Aug 9, 1976 - background light in the chamber, and a full parabolic mirror is fitted to maximize light collection. ... shot) noise level read from the signal meter. A polished .... (arrow, Fig. 4) rather .... specimen, whether air dried or frozen dried.
&Journal of Microscopy, Vol. 108, Pt 2, Noaember 1976, pp. 177-184. Re&ed paper accepted 9 August 1976
Improved resolution in cathodoluminescent microscopy of biological material
by G. H. HAGGIS, E. F. B O N Dand R. G . FULCHER*, Chemistry and Biology Research Institute, Canada Department of Agriculture, Ottawa and *Department of Biology, Carleton University, Ottawa, Canada SUMMARY
This paper continues work reported in an earlier paper? on modification of a Cambridge Stereoscan Mk IIA to improve the quality of cathodoluminescent micrographs of biological material. In the work presently described the microscope gun has been offset laterally by 2 mm, to prevent light from the filament passing down through the column to the specimen chamber. The electron beam is brought onto the column axis by deflection coils. This modification effectively eliminates background light in the chamber, and a full parabolic mirror is fitted to maximize light collection. Results for yeasts and wheat seed sections are described. INTRODUCTION
In an earlier paper (Bond et al., 1974) we showed that for study of the relatively weak cathodoluminescence (CL) of biological materials it was essential to collect light emitted from the sample from a wide solid angle. We used a half parabolic mirror to increase light collection efficiencyover that of the standard CL attachment supplied by Cambridge Scientific Instruments Ltd for the Stereoscan Mk IIA scanning electron microscope and were able to obtain CL images of autoluminescent plant material at magnifications up to 400 diameters (resolution 0.5 pm). Horl & Miigschl(l972) had described an elliptical mirror which achieved the same purpose of efficient light collection, and recently Broecker et al. (1975) have used a mirror of this type to show the distribution of fibrin in kidney sections stained with fluorescein-labelled fibrin antibody. They also demonstrate resolution 0.5 pm, sufficient to give a useful magnification of about 400 diameters. One aim in CL research is to obtain better resolution than is possible with U.V. or visible light fluorescence microscopy, that is, a useful magnification of greater than 1000 diameters, and resolution -0.1 pm. We showed in the earlier paper (Bond et al., 1974) that when an efficient light collection mirror is fitted to a Cambridge Stereoscan Mk IIA the chief limitation to the detection of weak CL signals is background light, coming down the column through the apertures from the glowing tungsten filament and reflected from the specimen. With the cooperation and help of Cambridge Scientific Instruments Ltd, we have displaced the gun of the microscope laterally by 2 mm to baffle this light, and fitted deflector coils to bring the beam down the column to a now dark chamber. This modification is described in the present paper. N
N
t Bond et al., 12
1974.
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G. H. Haggis et al. Further, to decrease losses in resolution caused by beam spread in tissue depths, we have examined plant material in thin section and microorganisms (yeasts) on thin support films. With these specimens we have used a full parabolic mirror to achieve increased efficiency of light collection. In this paper we report the improvement in C L image formation achieved by these means. The beam voltage used throughout was 20 kV. METHODS
The offset gun An offsetting ring was inserted into the microscope column immediately below the gun to displace it laterally 2.0 mm off the column lens centre line, and double deflection coils (displaced 1.0 mm from the column centre line in the same direction as the gun offset) were inserted above the first condenser lens (Fig. 1). Voltages applied to the deflection coils allowed bending the beam twice on both 3c and y axes, so that the beam below the coils could be aligned on the column centre line. The coils and offsetting ring designed for this purpose were supplied by Cambridge Scientific Instruments Ltd. Optimum coil voltages, to maximize the beam current at the specimen, were determined empirically as : vZl=4.15v
vg1=2*33v
Vzz= 4.15 V
Vgz = 2.25 V
Measurements were made to determine the decrease in background noise arising from the filament light with the offset gun modification. The CL-detecting photomultiplier (PM) was blanked off, the maximum PM voltage was applied (2.2 kV) the microscope filament heater current was set to 2.6 A and the (dark current and shot) noise level read from the signal meter. A polished aluminium stub was placed under the half parabolic mirror, the blanking plate was removed and the noise level was compared for the offset gun and normal column configuration by decreasing the PM voltage to give the same meter reading.
