.
Digital Camera. 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. With pixels that are 6.45 microns2, an effective pixel area of 8.67mm x 6.60mm, a dynamic range of 3000:1, and a quantum efficiency exceeding 70% in the visible spectrum, this camera delivers excellent spatial resolution and excellent sensitivity in low-light imaging situations. Images are previewed and acquired using Improvision’s Volocity and are processed and analyzed using either Volocity Visualization and Quantification or Media Cybernetic’s Image Pro Plus. The Orca digital camera technical characteristics are available in PDF format.
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. With pixels that are 6.45 microns2, an effective pixel area of 8.67mm x 6.60mm, and a dynamic range of 1384:1, this camera also delivers excellent sensitivity in low-light situations. Images are previewed and acquired using Improvision's Volocity and analyzed using either Volocity or Image Pro Plus. Volocity software allows spectral separation and analysis of distinct color channels with individual images and volumes. The Orca-3CCD technical characteristics are available in PDF format.
Imaging Software. Volocity by Improvision is a complete imaging software package providing 3D or 4D acquisition, restoration, fully interactive volume visualization, and quantification. Automated acquisition protocols controlling filter, camera, microscope, and shutter settings can be saved for individual users. An experiment log is generated for each acquired image, creating a permanent record of the protocol, hardware and time settings, and ensuring that vital methodological data is saved. The restoration module uses iterative restoration algorithms based upon maximum entropy techniques to remove noise and blur, creating significant improvements in XY and Z resolution of both widefield and confocal data stacks. A table is available to assist with the restoration of confocal data stacks. With the visualization module, 3-D objects can be explored interactively, including real time, rotation, zooming, animation, and fly-through. Adjusting the point of view inside or outside the object allows unique and realistic perspectives on biological structures. Using the Movie Sequencer tool, volume animations can be created and exported as movie or virtual reality files. With the Quantification module, image analysis can be done. Restoration, Visualization, and Quantification are available on a Dell 64-bit workstation, providing the ability to work on much large volumes, and a 36% increase in processing time. The Volocity software package brochure is available in PDF format.
Funding Support.
The acquisition of these insturments and software for 3 and 4-D epi-fluorescent imaging has been made possible by support from the National Science Foundation (Susan Fahrbach; Anita McCauley), the U.S. Department of Agriculture (Gloria Muday), the Cannon Foundation of North Carolina (Anita McCauley), and Wake Forest University.
Applications
Experience-Dependent Brain Growth in Honey Bees.
The mushroom bodies of the insect brain are multi-modal sensory integration centers that have been compared to the hippocampus and cortex of the vertebrate brain. This brain region is larger in forager honey bees than in bees that work in the hive. The laboratory of Susan Fahrbach is studying the mechanisms responsible for experience-dependent brain growth using low-density primary cultures of mushroom body neurons. The entire surface of insect neurons is labeled with an antibody raised in rabbits directed against horseradish peroxidase; application of a secondary antibody (goat anti-rabbit) tagged with a fluorophore (fluorescein or AlexaFluor488) permits us to visualize entire neurons and major processes (axons and dendrites) using routine fluorescence microscopy. Counting and measuring the fine branches of our cultured neurons, however, requires collection of a stack of images, 3-D reconstruction, and removal of image blur by application of the computational method of deconvolution. The Zeiss AxioObserver paired with Volocity software makes possible the efficient collection of digital images for subsequent quantitative analysis.
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