The Microscope

Four microscope settings are useful in crystal analysis and in the training process: ordinary microscopy, simple polarized microscopy, compensated polarized microscopy ( Fig. 2-1 , A and B ), and phase microscopy. These four settings are usually achieved with the same microscope and different filters, except phase microscopy, which requires specific lenses and condenser. The basic laboratory microscope of most brands can adapt polarized filters for simple polarized microscopy, and many can also incorporate a first-order compensator (also known as retardation plate or lambda [λ] plate) to allow for compensated light microscopy. Many microscopes can also be fitted with phase lenses and condenser; phase microscopy is good to have, especially at the learning stage, but it is not necessary for crystal analysis.

A, Compensated polarized light microscope, set up in the standard manner, with two crossed polarized light filters (termed the “polarizer” and the “analyzer”) and a first-order red plate compensator filter. B, Compensated polarized light microscopy used to detect and identify synovial fluid crystals. The crystals, interposed in a darkfield of crossed polarizing filters, bend light into slow and fast components. The figure illustrates that the slow vibration of light by the crystal allows the crystal to be detected in the darkfield, and this phenomenon is termed “birefringence.” Comparison with the axis of slow vibration of polarized light by a compensator filter (see A ), typically using a first-order red plate compensator, allows definition of whether the crystal is negatively or positively birefringent.

(Redrawn from American College of Rheumatology Image Bank © 2009. A , Available at http://images.rheumatology.org/viewphoto.php?imageId=2861705 . B, Available at http://images.rheumatology.org/viewphoto.php?imageId=2861704 .)

Before starting training on crystal analysis, it is necessary to become acquainted with the use of the ordinary light microscope. The initial tool for crystal analysis was the geological polarized microscope, fitted with a rotating stage. It allows measurement of the angle of extinction of the birefringence (see later) and allows orientation of the crystals in relation to the axis of the compensator through rotation of the stage. These measurements are especially useful when there is a need to distinguish among a number of different crystal types, as a geologist may need to do, but in the joint we are essentially concerned with MSU or CPPD crystals, from which artifacts or the sporadic previously injected corticosteroid crystals are readily distinguished. In this context, corticosteroid crystals such as those of triamcinolone often have bright positive birefringence rather than the weak positive birefringence of CPPD. We believe that an ordinary microscope fitted with the appropriate lenses and filters is an adequate tool for crystal analysis.

To better understand our observations, the basic tool is described—the ordinary brightfield microscope—and then built-in complexity.

• 1.
  

  Brightfield microscopy: This is the illumination of conventional microscopes. Because SF preparations are fresh and unstained, detection of crystals with this illumination is based solely on shape. Cells and crystals are essentially transparent, so the height of the microscope condenser and aperture of the diaphragm must be regulated to obtain the best detail and contrast of the SF elements. Crystals are seen as regular structures with sharp edges, which can be found both inside and outside cells.

MSU crystals are always seen as thin needles of different sizes, intracellular or extracellular, and occasionally forming groups (with spherulites of MSU an infrequent finding). MSU crystals observed in SF from inflamed or uninflamed joints are smaller than those obtained by needling a tophus, which can be very large in comparison ( Fig. 2-2 ).

Monosodium urate (MSU) crystals. A–C, From synovial fluid. Brightfield microscopy, ×400. D, From synovial fluid. Brightfield microscopy, ×600. E, From an aspirate of a tophus. Brightfield microscopy, ×400. F–H, From synovial fluid. Same microscope fields as A–C, simple polarized microscopy, ×400. I, From synovial fluid. Same microscope fields as D, simple polarized microscopy, ×600. J, From an aspirate of a tophus. Same microscope field as E, simple polarized microscopy, ×400. K, From an aspirate of a tophus. Simple polarized microscopy, ×400. MSU crystals from tophus aspirates can be very large. L, From synovial fluid. Same microscope field as D and I. Compensated polarized microscopy, ×400. M, From synovial fluid. Compensated polarized microscopy, ×400. N, O, From an aspirate of a tophus. Same microscope fields as E, J, and K. Compensated polarized microscopy, ×400. P, From synovial fluid. Compensated phase microscopy ×1000. These figures show that brightfield allows very close detection and identification of MSU crystals by shape and that simple polarized microscopy shows them well by their bright shine. Compensated polarized microscopy remains the standard for crystal identification. Compensated phase microscopy shows well the acicular MSU crystals but do not add to detection or identification.

