J. Cell Sci. a, 163-168 (1967) 163 Printed in Great Britain
THE LIGHT MICROSCOPE AS AN OPTICAL DIFFRACTOMETER
J. G. GALL
Department of Biology, Yale University, Netv Haven, Connecticut, U.S.A.
SUMMARY
The analysis of electron micrographs by optical diffraction was introduced recently by Klug
& Berger (1964). Their experiments were conducted with a special diffractometer designed for use with diffracting masks up to several inches in diameter. A method is described here for using a conventional light microscope as an optical diffractometer which can accept masks up to 5 mm in diameter. A 100-/4 electron-microscope aperture is used as a pinhole source of illumination, and the micrograph to be studied is introduced above the objective. The diffrac- tion pattern produced by the micrograph appears in the usual image plane of the microscope within the eyepiece.
The technique of optical diffraction is widely used in crystallography for the analysis of proposed molecular structures (Taylor & Lipson, 1964). Recently Klug & Berger (1964) and Finch, Klug & Stretton (1964) have shown that useful information can be obtained from conventional electron micrographs by subjecting them to optical diffraction. In practice the electron micrograph is placed in a beam of coherent, monochromatic light and the focused diffraction pattern is photographed. An analysis of the resulting spots permits deductions about periodic spacings in the original micrograph. In their study Klug and co-workers demonstrated diffraction patterns from virus particles, actin filaments, and catalase crystals. Morgan &
Uzman (1965) applied the technique to the analysis of ribosome-like aggregates in the Protozoan, Entamoeba.
An impediment to the widespread use of this interesting technique is the complexity of existing optical diffractometers (Hughes & Taylor, 1953; Taylor & Thompson, 1957; Wyckoff, Bear, Morgan & Carlstrom, 1957). These instruments were designed with the needs of X-ray crystallography in mind, and their construction would be impracticable for the average biological laboratory. Experiments with simpler designs led to the realization that a conventional light microscope will produce optical diffrac- tion patterns of high quality.* The only accessory needed is a 100-/4 platinum aperture from an electron microscope.
Fig. 1 shows the optical system diagrammatically. The microscope is used with all
• C. W. Bunn describes another way of using a microscope as an optical diffractometer in Chemical Crystallography (Oxford, 1945). In his system the pinhole was located several feet from the condenser, which consisted of a 50-mm objective screwed into the substage. The mask was placed between the source and the condenser. However, the potentiality of a microscope as a precision diffractometer has not been widely exploited.
lenses in their normal positions, and the diffraction pattern appears in the usual image plane within the eyepiece. The ioo-/i aperture is placed in or near the plane of the field diaphragm. Most research microscopes with built-in illuminators have a glass window in the base plate immediately above this diaphragm, on which the aperture can be placed. The field diaphragm is closed as far as possible, so that the pinhole becomes the sole source of illumination. The condenser forms a demagnified image of the pinhole in the specimen plane and a low-power objective is focused on this demagnified image. Looking into the eyepieces, therefore, one sees a black field with
Diffraction pattern
Diffraction mask
Objective
Condenser
Diaphragm
E.M. aperture - Field diaphragm
Fig. i. Diagram of the optical system of a light microscope, showing its use as an optical diffractometer.
a very small spot of light in the centre. The micrograph to be studied (the diffraction mask) can be placed at any convenient point in the convergent beam above the objec- tive. The Zeiss microscope which I use for this work has a plate which slides into the body tube about 50 mm above the objective. This plate has a large hole in it into which I have fitted a disc of aluminium. In the centre of the disc is a 6-mm circular hole over which the diffraction mask is taped. When the sliding plate is pushed into place the diffraction mask is automatically aligned. Exact alignment on the optic axis is not critical, since lateral movement of the mask does not alter the position of the diffrac- tion spots. The condenser diaphragm should be closed to illuminate an area slightly
Optical diffractometer 165 less than the full aperture of the objective. A short focal length telescope ('phase telescope') is useful in centering the mask and aperturing the objective. A beautiful demonstration of the system can be made by placing a 100-mesh electron-microscope grid above the objective; sixteen orders of diffraction are visible.