Photomultiplier calibration The photomultiplier was calibrated against light intensity by the method described in the earlier paper (Bond et al., 1974) with the calibration extended to lower light levels (Fig. 2). The full parabolic mirror A parabolic mirror, of full 360" around the axis of revolution, and of 2.0 mm focal length was machined from aluminium and a hole, 2.0 mm diameter, drilled through the mirror at the focus parallel to the directrix (Fig. 3). A slot 2 mm wide was milled through the vertex, normal to the hole, and extending 3 mm into the mirror to allow positioning at the focus of a grid holder carrying a transmission electron microscope grid 3 mm in diameter. The grid holder was attached to the microscope stage, to allow a specimen movement of 2.5 mm along the X and Y axes and a small 2 axis movement ( _+ 0.5 mm) limited by the mirror slot, using the normal X Y 2 external controls. The mirror was made with a parabolic formed tool and polished to a micro finish (estimated accuracy within & 25 pm) and was bolted to a heavy brass bracket which slid into a dove-tail mount fitted to the fixed part of the SEM stage. The hole through the mirror was centred on the electron beam, and the mirror axis aligned with the CL detecting photomultiplier, mounted in the side wall of the specimen chamber. The CL detection system of quartz lens, quartz window, and 9635 QB photomultiplier remained as previously described
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Cathodolurninescence of biological material
E l ect3, b eam
Electron gun
7
Offsefting r ng tdation valve
/ "'per Deflection. / Lower
COllS
Condenser lens
/ kdc2fer
Fig. 1. The arrangement of offsetting ring and deflection coils. 2000
1500
0
0 Is) + -
9 1000
500 Intensity
(Candles)
Fig. 2. Photomultiplier calibration curve.
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I
1
F/PP
n To electron channel
10 m m 1
Fig. 3. Scale diagram showing the parabolic mirror and its position relative to the final lens. F/PP = final lens pole piece; SjLG = scintillatorlight guide positioned to collect transmitted electrons; X = position of specimen.
(Bond et al., 1974) and is not sensitive to minor imperfections in the mirror surface. To allow simultaneous CL and transmitted electron imaging of grid-mounted sections, a Cambridge scintillator disk was positioned beneath the mirror, under the hole and linked with a light guide to the photomultiplier at the back of the specimen chamber normally used to record the secondary electron image. Particulate specimens (yeasts) mounted on formvar-coated grids could be examined simultaneously in the CL and secondary electron imaging modes using the full parabolic mirror and the standard electron detector, collecting secondary electrons from the open end of the mirror. When recording CL images, whether with a half or full parabolic mirror, and whether the scintillator was positioned to collect transmitted or secondary electrons, it was our standard practice to apply - 250 V to the electron collector (achieved by setting the collector switch on the Cambridge Stereoscan Mk IIA to the ‘reflectedcollector’ position) while recording the CL image, to reduce the risk of a spurious CL signal arising from electrons reaching the collector scintillator. To check spurious imaging in the CL mode a torn formvar-coated grid with holes and gaps of 1-100 pm, was coated with 30 nm of gold-palladium, mounted in the grid holder and, with the full parabolic mirror installed, examined for CL with - 250 V applied to the electron collector. With maximum voltage applied to the PM tube no image of the tears was seen on the CL channel. If the precaution were not taken of applying this negative voltage to the collector, and it was left at + 250 V (the ‘secondary-collector’ position on the Stereoscan Mk IIA) while recording on the CL channel, then the holes and tears in the formvar-coated grid were seen as a spurious C L image, using the half parabolic mirror, though not with the full parabolic mirror. Using as a test sample a small crystal of zinc sulphide supported on a formvarcarbon film on a transmission electron microscope grid, the light collecting efficiency 180
Cathodoluminescence of biological material of the full parabolic mirror was found to be three to four times greater than that of the half parabolic mirror.
