Urine Sediment of the Month: Oblique Illumination in Microscopy

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Urinary sediment microscopy is a diagnostic technique designed to acquire 2-dimensional images of structures present in a slide. However, 2-dimensional views may lack sufficient contrast to allow for optimal visualization. One way to enhance the images and create a 3-dimensional view is by applying oblique illumination.

By allowing the light to project onto the field through only 1 side of the condenser, structures cast a shadow and create a 3-D-like image. Standard microscopes do not routinely come from the manufacturer set up to provide this type of illumination unless they get customized. However, oblique illumination can be easily achieved by taking advantage of the artifactual effect of partially rotating the condenser diaphragm turret between 2 fixed settings.

Figure 1. Condenser turret illustrating settings at (A) bright field (BF) and (B) dark field (DF). By rotating the turret ¾ of the way between BF and DF (C), oblique illumination can be achieved.

The 2 approaches that offer the best balance of light and contrast to generate a “relief” or “satellite view” effect are by rotating the condenser turret between bright field and dark field illumination (Figure 1) or between phase contrast and bright field illumination settings.

Figure 2. Bilirubin-stained renal tubular epithelial cell casts (RTECC) under bright field microscopy (A and C). The cast matrix is better visualized under oblique illumination (B and D) Images at 400x magnification.
Figure 3. White blood cell cast under bright (A) and dark field (B) illumination (Sternheimer-Malbin stain). While the cast matrix is noticeable along half of the cast (right side of the images), it is difficult to visualize its full length. Oblique illumination (C) allows better distinction of the cast matrix. In addition, other hyaline casts (some containing cells) become more apparent. Images obtained at 400x magnification.

Oblique illumination can be particularly helpful to visualize a cast matrix that may not be very visible on bright or dark field illumination, particularly when phase contrast microscopy is not available (Figures 2-4).

Figure 4. Oblique illumination accentuates (A) the granularity of coarse granular casts. Yeast (pseudo-hyphae) can be more noticeable; (B) nuclei from renal tubular epithelial cells (RTEC) can be nicely outlined; texture of broad (C) cracked and (D) smooth waxy casts becomes more visible.

It also allows for crisp visualization of cell nuclei and thus better characterization of the type of cells or cells free-floating in the urine sediment or embedded within casts.

Figure 5. Dysmorphic erythrocyturia, scattered acanthocytes and leukocytes from a patient with crescentic glomerulonephritis (A). Crenated erythrocytes from a urine specimen contaminated due to metrorrhagia (B). Squamous epithelial cells are also seen in the upper side of the image. As seen in both panels, red blood cell morphology can be clearly appreciated under oblique illumination.

The characteristic dysmorphism of acanthocytes is also nicely revealed by oblique illumination (Figure 5).

Post by Juan Carlos Velez

Edited by Anna Gaddy

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