Optical Sectioning with de Sénarmont DIC Microscopy

The ability to image a specimen in de Sénarmont DIC microscopy with large condenser and objective numerical apertures enables the creation of remarkably shallow optical sections from a focused image plane. Without the disturbance of halos and distracting intensity fluctuations from bright regions in lateral planes removed the focal point, the technique yields sharp images that are neatly sliced from a complex three-dimensional phase specimen. This property is often utilized to obtain crisp optical sections of cellular outlines in complex tissues with minimal interference from structures above and below the focal plane.

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The tutorial initializes with a randomly selected image appearing in the DIC Specimen Image window and a model of an objective and specimen on the right-hand side of the window. In order to operate the tutorial, use the Objective Focus slider to focus the specimen through a range of planes starting at the upper surface and progressing through the central region to the lower surface. As the slider is translated, various image planes in the specimen will be brought into sharp focus. Simultaneously, the microscope objective model will move upward and downward with respect to the specimen to simulate the actual relationship in the microscope. A new specimen can be examined by selecting from the palette in the Choose A Specimen pull-down menu.

In all traditional forms of transmitted and reflected light microscopy, the condenser aperture iris diaphragm plays a major role in defining image contrast and resolution. Reducing the aperture size increases the depth of field and overall image sharpness while simultaneously producing enhanced contrast. However, if the condenser diaphragm is closed too far, diffraction artifacts become apparent and resolution is sacrificed. Often, the optimum aperture diaphragm setting is a compromise between accurately rendering specimen detail in sufficient contrast and retaining the resolution necessary to image minute features while avoiding diffraction artifacts.

When the condenser iris diaphragm is adjusted to approximately 70 percent of the objective rear aperture size, most high-performance de Sénarmont DIC optical systems produce excellent contrast. However, these microscopes also perform superbly when the condenser diaphragm is opened to match the objective rear aperture diameter. In order to achieve the optimum balance between resolution and contrast for optical sectioning experiments, it is critical that the microscope be properly configured for Köhler illumination and that the Nomarski prism components, analyzer, and de Sénarmont compensator be accurately aligned.

Optical sections taken of a living Volvox colony using de Sénarmont DIC to achieve bias retardation on an inverted tissue culture microscope are illustrated in Figure 1. The aquatic microorganism is composed of hundreds to thousands of identical green algae cells having approximately the same diameter, but organized into several morphological motifs. On the periphery of the colony, individual cells are arranged in a semi-transparent widely spaced layer termed the mucilage, as illustrated in Figure 1(a). Farther into the mass, the colony forms several concentrated spherical groups of reproductive cells, termed gonidia, which produce small daughter colonies within the parent colony (Figure 1(b)). As the microscope is focused on the uppermost layer of the colonies in the mucilage (Figure 1(c)), structural detail in the individual cells becomes visible, but many cells are masked by the daughter colony.

Thin biological specimens (10 to 20 micrometers in thickness) usually produce poor optical sections at lower magnifications, but often reveal substantial internal detail when visualized with high magnification objectives having large numerical aperture (60x and 100x). Thicker specimens can be readily sectioned at low magnifications where aberration is minimized. Collection of optical sections from thicker biological specimens, especially those immersed in aqueous saline or buffered solutions, is often hampered by spherical aberration produced by refractive index discontinuities at the interface between the cover glass and mounting medium. This artifact will reduce resolution at higher penetration depths in the optical section series.


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Contributing Authors

Stanley Schwartz - Bioscience Department, Nikon Instruments, Inc., 1300 Walt Whitman Road, Melville, New York 11747.

Douglas B. Murphy - Department of Cell Biology and Anatomy and Microscope Facility, Johns Hopkins University School of Medicine, 725 N. Wolfe Street, 107 WBSB, Baltimore, Maryland 21205.

Kenneth R. Spring - Scientific Consultant, Lusby, Maryland, 20657.

Matthew Parry-Hill and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.