The polymorphic CPPD crystals may pose more difficulties. Observed under brightfield microscopy, typical crystals are rhombi or parallelepipeds. Some very thin and long parallelepipeds can look like rods or even needles; these are the only CPPD crystals that can be mistaken for MSU crystals. Often, CPPD crystals show a less regular shape, likely because of their position (it is a learning experience to observe typical crystals in a fresh preparation while the cells are still moving and watch the apparent shape of the crystal change with cell movement). In addition, some crystals may have broken off of larger crystals and therefore have a more irregular shape. Very small intracellular CPPD crystals are common and are seen as distinct inclusions that may have one or more linear borders or angles; tiny needles are also common. These smaller crystals are much better seen under a higher magnification with the ×1000 oil lens. The ×600 or ×630 lens (a good magnification level for crystal analysis) also shows them well. CPPD crystals are quite frequently seen inside a vacuole, while this appears not to be the case with MSU crystals ( Figs. 2-3, 2-4, A–C , 2-5, A , and 2-6, A ). The particularly inflammatory osmotic and membranolytic effects of MSU crystals discussed in Chapter 5 may account for this phenomenon.

Calcium pyrophosphate dihydrate (CPPD) identification by shape with a brightfield microscope. A–D, CPPD crystals intracellularly and extracellularly, ×400. E, Two needle-shaped CPPD crystals (intracellular and extracellular) ( left ); the shape of the crystals ( right ) is important clue for identification; absence of birefringence or faint one, another. Compensated polarized microscopy helps to determine the coincidence of MSU and CPPD crystals, ×600. F, Rhomboidal crystal at ×600. G, H, CPPD at ×1000. Smaller crystals are also seen. These figures show that brightfield allows very close detection and identification of CPPD crystals by shape: ×400 allows good distinction of the crystals, especially the large ones, but ×600 and ×1000 in particular show much better detail and are important means of observation, allowing beginners to become closely acquainted with the different shapes and sizes of CPPD crystals.

Calcium pyrophosphate dihydrate (CPPD) by brightfield and simple polarized microscopy. A1 and A2, CPPD crystals seen by brightfield microscopy (A1, ×400; A2, ×600). B1 and B2, Same microscope fields by simple polarized microscopy: the crystals do not show birefringence—an out-of-focus crystal on E , top right, does. C, Abundant CPPD crystals brightfield ×400. D, Same microscope field seen by simple polarized microscopy—and showing that only a minority of crystals show any birefringence. These figures illustrate that CPPD crystals often do not show birefringence under simple polarized light. For beginners it is convenient to carefully observe the lesser to very faint to absent birefringence of CPPD crystals and become acquainted with it.

Calcium pyrophosphate dihydrate (CPPD) compensated polarized microscope. A, Rhomboidal CPPD crystal seen by brightfield microscopy, ×1000. B, Same as A by simple polarized microscopy (×1000) showing faint birefringence (taken in reference to the birefringence shown by monosodium urate crystals). C, Same as A by compensated polarized microscopy, ×1000. The shape of the crystal does not allow determination of its orientation in relation to the compensator axis (λ marked by an arrow ). D, Parallelepipedic CPPD crystal by compensated polarized microscopy, ×1000. The long axis of the crystal lies parallel to the axis of the compensator (λ marked by an arrow ); the blue color shown by the crystal indicates positive elongation (also referred as birefringence) characteristic of CPPD crystals. These figures show the positive birefringence shown by CPPD crystals. Only parallelepipedic and rod-shaped crystals can be oriented in relation to the compensator axis.