Biological materials The fission yeast, Schizosaccharomyces pombe NCYC 132, and the budding yeast, Saccharomyces cerevisiae 3483, were incubated in 2O(, malt extract broth (Difco Laboratories, Detroit, Michigan, U.S.A.) for 8 h at 35°C to produce a log phase culture. T o 9 ml of log phase culture was added 1 ml of a 1% solution of Calcofluor White M2R New (American Cyanamid Co. Bound Brook, New Jersey, U.S.A.) in 0.1 M NazHP04. (The Calcofluor, kindly supplied by American Cyanamid Co., is a water soluble diaminostilbene disulphonic acid laundry brightener which shows an affinity for active growth centres, Darken, 1961). The cultures were shaken and let stand for 1 min, gently centrifuged and twice resuspended in distilled water to remove excess stain. After a final concentration of the yeast by centrifugation a droplet of the heavy suspension was frozen dried at - 80°C and dusted onto formvar-coated index grids. The grids were mounted on cuptyped stubs (Soni et al., 1975) painted internally with carbon dag to give a structureless, nonluminescent background for examination under the half parabolic mirror, or mounted in the grid carrier for CL examination using the full parabolic mirror. After CL examination, the yeast-bearing grids were coated with gold for subsequent examination in the secondary electron mode. Ramsay wheat seeds, Triticum aestivum, were imbibed for 24 h in water, hand sectioned at 0.75 f 0.25 mm into Karnovsky's fixative (Karnovsky, 1961) for 2 3 h, dehydrated, and embedded in Spurr's medium (Spurr, 1969). Thin sections (- 0.3 pm) cut from these blocks were mounted on formvar-coated grids, stained for 1 min with the 1"',solution of Calcofluor described above, at room temperature, rinsed with distilled water and the grids mounted on the carrier and examined for CL using the full parabolic mirror. Sections from the same block and of comparable thickness (0.3 pm) were mounted on slides for fluorescence microscopy, stained 1-5 min with the 1O i l Calcofluor solution at room temperature, rinsed with distilled water, water-mounted under quartz cover slips sealed with lacquer, and examined in a Zeiss microscope equipped with epifluorescence optics and Neofluor objectives (illumination HBO 200 W mercury arc, exciter filter UG5, barrier filter 41). RESULTS AND D I S C U S S I O N
The 2 mm gun offset decreases the background light to a level where it cannot be distinguished from electronic noise and is effectively eliminated (Table 1). The calibration of the photomultiplier (Fig. 2) becomes less accurate at low light intensity but we estimate an increased CL sensitivity of 50-100 times with the offset gun configuration. It is not possible to align the beam below the deflection coils perfectly on the column centre and some resolution is sacrificed. With the offset configuration it is possible to image the 30 nm spacings on an IBM resolution standard when the column is clean. However, microscope performance deteriorates more rapidly with the offset gun fitted, as the column becomes contaminated, than it does with the normal column configuration. Table 1. Photomultiplier voltages for a constant signal meter reading
PM (V) Photomultiplier blanked off Photomultiplier unblanked, offset gun configuration Photomultiplier unblanked, normal configuration
2200 2080 1044
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Figs. 4 and 5. Secondary electron (Fig. 4) and CL micrographs (Fig. 5) of Schizosaccharomyces pombe. A luminescent band (arrow, Fig. 5) can be seen to be at the level of the constriction between the dividing cells (arrow, Fig. 4) rather than at the level of the fission scar ridge. x 1800. Figs. 6 and 7. Secondary electron (Fig. 6) and CL micrographs (Fig. 7) of Saccharomyces cerevisiae. Figure 6 is an enlarged secondary electron view of the bottom left area of Fig. 7. A luminescent band (arrow Fig. 7) can be seen at the bud constriction. A small proportion of the cells in these preparations show intense luminescence, an example being included in this micrograph (left of arrow in Fig. 7). We assume these to be ruptured or damaged cells which strongly bind the Calcofluor stain, or are perhaps initially empty and become filled with stain. Part of this cell is included at upper left in Fig. 6. Figure 6 x 7200, Fig. 7 x 1800. Figs. 8 and 9. Saccharomyces cerevisiae (Fig. 8) and Schizosaccharomyces pombe (Fig. 9) examined by epifluorescence. x 1800.
