A Zoom Fluorescence Research Microscope for Macro- to Microfluorescence Imaging

Scientists are increasingly interested in viewing both ends of the research spectrum—from cellular and subcellular level observations to tissue, organ, and whole animal studies. New hybrid instruments are a meaningful step toward a deeper understanding of how macro- and microlevel life events are intertwined.

Scientists who work with specimens as varied as Zebrafish, mouse, mammalian embryo, Arabidopsis, rat brain, C. elegans, and Drosophila have a common challenge. These researchers must capture vivid fluorescence images of entire tissues, organs, systems, or organisms at low magnification. They also need to capture higher-magnification images of the cellular events that are behind the macrolevel changes. Technology has not made this easy for developmental biologists, cancer researchers, cell biologists, genetics researchers, molecular biologists, ophthalmologists, neuroscientists, botanists, and others who perform this kind of imaging.

Figure 1 - Optical paths of compound (left), macrozoom (center), and stereo (right) microscopes. (Figures 1 and 2 courtesy of Michael W. Davidson, The Florida State University, Tallahassee, FL.)

Historically, stereomicroscopes have been used to view specimens that are too large or thick for compound microscopes. Stereoscopic instruments have many useful features for researchers studying larger samples in Petri dishes or as whole animal preparations. In vivo processes can in some cases be followed in real time in living specimens without sacrificing the animals. As the name indicates, stereo instruments offer three-dimensional viewing, a benefit for those performing dissections or animal surgery. The stereo view is produced by observing the same visual field from two slightly divergent positions, much as our eyes view nearby objects, resulting in the brain’s ability to distinguish depth (Figure 1). Most stereomicroscopes also conveniently allow researchers to adjust magnification, giving them the ability to zoom in and out on the specimen without extensive refocusing or repositioning. However, most stereomicroscopes have severe limitations for the researcher using fluorescence. Stereo optics have a relatively low capacity to gather light, as measured by the numerical aperture (NA) of their lenses. Low NA limits the amount of signal that can be captured from the sample, making it extremely difficult to view or capture fluorescence images with a high signal-to-noise ratio, the chief indicator of a good fluorescence image.

Figure 2 - The common main objective of a stereomicroscope (left) makes it impossible for either of the instrument’s two light paths to receive even half of the emitted light coming from the specimen. The objective (right) of the MVX10 MacroView zoom fluorescence research microscope (Olympus Corp., Tokyo, Japan) is shown for comparison.

The light gathering limitation is critical and can be further understood by looking at the two principal designs of the stereomicroscope. The simpler one is the Greenough design, in which two narrow, conventional microscope optical paths are angled within a single zoom body to produce a stereo view. Higher-performance stereomicroscopes typically are of a common main objective (CMO) or Galilean design. Light from the sample passes through the CMO and is imaged by two parallel zooming “telescopes.” Because the two light paths view opposite sides of the CMO, each receives less than half the light captured from the specimen (Figure 2). Since a camera can only be attached to one light path, a great deal of signal from the specimen cannot be captured for imaging purposes. Thus, stereomicroscopes, limited in both NA and by separate light paths, are not optimized instruments for fluorescence microscopy.

Because of the relatively low light gathering ability of stereomicroscopes, many biologists have chosen traditional compound microscopes for fluorescence imaging. However, the design parameters that make compound microscopes flexible and functional for high-magnification imaging tend to also limit their performance for low-magnification, large-field-of-view imaging. Limitations in NA and working distance can frustrate low-magnification fluorescence imaging, manipulation, and dissection on compound instruments.

Figure 3 - The MVX10 MacroView microscope combines the best features of both stereo and compound microscopes for bright, crisp fluorescence images from 4× to 250×.

In the end, a number of researchers find themselves switching between stereo and compound microscope systems to meet the requirement of associating cellular and molecular phenomena with specific events at the organism level. But combining the best of both stereo and compound microscopes would theoretically be the optimum way to address the need for low- to high-magnification imaging of live, fluorescing specimens. The optimal system for fluorescence observation and recording in intact organisms must combine maximum detection sensitivity at the lowest magnifications with a high-magnification zoom for the resolution of fine details within organs, tissues, and cells. The MVX10 MacroView zoom fluorescence research microscope addresses the tradeoffs between stereo and compound imaging systems, and was designed specifically for macro- to microfluorescence imaging (Figure 3). It offers the same working distances and large fields of view as a stereomicroscope, but with a single, full-objective optical path for imaging devices, and much higher NAs for improved resolution and light gathering ability.

Fluorescence observations ranging from low-magnification viewing of whole organisms like Zebrafish to high-magnification imaging of gene expression at the cellular level are possible (Figure 4). With features such as a two-objective nosepiece, 2× objective lens, and 2× magnification changing lens, the microscope can zoom from 4× to 250× (a zoom factor of 31 times) with high image quality, regardless of the specimen medium. Moreover, the 2× objective has a correction collar that adjusts for up to 5 mm of aqueous medium above the specimen. The instrument also has working distances (WD) that are much greater than compound microscopes (for instance, 65 mm WD for the 1×/0.25-NA objective) and its field of view ranges up to 55 mm, making it easy to observe, track, or work on large or fast-moving specimens.

Figure 4 - Images (including zoom image) of transgenic green fluorescence protein (GFP) Zebrafish with expression in the brain and spinal cord, captured using the MVX10. (Courtesy of Richard Dorsky, Dept. of Neurobiology and Anatomy, University of Utah, Salt Lake City, UT.)

Its three parfocal objectives—0.63×/0.15 NA, 1×/0.25 NA, and 2×/0.50 NA—are plan apochromatic to produce the highest-level image quality with better-quality chromatic aberration correction. They offer good transmission and high signal-to-noise ratios, so that living samples can be exposed to intense illumination for much briefer time periods. The objectives can deliver up to 10 times the light intensity from a fluorescing specimen to an image capture device, when compared with many high-end stereomicroscopes. The objectives offer high optical performance through the near-IR range, making the instrument versatile enough to address the wide variety of live cell applications for current and future use.

For visual stereoscopic viewing, the patent-pending pupil separation mechanism mimics the natural offset of the human eyes at a somewhat lower degree of separation—all by just moving a slider. The mechanism is not used when imaging with a camera, so that 100% of the light output is always delivered to the image capture device at the moment of exposure. Ergonomic features such as tilting eyepieces make it comfortable for users of varying heights.

With novel imaging capabilities becoming available to researchers through instrumentation, software, probes, and new methodologies, scientists will move to the next level in their understanding of how events on the cellular and organism levels are related.

Mr. Higgins is Group Marketing Manager, Olympus America Inc., Scientific Equipment Group, Two Corporate Center Dr., Melville, NY 11747, U.S.A.; tel.: 631-844-5066; fax: 631-844-5111; e-mail: [email protected].