Calcium pyrophosphate dihydrate (CPPD) as seen with phase contrast microscopy. A1, Brightfield microscopy, ×1000. Several rod-shaped CPPD crystals. A2, Phase contrast microscopy, ×1000. Same microscope field. B1, Brightfield microscopy, ×1000. A single small intracellular CPPD crystal in a vacuole. B2, Phase contrast microscopy, ×1000. Same microscope field. The crystal and vacuole are now more clearly distinguished. C, Simple polarized microscopy, ×1000. Only one parallelepipedic CPPD crystal shows clear birefringence. D, Compensated polarized microscopy, ×1000. Same field as C . Only two crystals (showing + elongation or birefringence—yellow when perpendicular to the compensator axis [λ] and blue if parallel) can be identified as CPPD crystals. E, Phase contrast microscopy. Same field as C and D. A larger number of crystals are now clearly seen. F, Compensated polarized microscopy, ×1000. Only two crystals can be identified as CPPD by their birefringence (+ elongation; yellow when parallel to the compensator axis [λ]). G, Phase contrast microscopy, ×1000. Same microscope field as F . More crystals are now distinguished. H–K , Phase contrast microscopy, ×1000. Several images showing the capability of phase contrast microscopy to distinguish CPPD crystals by shape, even if small. All these figures show the clear vision obtained by phase contrast microscopy, ×1000. It may help to identify small crystals, but its advantages for routine synovial fluid analysis remain unproved. Our practice is to look for CPPD crystals by brightfield microscopy, and for long rod-shaped crystals, to check (absence or faint birefringence) under simple polarized microscopy. Compensated polarized microscopy remains useful for occasional doubtful crystals.

The morphologic identification under ordinary light of CPPD crystals should be done only when enough typical crystals are seen; the finding of very small or less “typical”-looking crystals should prompt the observer to keep searching until he or she can identify an unquestionable crystal. In a small trial, both types of crystals were reasonably well distinguished with brightfield microscopy by their shape—63 SF samples (13 from patients with gout; 14, CPPD arthopathy; 1, both MSU and CPPD crystals; 35, different noncrystalline inflammatory arthritides) were blindly examined with brightfield microscopy by two different observers. The observers detected the presence of crystals in 100% (observer 1) and 97% (observer 2, who was a resident) of the samples; the crystals were properly identified with the brightfield microscope (in relation with the compensated polarized microscope) as MSU or CPPD in 92% (observer 1) and 87% (observer 2) of the occasions. In the sample containing MSU and CPPD crystals, both observers perceived the presence of both crystals.

• 2.
  

  Simple polarized light microscopy: Simple polarized light can identify crystals due to the intensity of their birefringence; it is often used to identify foreign material in pathologic specimens. Simple polarized microscopy is obtained when two polarized filters are added to the brightfield microscope. One of the filters is placed between the light source and the microscope slide (polarizer), while the second is placed above the lens (analyzer). A polarized filter is one that allows only the light vibrating in a plane parallel to its axis to pass through. In essence, you can imagine a polarized filter as a comb whose teeth are very closely packed and only the light vibrating parallel to its teeth is allowed to pass through. When the axes of the two polarized filters are placed perpendicular to each other, the light that passed through the polarizer is retained by the analyzer and the observer sees a dark microscope field (also known as crossed polarization). When the axes of the two filters are parallel to each other, observations can be made as with ordinary light. In an optic microscope, one of the filters is movable, allowing a change between brightfield and simple polarized light. The mechanism for crossing the filters is different depending on the microscope.

When a birefringent crystal is placed on the stage, the already polarized light beam that reaches it is split into two perpendicular components, none parallel to the incident ray, and with different velocities (fast ray and slow ray) and therefore out of phase with each other. The result is elliptically polarized light; the portion of this light vibrating parallel to the axis of the analyzer passes through it as plane polarized light, producing an image of a white crystal on a darkfield. In short, birefringent crystals seem to shine on a dark background because the microscope light can be seen through the crystal. By partially uncrossing one of the filters, less polarization is obtained; this allows the viewer to simultaneously distinguish the SF elements and the birefringence of crystals, which can occasionally be useful, especially for photography.

When seen under simple polarized light, MSU crystals shine brightly over the dark background. Brilliance, however, also depends on the intensity of the light source of the microscope—showing higher luminosity with the microscopes fitted with a stronger light source such as halogen lighting—and the height of the microscope condenser, which should be graduated to obtain the best image. Occasional MSU crystals may show no birefringence; this depends on crystal mass (very small crystals may be anisotropic) but also on the position of the axes of the crystal in relation with the axis of either the polarizer or the analyzer filters. When the crystal axis is positioned parallel to the axes of crossed polarized filters, birefringence disappears and the crystal is said to be in a position of extinction. On occasion, a crystal moving in the microscope field can fall in and out of the position of extinction and therefore show oscillating birefringence. Spherical aggregates of MSU crystals, all radiating from a center, have been reported, and incomplete radiating appearances are occasionally seen (an irregular radiating structure seen in a tophus is shown in Fig. 2-6 D–F ). After the viewer gains some experience, the bright MSU crystals are usually well detected at a magnification of ×200 with simple polarized microscopy. A better image is obtained at ×400; when searching for MSU crystals, there is rarely a need to use higher magnifications (see Fig. 2-2 , F–K ).

