Scatter Refractive Index and the Becke Line

Nonspecific light loss resulting from scatter from differences in refractive index (nD) of a specimen and its mounting medium is often one of the most serious errors affecting cytophotometric determinations of DNA-Feulgen amounts in tissue samples (12,38,41,49,64). Refractive index is a physical property of matter and is a quantitative expression of the degree of change in the velocity of a ray passing through a substance of one refractive index (nD) to another of a different refractive index. The ray of light is bent from the substance of lower refractive index toward the medium of higher refractive index. That is to say, light is deviated from its initial path either in toward or out from the object of interest if the object and its mounting medium do not have the same refractive index. Look at the fine print on the label of a bottle of microscope immersion oil for which nD = 1.515. The refractive index of the glass used in making oil immersion objectives, glass slides, and cover slips is 1.515. This means that there will be minimal distortion resulting from light scatter as the light from the substage illuminator traverses the condenser and passes through the slide, the object, the cover slip, and microscope optics to the retina of your eye without significant retardation of the incident light (see Note 13).

Immersion Method Glass Becke Line

Fig. 10. A crystal of ascorbic acid (vitamin C, nD = 1.588) mounted in a liquid with nD = 1.744. (A) The specimen is within the focus of the microscope; (B) the same specimen, but the focus of the microscope has been raised. The Becke line (bright "halo") appears in both images but is nearer the substance of higher refractive index. The focus of the microscope is raised to show the Becke line (light "halo") bent toward the substance of higher refractive index; that is, it has been displaced outward from the crystal. If the microscope tube is lowered, the Becke line will move inward and the crystal will appear brighter. (Adapted from ref. 67.)

Fig. 10. A crystal of ascorbic acid (vitamin C, nD = 1.588) mounted in a liquid with nD = 1.744. (A) The specimen is within the focus of the microscope; (B) the same specimen, but the focus of the microscope has been raised. The Becke line (bright "halo") appears in both images but is nearer the substance of higher refractive index. The focus of the microscope is raised to show the Becke line (light "halo") bent toward the substance of higher refractive index; that is, it has been displaced outward from the crystal. If the microscope tube is lowered, the Becke line will move inward and the crystal will appear brighter. (Adapted from ref. 67.)

The appearance of bright "halos" around or above nuclear outlines can significantly affect the net amount of light impinging on the light-sensing elements of the system. This bending of light is manifest as the Becke line (67). Although conditions are not always favorable for its recognition, the phenomenon is clearly evident, when the specimen is of a crystal nature and has straight sides, as shown in Fig 10. In this case, taken from Shillaber (67), a crystal of vitamin C (ascorbic acid), nD = 1.588, is mounted in a liquid for which nD = 1.740. In Fig. 10A, the specimen is within the focus of the microscope. In Fig. 10B, the focus is high to show the Becke line (the bright "halo"), which is nearer the substance of higher refractive index. It is displaced outward from the crystal. If the microscope focus is lowered, the Becke line will move inward and the crystal interior will appear brighter.

The consequences of light refraction in an optical path are clearly illustrated by the changes in appearance of microscopic glass spheres (nD = 1.468)

Fig. 11. Photomicrograph of clear glass spheres. The refractive index of the spheres is about 1.468. They are mounted in ethylene glycol (nD = 1.400). In this micrograph, the focus of the microscope was made as accurately as possible to show how the spheres actually appear under normal lighting conditions. (Adapted from ref. 67.)

Fig. 11. Photomicrograph of clear glass spheres. The refractive index of the spheres is about 1.468. They are mounted in ethylene glycol (nD = 1.400). In this micrograph, the focus of the microscope was made as accurately as possible to show how the spheres actually appear under normal lighting conditions. (Adapted from ref. 67.)

mounted in ethylene glycol (nD = 1.400). In Fig. 11, the focus of the microscope was made as nearly perfect as possible to give a true idea of how the spheres appear under these conditions. Compare this image with Fig. 12, where the microscope focus was raised from the optimum focus. The centers of the individual spheres become brighter as the Becke line bends inward toward the higher refractive index. In Fig. 13, the same objects are viewed at a slightly lower optical level and the centers of the spheres appear darker than the surrounding field. Although the amount of incident light is the same for these three views of the spheres, the light bending in and out of the main beam can lead to erroneous transmittance values for small objects such as nuclei.

Refractive index matching is an important part of preparing a specimen for cytophotometry because it determines what a photosensing element will "see" if the specimen and the mounting liquid have the same refractive index, and if that is the same as the microscope slide (nD = 1.515), the light rays are not refracted and the Becke line does not exist. The specimen itself will be invisible, save for the magenta staining of chromatin by the DNA-Feulgen reaction. For example, the cross-bands of Drosophila giant salivary gland chromosomes will appear as pink rungs of a ladder. When a transparent, colorless specimen has low visibility and is neither very small nor very thin, it is generally safe to

Fig. 12. Photomicrograph of same subject as in Fig. 11, but the microscope tube has been raised from the optimum position of focus. Note how the centers of individual spheres become brighter when they are mounted in a liquid with refractive index lower than the nD of the test objects used here. (Adapted from ref. 67.)

