Epi-Fluorescent Stereomicroscope Imaging System
Microscope
The Leica MZ16 FA is a motorized and automated stereo fluorescence microscope with a highly-corrected apochromatic optics system based upon the common main objective (CMO) design. Motorized zoom, focus, filter changer, condenser aperture, and fluorescence shutter controls create an ergonomic working environment with more precise and repeatable imaging conditions. Programmable zoom and magnification settings allow users to easily move between multiple objectives and samples. This microscope is equipped with the Fluo Combi attachment, enabling the use of stereo and micro objectives. With .63x, 1.6x, and 5x objectives, this microscope delivers a magnification range of 4.5x–460x due to its 16:1 zoom range. The transmitted light base provides a range of contrast-enhancing options including positive/inverted/dynamic relief contrast, vertical/oblique illumination, and single-sided darkfield. Reflected light illumination is also available using bifurcated pipes equipped with focusing lens and polarizing filters. An analyzer attaches to the objectives to eliminate unwanted glare. A gliding stage allows fine, controlled movement of the sample during viewing. Manuals for technical issues, the fluorescent lamp house, the hand controller, the combi objectives, and the base are availble in PDF format.
Accessories
Epi-Fluorescence Filter Sets. Filter cubes made by Chroma Technology Corp. for UV, GFP, and Rhodamine/DiI provide a range of opportunities for single or multi-label fluorescent samples. Filter cube specifics are available in PDF format.
Digital Camera. A Q-Imaging Retiga 4000R digital camera provides high-performance 12-bit monochromatic or 36-bit color digital image capture. This camera has a 4.19 megapixel progressive-scan interline CCD chip. With pixels that are 7.4 microns2 and an effective pixel area of 15.15mm2, this camera is capable of excellent spatial resolution at low and high magnification, and is especially well-suited for the large fields-of-views typical of stereomicroscopes. Color images are acquired using a liquid-tunable filter which captures red, green, and blue exposures sequentially. This acquisition method, which gives better color resolution than the Bayer Mosaic method, combined with the 36-bit dynamic range produces high-fidelity color images. The pixel size, dynamic range of 3333:1, and quantum efficiency near 60% in the visible spectrum gives this camera excellent sensitivity in normal and low-light imaging situations. Images are previewed, acquired, processed and analyzed using Media Cybernetic’s Image Pro Plus. Movies of live-cell processes can be created using the image acquisiton software to control camera parameters. The Retiga 4000R technical characteristics are available in PDF format.
Imaging Software. Media Cybernetic’s Image Pro Plus 6.2 is used for image acquisition, processing, and analysis. Images can be captured as a single or sequence of files, allowing both static and time-lapse capture and analysis. Processing functions including spatial filtering, background subtraction, and pseudo-coloring, as well as analysis functions including counting, intensity, and area measurements are available. Stitching tools allow a montage of images to be combined into a large, single image file.
Auto-Montage Essentials by Syncroscopy is also available. This software provides a solution for problems encountered when imaging a sample with a large Z-dimension. Using a stack of images acquired from throughout the full depth of the sample, this software converts the three-dimensional stack into a perfectly-focused two-dimensional image. Additional functionality includes creating depth and color relief maps, anaglyphs, and 3-D surface views.
Funding Support.
The acquisition of this entire imaging system was made possible by funding from the National Science Foundation (Anita McCauley) and Wake Forest University.
Applications
Role of Resilin in Coremata Inflation in Moths.
Ph. D. candidate Alex Jordan and his graduate advisor Bill Conner explored the use of resilin, a natural insect protein with rubber-like qualities, to provide elasticity to inflatable androconial structures in male tiger moths (Lepidoptera: Arctiidae). Resilin fluoresces when illuminated with ultraviolet light (360nm.). When they scanned the coremata of the salt marsh moth, Estigmene acrea, they found resilin in the socket at the base of each androconial scale. Resilin in this location insures that the scales projects perpendicularly from the surface of the coremata and promotes retraction and folding of the structure when it is not in use.
Examination of Functional Characters for Erica Systematics.
The laboratory of Kathy Kron is interested in studying the phylogenetic relationships of flowering plants, including the blueberry family (Ericaceae). Despite a major shift towards using molecular techniques for the classification and systematics of plants, morphological features remain a valuable resource for the classification of taxa and the study of their evolutionary relationships. It has been shown that a “total evidence” approach, using both molecular and morphological data generally give the best results. Many of these characters which are important for the identification of plants, such as hair type, the structure of anthers and other floral features, can only be observed under magnification as shown by this example of Erica innovans (sample by E.G.H. Oliver; image by Tanja Schuster). These images can be stored in databases and immediately made available to anyone who may not have direct access to a specimen.
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