Biomedical Engineering Reference
In-Depth Information
called retardance (see Section 15.7 ). If the retardance is less than 275 nm, the object
appears gray between crossed polars, while thick or highly birefringent objects that induce
a retardance of more than 275 nm appear colored. With increasing retardance, often
associated with increasing thickness of an object, it appears yellow (retardance B 300 nm),
orange (500 nm), red (530 nm), purple (560 nm), blue (650 nm), green (750 nm), and
yellow again (900 nm). The color of the objects of even higher retardance roughly repeat in
the same order, albeit the colors become less saturated. To identify the retardance of a
birefringent object based on its interference color, one can use a so-called Michel-L ยด vy
chart, which graphically represents the sequence of colors and their associated retardance
values. The chart often includes additional lines that help relate the retardance to the
birefringence of a specimen and its thickness measured in the direction of the light path.
Aside from the preceding remarks, this chapter discusses the instrumentation and methods that
are optimized for analyzing the objects of low birefringence and retardance of less than
275 nm. When viewed in white light between crossed polars, objects of low retardance remain
gray but appear increasingly brighter, the higher their retardance is. Biological specimens, such
as the ones presented in Section 15.1 , induce a retardance that is often only a few nanometers
and therefore require sensitive methods to observe them and measure their retardance
accurately. In this chapter, I discuss the methods for detecting, imaging, and measuring low
retardance with high fidelity and sensitivity. I refer to the topics by Hartshorne and Stuart [5,6]
for a discussion of quantitative measurements of more highly birefringent objects and mention
the chapter on microscopes in the Handbook of Optics [21] .
15.2.1 Basic Setup
The polarized light microscope (also called polarizing microscope or polarization
microscope, Figure 15.4 ) generally differs from a standard transilluminating microscope by
the addition of a polarizer before the condenser; a compensator and analyzer behind the
objective lens; strain-free optics; a graduated, revolving stage; centrable lens mounts;
cross hairs in the ocular aligned parallel or at 45 to the polarizer axes; and a focusable
Bertrand lens that can be inserted for conoscopic observation of interference patterns in the
back aperture of the objective lens. (For a definition of polarization optical terms, such as
polarizer, compensator, and birefringence, see Section 15.7 .)
Polarizers
Most light sources (halogen bulb, arc burner, and light-emitting diode) generate
unpolarized light, and hence the first polarizer located before the condenser optics
polarizes the light that illuminates the specimen. The second polarizer serves to analyze
the polarization of the light after it passed through the specimen; therefore, it is called
the analyzer. In its most basic configuration, the polarizing microscope has no
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