Fig. 12. Photomicrograph of same subject as in Fig. 11, but the microscope tube has been raised from the optimum position of focus. Note how the centers of individual spheres become brighter when they are mounted in a liquid with refractive index lower than the nD of the test objects used here. (Adapted from ref. 67.)

assume that its refractive index is very close to that of the mounting liquid and that of glass.

Awareness of the influence of refractive index on estimating DNA content by cytophotometry is an important variable to evaluate before measuring an unfamiliar tissue. The nD of most biological material ranges from 1.520 to 1.570. For example, refractive index liquid oils of 1.568 and 1.524 were used to compare C values for the very large nuclei of the larval fat body with the DNA levels of the small nuclei of the adult fat body and of oenocytes in Droso-phila (Fig. 3 vs Figs. 1 and 2). DNA values for individual Feulgen-stained sperm of Drosophila are useful as a reference standard for organisms with small genome sizes, but may be significantly underestimated if not matched with refractive index liquids because of their staining density and high refractive index in usual mounting media. Table 5 lists many commonly used mounting media and their refractive index values.

Sets of oils of different refractive indices are available commercially for use in mounting cells and tissues for cytophotometry (see Note 14). As stated so simply and succinctly in the procedure outlined by Hardie et al. (12), "the following steps can be taken to select an oil with the appropriate refractive index for the cell type being analyzed:

Fig. 13. Photomicrograph of the same object as in Fig. 11, but taken at a slightly lower optical level than in Fig. 12. The centers of the spheres now appear darker than the surrounding field. The relative density values obtained here would be significantly higher here than those obtained from the same objects mounted in a matching refractive index liquid that would minimize light loss resulting from scatter. (Adapted from ref. 67.)

Fig. 13. Photomicrograph of the same object as in Fig. 11, but taken at a slightly lower optical level than in Fig. 12. The centers of the spheres now appear darker than the surrounding field. The relative density values obtained here would be significantly higher here than those obtained from the same objects mounted in a matching refractive index liquid that would minimize light loss resulting from scatter. (Adapted from ref. 67.)

1. Begin by placing a drop of oil with a mid-range refractive index (e.g., nD = 1.54) on the slide and add a cover slip.

2. Focus on a nucleus, which should appear as a pink jewel suspended in space. If membranes are visible, the refractive index is incorrect. (Note that 100x objectives require a second drop of immersion oil, usually nD = 1.515, between the cover slip and the lens.)

3. To determine whether a higher or lower refractive index is needed, focus up and down through the nucleus. In one direction, a bright outline of pink light will be seen on the perimeter of the nucleus. In the other direction, the nucleus will appear to enlarge as the focus moves through it, without any bright light. Take note of which direction of focus (i.e., "up," moving the lens away from the slide, or "down," toward the slide) produces which effect.

4. If the bright light appears when focusing up, then a higher refractive index is required. If the bright light appears during the down focus, then a lower refractive index is needed.

In properly matched preparations with very high transmittance values, or conversely with very low IOD values, the relative photometric error, with regard

Table 5

Refractive Indices of Common Reagents and Mounting Media for Biological Specimens

Listing according to ascending Alphabetical listing values of nD

nD nD

Temperature Temperature

1.000294 Aira 1.000294 Aira

1.358 Ethyl alcohol 1.333 Water

1.323 Methyl alcohol 1.323 Methyl alcohol

1.535 Canada balsam (hard) 1.358 Ethyl alcohol

1.477 Castor oil 1.452 Paraffin oil (light)

1.54 Clarite 1.463 Glycerol

1.57 Clarite X 1.468 Olive Oil

1.529 Clove oil 1.477 Castor oil

1.48 (approx) Glycerin jelly 1.48 (approx) Glycerin jelly

1.463 Glycerol 1.480 Linseed oil

1.515 Immersion oilb 1.491 Xylene

1.480 Linseed oil 1.515 Immersion oilb

1.468 Olive oil 1.529 Clove oil

1.452 Paraffin oil (light) 1.535 Canada balsam

1.333 Water 1.54 Clarite

1.491 Xylene 1.57 Clarite X

aThe index of air is based on that of a vacuum as unity; it is 1.000294. The index of refraction of a substance other than air is usually based on air with an assumed index of unity. To convert an index based on air to an index referred to a vacuum as unity, multiply by 1.000294.

fcThe index of flint glass is 1.515. Hence the value of the majority of commercially available immersion oil is 1.515.

Source: Adapted from ref. 67.

to the accuracy of readings by the photosensing component of the system, may be in error by as much as 50% or more, simply because of the inaccuracy in readings below IOD values of 0.20 or above 0.80 (see Fig. 5). It is important to be aware of nonspecific light loss resulting from scatter and the relative photometric error inherent in measuring objects that are very pale or very densely stained. These issues also apply to microdensitometry by image analysis (12).

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