Compensated Polarized Light Microscopy Using Cellophane Adhesive Tape

Transcription

1 Compensated Polarized Light Microscopy Using Cellophane Adhesive Tape Terrence J. Fagan and Martin D. Lidsky Compensated polarized light microscopy is a useful diagnostic tool whose basis is poorly understood by the clinician. The elementary physics of compensated polarized light are reviewed. A simple method of converting the ordinary light microscope into a compensated polarized light microscope, using cellophane adhesive tape and polarizing sunglass, is described. In 1961 McCarty and Hollander described the use of polarized light microscopy in the identification of monosodium urate crystals in gouty synovial fluid (1). The use of the polarizing microscope led to the discovery of a second type of crystal, calcium pyrophosphate dihydrate (23) in joint fluids from patients with pseudogout. The negatively birefringent urate crystal was shown to be readily distinguished from the positively birefringent calcium pyrophosphate crystal with the use of compensated polarized light (4). Thus the polarizing microscope became established as a vital diagnostic tool in rheumatology. The more widespread use of compensated polarizing microscopy has been hampered by lack of understanding of the physics involved and the notion that complex equipment is required. This paper reviews From the Section of Rheumatology, Department of Medicine, Veterans Administration Hospital, and Baylor College of Medicine, Houston, Texas. TERRENCE J FAGAN, MD: Department of Medicine, Veterans Administration Hospital, Houston, Texas: MARTIN D LIDSKY: Baylor College of Medicine, Houston, Texas. Reprint requests should be addressed to: Dr Martin D Lidsky, 2002 Holcombe Boulevard, Houston, Texas Submitted for publication July 18, 1972: accepted December 4, the elementary physics of compensated polarized light and describes the use of easily available, inexpensive materials which can convert the ordinary light microscope into a compensated polarizing microscope of sufficient quality to provide accurate recognition of joint fluid crystals and other substances encountered in medicine. A similar conversion was developed independently and described in a letter to an editor (5). THE PHYSICAL BASIS OF POLARIZED LIGHT (6) Ordinary light is a random collection of electromagnetic waves. Each wave is characterized by an electric field vector which lies in a plane perpendicular to the propagation of the wave (Figure 1). The magnitude of the electric vector varies with time and oscillates from positive to negative with a frequency which is constant regardless of the medium through which the wave travels. The velocity (V) of propagation of the wave is equal to the frequency multiplied by the wavelength (A). Since the frequency of any given wave is constant, any change in the velocity must be accompanied by a change in wavelength. Velocity of light is also related to the 256 Arthritis and Rheumatism, Vol. 17, No. 3 (MayJune 1974)

2 CELLOPHANE AND POLARIZED LIGHT Fig 1. Diagrammatic representation of plane polarized light wave. index of refraction of the medium (n) through which light travels and is ex- C pressed as V =-when C is the velocity n of light in a vacuum. A polarizing lens has a fixed axis of transmission. Ideally, only waves with electric vectors parallel to the axis of transmission will pass through the polarizing lens unattenuated. Waves with electric vectors perpendicular to the axis will not be transmitted. When light with an electric vector between these two extremes is incident on a polarizing lens, only the component of the vector parallel to the axis of transmission will pass through the lens (Figure 2). Light passing through the polarizing lens is termed plane polarized. When two polarizing lens are crossed so that their axes of transmission are perpendicular to each other, light which is plane polarized by the first lens (polarizer) will not be transmitted by the second lens (analyzer) and hence, the background will appear dark. BIREFRINGENCE (6) The speed of light in ordinary materials is independent of electric vectors. In birefringent materials, the velocity of light is related to the direction of electric vectors and optic axes. Birefringent materials are characterized by having one or more axes of preferential direction called optic axes. This discussion deals only with uniaxia2 birefringent material. Consider a flat plate of such material cut parallel to the optic axis (Figure 3). Light waves traveling parallel to the optic axis possess the same velocity regardless of the direction of electric vectors. A wave striking the plate (and optic axis) perpendicularly and having an electric vector at 45O to the optic axis, is split into two separate waves traveling in the same direction with different velocities and having electric vectors which are mutually perpendicular. The wave whose electric vector is parallel to the optic axis is termed the extraordinary (e) wave and the wave travels C through the plate with velocity of V, =- ne The wave whose electric vector is perpendicular to the optic axis is termed the ordinary (0) wave and has a velocity of v, =-. Birefringence (B) is a quantity n0 expressed by n, - no. For positively birefringent material the difference is positive. If n, < no the material is negatively bire- Arthritis and Rheumatism, Vol. 17, No. 3 (MayJune 1974) 257