The production of suitable masks is the most critical part of the procedure (Taylor
& Lipson, 1964). These must be small, very sharp, and of high contrast. In addition they should contain only the area of interest, all else being blacked out. The following steps have proved convenient. The electron micrograph is printed in the normal manner on 8 x 10 in. photographic paper. A rectangular mask is constructed by taping 4 strips of white paper on to the micrograph so that they include only the area to be investigated. To provide reference axes for the object, opposite sides of the rectangle should be strictly parallel, and all corners 900. If the specimen has a clearly defined morphological axis, this should be oriented in a known fashion relative to the sides of the mask. The masked area is then photographed with a 35-mm camera from such a distance that the longest side of the rectangle will not exceed 5 mm on the negative.
It is convenient to lay a millimetre ruler near the mask, so that it will appear on the side of the negative for calibration. The 5-mm maximum is imposed by the micro- scopical system, since the cone of light leaving the objective has approximately this diameter. The relevant spacings should be between 25 and 500 /i on the negative.
Resolution of the photographic film becomes a problem below this range; if wider spacings are used, too few of them are included in a 5-mm mask, and the resulting diffraction spots are also too near the centre of the pattern. Good masks have been obtained from Kodak High Contrast Copy Film developed in Dektol.
The negative containing the diffraction mask is cut to a convenient size and taped in position on the sliding plate above the microscope objective (Fig. 1). It has not proved necessary to mount the mask in oil between optical flats, as recommended by Klug & Berger (1964). The diffraction pattern is produced in the usual image plane within the eyepiece, and is therefore observed as if it were a microscopic specimen.
Contrary to one's first expectation, spacings in the diffraction pattern are not altered by changing objectives. However, the size of the individual spots depends upon the objective magnification, since the spots are merely images of the pinhole. Conse- quently the separation of spots in the diffraction pattern is improved by using the lower-power objectives. The x 10 objective offers a good compromise between spot size and light-gathering power.
A standard 6-V tungsten bulb gives adequate illumination for many purposes. For photography with monochromatic light an interference filter with maximum trans- mission at 546 m/i is used, although visual observation of weak patterns is often more conveniently carried out in white light. Photographs have been recorded on Kodak Tri-X film developed in Diafine, giving a nominal ASA film speed of 2400. Exposure times will vary from several minutes to an hour or more with the tungsten bulb and interference filter.
Considerable improvement in both visualization and photography is afforded by a high-pressure mercury arc. The patterns shown in Figs. 2, 4 and 6 were made with an Osram HBO 200 source on the Zeiss Photomicroscope. The spots were readily
visible in monochromatic light, despite the use of a binocular head which reduces the image intensity. Exposures of I - I O min are possible with the built-in camera, which transmits only a fraction of the available light to the film. With a straight (monocular) microscope tube giving ioo % light transmission to the film, exposures of not more than 1-2 min are sufficient. Although a direct comparison of instruments has not been made, the microscope apparently gives a brighter image than the larger diffractometers.
Berger, Zobel & Engler (1966) state that even with a mercury arc visual observation of the patterns is difficult, and exposures of several hours duration may be necessary;
the pattern shown in Morgan & Uzman (1965) required 18 h exposure.
The overall performance of the system can be judged by examination of Fig. 2, which shows the diffraction pattern obtained from an electron micrograph of a catalase crystal. The original micrograph and its diffraction pattern, obtained on the large diffractometer at Manchester, were published as plate 1 in the article of Klug &
Berger (1964). To produce the pattern shown here Klug & Berger's micrograph was photographed from the journal and the resulting negative used in the microscope diffractometer. Berger et al. (1966) have recently published another diffraction picture of the same micrograph, using a gas laser for illumination. Their pattern is similar to that shown here, but was obtained with o-i sec exposure.
