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 0.63x, 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.
Epi-Fluorescence Filter Sets. Filter cubes made by Chroma Technology Corp. for UV, GFP, andRhodamine/DiI provide a range of opportunities for single or multi-label fluorescent samples. Filter cube specifics are available in PDF format.
Digital Camera. Two digital cameras are available for image capture on this microscope. For monochromatic digital capture, a Q-Imaging Retiga 4000R digital camera provides high-performance 12-bit monochromatic image capture using 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. This camera can also be used for 36-bit color digital capture via a liquid-tunable filter which captures red, green, and blue exposures sequentially. 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.
For most color image capture, the Infinity 2-2 (Lumenera, Ontario, CA) is recommended. This camera has a 2.01 megapixel progressive-scan interline CCD chip with pixels that are 4.4 microns2. Images can be acquired in 8-bit or 12-bit mode using the full 1616×1216 pixel area or a sub-region of the sensor.
Imaging Software. Media Cybernetic’s Image Pro Plus 6.2 is used for acquisition, processing, and analysis of images acquired with the Retiga 4000 camera. 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. Images are acquired and analyzed using the Infinity Capture and Analyze software for the Infinity 2-2 camera.
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 (DBI-0500702; PI: McCauley) and Wake Forest University.