3 FAGAN AND LIDSKY Axis of Transmission light Wave Fig 2. Production of plane polarized light wave. fringent. The optic axis of positively birefringent material is often called the slow axis because V, < V,. COMBINED USE OF POLARIZED LIGHT AND BIREFRINGENCE The extraordinary and ordinary waves enter the birefringent material in phase with each other. However, because they have identical frequencies and different velocities, the extraordinary and ordinary waves leave the material with a phase difference. By considering the time necessary for each wave to pass through the birefringent plate, it can be shown that the phase difference (6) (Q) is expressed by the relation, cp = 560' Bt where B = birefring- x ence and t = thickness of the plate. In gen- eral, the emerging extraordinary and ordinary waves recombine when they are out of phase, thus resulting in an elliptically polarized wave whose electric vector is no longer parallel to a single plane. The human eye detects frequency (color) and intensity (brightness) but cannot detect polarization. Since frequency and intensity are not altered by passage through birefringent material, the eye perceives no change. The result differs when the birefringent material is interposed between two crossed polarizing lenses. Since the wave emerging from the birefringent material is not plane polarized, the wave cannot be totally blocked by the second polarizing lens. In this situation, the intensity of transmitted light is altered depending on the phase difference. The intensity of light transmitted through the second polarizing lens 258 Arthritis and Rheumatism, Vol. 17, No. 3 (May-June 1974)

4 CELLOPHANE AND POLARIZED LIGHT of Fig 3. Behavior of plane polarized light wave perpendicularly striking a uniaxial birefringent plate. The electric vector of the incident wave is at 45" to the optic axis. E represents the electric vector of the extraordinary wave. 0 is the electric vector of the ordinary wave. Thickness of plate is represented by t. (analyzer) is expressed as the percent transmission (% T) (7), yo T = sin? - = sin' 180' Bt 2 h For a plate of birefringent material with a Bt product of 565 nm, the percent transmission of the different frequencies of the visible spectrum ( nm) is shown in Figure 4. If white plane polarized light enters a birefringent plate (Bt = 565 nm) and then passes through a second crossed polarizing lens, the emerging light will appear redviolet because of the attenuation of the frequencies around 565 nm. Such a birefringent plate is called a first-order red plate. When the Bt product is different from 565 nm, a different transmission curve is obtained and a different emerging color is observed (Figure 5). COMPENSATED POLARIZED LIGHT Compensated polarized light microscopy involves the use of two polarizing lenses and a birefringent plate. One polarizing lens (polarizer) is placed between the source light and specimen. A second polarizing lens (analyzer) is placed between the specimen and the examiner's eye. When the axes of transmission of the two polarizers are perpendicular to each other, the background appears dark. Any birefringent specimen placed in the microscopic field will appear bright against the dark back- Arthritis and Rheumatism, Vol. 17, No. 3 (MayJune 1974) 259

5 FAGAN AND LIDSKY ( Vief he Green Ydbw 0r-e Red [m] Fig 4. Relation between intensity of light (percent transmission) and wavelength when a first-order red birefringent plate (Bt product = 565 nrn) is interposed between two crossed polarizing lenses. ground. The addition of a positively birefringent plate with a Bt product of 565 nm (first-order red plate) in a slot below the analyzer in the barrel of the microscope produces a change in the color of the background from black to first-order red. The position of the slot insures the optic axis of the birefringent plate to be at 45O to the axis of both polarizing lenses. IDENTIFICATION OF CRYSTALS IN SYNOVIAL FLUID WITH COMPENSATED POLARIZED LIGHT It is generally assumed that the optic axes of crystals are parallel to the long axes. Hence, if a crystal lies parallel to the optic axis of the first-order red plate (the direction of the optic axis is indicated on the plate) and appears blue on the red background, the crystal is positively birefringent (4). The Bt product of such a crystal is added to that of the first-order red compensating plate to give a color consistent B t PRODUCT (nrn] I I ST ORDER I 2 ND ORDER Fig 5. Emerging colors produced by birefringent plates with different Bt products when placed between two crossed polarizing lenses. with a larger Bt product. It should be noted that there is no blue color to the left of the first-order red color in Figure 5. If the positively birefringent crystal lies in a direction in which its optic axis is perpendicular to the optic axis of the red plate, the crystal appears yellow because, in essence, its Bt product is subtracted from that of the plate. It is important to note (Figure 5) that the two colors (blue and yellow) are symmetrically located about the first-order red background. Thus the behavior of the positively birefringent calcium pyrophosphate crystal is described. A negatively birefringent crystal aligned parallel to the axis of the first-order red plate behaves in a manner opposite to that of the positively birefringent crystal; hence the negatively birefringent crystal is yellow (4). At right angle to the optic axis of the plate the negatively birefringent crystal ap pears blue. Thus the behavior of the nega- 280 Arthritis and Rheumatism, Vol. 17, No. 3 (MayJune 1974)