Another example is shown in Figs. 3 and 4, once again from the paper of Klug &
Berger (1964). Fig. 3 is a mask representing a helix with the parameters of the tobacco mosaic virus; Fig. 4 is the diffraction pattern of this mask given by the microscope diffractometer.
A final example is shown in Figs. 5 and 6. In this case an electron micrograph of a negatively stained flagellar doublet was used as the diffraction mask (Fig. 5). The diffraction pattern (Fig. 6) shows two pairs of spots near the meridian, one more intense than the other. These presumably result from a helical arrangement of sub- units in the original fibre. Transverse striations can be seen in the micrograph which correspond to the more intense pair of spots; their spacing, deduced from the diffrac- tion pattern, is 40 A. The weaker pair of spots probably corresponds to striations on the side of the particle not so deeply embedded in the stain (Finch et al. 1964), but these striations cannot be seen clearly in the original micrograph. A detailed analysis of flagellar doublet fibres has recently been published by Grimstone & Klug (1966).
Their figure 17 shows a diffraction pattern very similar to that reproduced here.
Supported by a U.S. Public Health Service Research Grant (GM 12427) from the National Institute of General Medical Sciences. The technical assistance of Mrs Diane Dexter is gratefully acknowledged.
REFERENCES
BERGER, J. E., ZOBEL, C. R. & ENGLER, P. E. (1966). Laser as light source for optical diffracto- meters; Fourier analysis of electron micrographs. Science, N.Y. 153, 168-170.
FINCH, J. T., KLUG, A. & STRETTON, A. O. W. (1964). The structure of the 'polyheads' of T4 bacteriophage. J. molec. Biol. 10, 570-575.
GRIMSTONE, A. V. & KLUG, A. (1966). Observations on the substructure of flagellar fibres.
J. CeUSci. 1, 351-362.
Optical diffractometer 167
HUGHES, W. & TAYLOR, C. A. (1953). Apparatus used in the development of optical-diffraction methods for the solution of problems in X-ray analysis. J. scient. Instrum. 30, 105—no.
KLUG, A. & BERGER, J. W. (1964). An optical method for the analysis of periodicities in electron micrographs, with some observations on the mechanism of negative staining. J. molec. Biol.
io, 565-569-
MORGAN, R. S. & UZMAN, B. G. (1965). Nature of the packing of ribosomes within chromatoid bodies. Science, N.Y. 152, 214-216.
TAYLOR, C. A. & LIPSON, H. (1964). Optical Transforms. London: Bell.
TAYLOR, C. A. & THOMPSON, B. J. (1957). Some improvements in the operation of the optical diffractometer. J. tcient. Instrum. 34, 439-447.
WYCKOFF, H. W., BEAR, R. S., MORGAN, R. S. & CARLSTROM, D. (1957). Optical diffractometer for facilitation of X-ray diffraction studies of macromolecular structures. J. opt. Soc. Am. 47,
1061-1069.
(Received 30 December 1966)
Fig. 2. Optical diffraction pattern obtained from an electron micrograph of a catalase crystal. The original micrograph was published as plate i in the article of Klug &
Berger (1964). Scale on this and other diffraction patterns reproduced here: 1 mm corresponds to 0^4 mm"1 (reciprocal millimetres). Exposure: 1 min.
Fig. 3. An enlarged negative of a mask representing a helix with the parameters of the tobacco mosaic virus.
Fig. 4. Diffraction pattern of this helix; 10 min exposure.
Fig. 5. Electron micrograph of a short segment of a flagellar doublet fibre, negatively stained with phosphotungstate. x 160000.
Fig. 6. Diffraction pattern of the same doublet showing two spots on the 40 A-layer line; 5 min exposure.
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J. G. GALL (Facing p. 168)