In the early descriptions of CPPD crystals, it was noted that under the simple polarized microscope they show only a weak birefringence, which is often absent. In a more recent trial, 10 SF samples from patients with CPPD crystal–proved acute arthritis were examined separately by two observers. First, a brightfield microscope was used; then, the same area was examined with a simple polarized microscope. Crystals showing any birefringence were annotated. Both observers found that less that 20% of CPPD crystals showed any birefringence. Ten SF samples from patients with crystal-proved gouty attacks were also examined; examination under simple polarized microscopy showed more crystals than observation by brightfield, indicating that some MSU crystals had passed unseen under the brightlight. Therefore, for CPPD, careful observation under brightfield appears to be the best method for detecting the crystals; when MSU is the suspected crystal, observation under simple polarized light is clearly better. It follows that before deciding that an SF sample has no crystals, it should be examined under both brightfield and simple polarized microscopy (see Figs. 2-4, D–F , 2-5, B , and 2-6, C ).

• 3.
  

  Compensated polarized microscopy: This method remains the current standard for crystal identification; it adds to the previously discussed system a first-order red compensator (λ or retardation plate), which helps to determine the amount of retardation in the wavelength of the compound ray emerging from the long dimension of the birefringent crystal. When this ray has a raised wavelength, it is viewed as yellow when parallel to the compensator axis (usually marked by a λ and an arrow) and as blue if perpendicular and the crystal is said to have negative birefringence or elongation. When the vibration of the slow ray is parallel to the long dimension of the crystal, it has a positive birefringence or elongation. Compensated polarized microscopy helps in the distinction between MSU and CPPD crystals, because the crystals have different birefringence: in MSU, it is negative (and bright) birefringence (see Fig. 2-2 , L and M ), while in CPPD, it is positive (and weak) birefringence (see Figs. 2-5, C–E , and 2-6, D and F ). The axis of the compensator can also be determined by keeping reference MSU crystals (a dried imprint of material needled from a tophi provides abundant MSU crystals for reference). Although it remains the standard tool for definitive crystal distinction, the crystal nature is already evident in most occasions based on the shape and intensity of birefringence under simple polarized light. Compensated polarized microscopy is most useful in the identification of very occasional needlelike artefacts or when MSU and CPPD crystals coincide in the same SF; this occurrence appears sporadic and no report on its clinical consequences has been published. A recent report examining SF from a patient with gout after cytocentrifugation also found a few CPPD crystals in patients with associated moderate to severe osteoarthritis, and possibly related to the osteoarthritis.

  
  
• 4.
  

  Phase contrast microscopy: This is a contrast-enhancing technique that produces high-contrast images of transparent specimens using ordinary light. When light waves that passed through the phase condenser pass through the preparation, some of its elements diffract and retard the phase (i.e., retard the wavelength). These retarded emerging waves are invisible to the naked eye but are transformed by the phase plate in the lens into amplitude differences observable at the oculars. Objects that modify the light phase—as do crystals—are seen lighter (see Fig. 2-6 , A2, B2, E, and G–K ). Phase microscopy was recommended by McCarty and shows the crystal’s shape particularly well; if available, phase microscopy and observation at ×1000 can be very good teaching instruments to familiarize a beginner with the different crystal shapes, particularly with CPPD crystals, including the very small intracellular ones. On occasion, it can also be useful for the expert analyst.

  
  

  An ordinary microscope can be polarized by fitting it with polarized filters (obtainable online from commercial vendors) and cellophane tape can be used as a makeshift compensator. These are improvised polarized and compensated polarized microscopes, although the image quality may be inferior. Polarized filters can be cut by opticians from a large photographic polarized filter and adapted to the filter lodges of the microscope. Two filters are necessary—one under the stage, in the usual filter lodge of the microscope, and the other over it (most microscopes have a filter lodge in the inside of the piece that holds the lenses).