6 CELLOPHANE AND POLARIZED LIGHT tively birefringent sodium urate crystal is described. It should be evident from Figure 5 that if the compensating plate is first-order orange, the crystals might be yellow and violet; if the plate is blue, the crystals might be red and green. CELLOPHANE FIRST-ORDER RED PLATE It is not important whether the birefringent compensator is placed inside the barrel or directly over the polarizing lens (polarizer) covering the light source. However the compensator must reside somewhere between the two polarizing lenses with its optic axis at 45" to both polarizing lenses. Most compensators are made of quartz, gypsum, or mica. Any birefringent substance with a fixed thickness and Bt product around 565 nm can be used. It is generally known that cellophane is positively birefringent. One thickness of ordinary cellophane adhesive tape has a Bt product of about 250 to 350 nm. A compensating plate is made by placing two thicknesses of tape, one piece directly on top of the other, on a glass microscope slide. 'The optic axis of this compensator is parallel to the streak lines in the cellophane. Some experimentation with different tapes may be necessary to obtain the red background color, but as a rule it takes two thicknesses. The cellophane compensator is placed directly over the polarizer covering the microscope's source light. An alternative method of making a compensator is to tape two thicknesses of cellophane adhesive tape onto the plarizer covering the source light. The optic axis (streak lines) is positioned at 45" to the axis of transmission of the polarizer. With the other polarizing lens in the barrel of the microscope, one can view the back- Fig 6. Appearance of a cholesterol-containing fat droplet under compensated polarized light. ground in black and white when the taped side of the polarizer faces the microscope light (the compensator is not between the polarizing lenses) and in color by simply turning over the polarizer so that the tape lies between the two polarizing lenses. CLINICAL USE The polarizing lenses of sunglasses have a transmission axis which is vertical as one wears them since most reflected glare is horizontally polarized. These lenses are inexpensive and can be used effectively by cutting them to fit into the nosepiece or barrel of most ordinary light microscopes. Birefringent substances other than synovial fluid crystals are encountered in clinical work, for example collagen and amyloid fibrils in tissue, cystine crystals in urine, cholesterol crystals in body fluids and cholesterol-containing fat droplets in urine. The characteristic maltese crosses of the latter are bicolored yellow and blue under compensated light (Figure 6). The described simple method of making compensated polarized light microscopy will make readily available to medical students, house staff, and practicing physicians Arthritis and Rheumatism, Vol. 17, No. 3 (MayJune 1974) 261

7 FAGAN AND LIDSKY a useful diagnostic tool which heretofore has been little used because of lack of understanding and equipment. REFERENCES 1. McCarty DJ, Hollander JL: The identification of urate crystals in gouty synovial fluid. Ann Intern Med 54:452460, McCarty DJ, Kohn WN, Faires JS: The significance of calcium phosphate crystals in synovial fluid of arthritic patients: the pseudogout syndrome. Ann Intern Med 56: , Kohn NN, Hughes RE, McCarty DJ, et al: The significance of calcium phosphate crys- tals in the synovial fluid of arthritic patients: the pseudogout syndrome. 4nn Intern Med 56: , Phelps PS, Steele AD, McCarty DJ: Compensated polarized light microscopy. JAMA 203: , Owen DS, Jr: Letter to editor: A cheap and useful compensated polarizing microscope. N Engl J Med 285:1152, Sears FW, Zemansky MW: University Physics. Second edition. Massachusetts, Addison- Wesley, 1955, pp Shubnikov AV: Principles of optical crystallography. New York, Consultants Bureau, 1960, p Arthritis and Rheumatism, Vol. 17, No. 3 (MayJune 1974)