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Cathodoluminescence of biological material The finding that the light collecting efficiency of the full parabolic mirror is three to four times greater than that of the half parabolic mirror is inconsistent with our previously reported (Bond et al., 1974) collection efficiency of 8704 for a hemisphere of light emission with the half parabolic mirror. If we assume that the full parabola collects, at best, about 909; of the light emitted into a sphere, then our half parabola cannot be collecting more than about 50% of the light emitted into the upper hemisphere. The measurements reported in our earlier paper remain valid, namely that the half parabola improves light collecting efficiency by about 17 times over the simple lens system supplied by Cambridge Scientific Instruments Ltd, but the theoretical assumption made in that paper must be wrong, that all light hitting the lens reaches the photomultiplier. If we assume that the effective diameter of the lens is about 45 mm (as compared with an actual diameter of 50 mm) then the results become consistent. The light collection efficiency of the simple lens system would then be about 37; (of light emitted into the upper hemisphere), rather than the 5"(, previously assumed. The efficiency of the half parabolic mirror would be about 50°', (of light emitted into the upper hemisphere) and that of the fullparabolic mirror about 90"; of the total light emitted into the full sphere around a small luminescent object supported on a thin carbon film, or a small luminescent area in a thin section. Results of CL examination of Schizosaccharomyces and Saccharomyces using the offset gun are shown in Figs. 4-7. Figures 8 and 9 show the same yeasts by
Figs. 10 and 11. CL (Fig. 10) and fluorescence (Fig. 11) micrographs
of thin sections from the aleuron layer of wheat seed. x 1350. Figs. 12 and 13. CL (Fig. 12) and fluorescence (Fig. 13) micrographs of thin sections from the pericarp of wheat seed. Figure 12 x 1250, Fig. 13 x 1350.
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G. H. Haggis et al. fluorescence microscopy. In comparing CL and fluorescence the advantage and potential of CL are seen most clearly in Figs. 6 and 7, which illustrate the possibility of correlating CL features seen at low magnification with surface structural detail seen at high magnification in the secondary electron mode. It is clear that for yeast stained with Calcofluor the quality of the CL image is not as good as that of the epifluorescence image. The resolution of the images are not so different; it is mainly the lower signal-to-noise ratio which limits the quality of the CL image. Comparative micrographs for thin sections of the aleuron layer and the outer coat of wheat seed are shown in Figs. 10-13. For thin sections, where spreading of the electron beam is reduced, the CL image shows improved resolution, now equal to that of the fluorescence image. This may be considered the main positive result of the present work, that CL resolution has now been demonstrated at least equal to that of the best fluorescent methods. These sections are approximately 0.2 pm thick, and the CL resolution could in principle be improved by use of thinner sections. However, with our present staining methods, the luminescent signal has not proved adequate for CL micrographs of sections thinner than 0.2 pm. If C L resolution is to be improved over that of fluorescence microscopy, more intensely luminescent stains must be found, or means of more heavily adsorbing the stain to specific structures. The ruptured or damaged cell in Fig. 7 shows the intense C L signal that can be obtained from a high local concentration of Calcofluor. We must now however, consider another problem inherent to CL work, namely the effect of drying of the specimen prior to, or during, examination by electron microscopy. We have been unable to visualize by CL the bud rings of Saccharmyces cerevisiae so prominent in the fluorescence image of Fig. 8. Investigation showed that these rings were not visible by fluorescence microscopy either on a dry specimen, whether air dried or frozen dried. A current requirement, then, for a CL stain is that it must retain luminescence after drying, though there remains the possibility that some form of hydration stage might be devised for work of this kind in the future. ACKNOWLEDGMENTS
The deflection coils and displacement ring for the offset gun arrangement were kindly supplied by Cambridge Scientific Instruments Ltd, Cambridge, England. The full parabolic mirror was made in the Science Faculty Workshop at Carleton University, Ottawa. We are grateful also to Mr A. Lapenste for the machine work involved in making the mirror mount, special specimen holders, etc. This work was supported by a National Research Council Grant (No. A 6801).
References Bond, E.F., Beresford, D. & Haggis, G.H. (1974) Improved cathodoluminescence microscopy. J . Microsc. 100, 271. Broecker, W., Schmidt, E.H., Pfefferkorn, G. & Beller, F.K. (1975) Demonstration of cathodoluminescence in fluorescein-marked biological tissues. Proc. 8th Annual SEM Symp., I I T R I Chicago, 244. Darken, M.A. (1961) Applications of fluorescent brighteners in biological techniques. Science, 133,1704. Horl, E.M. & Miigschl, E. (1972) Scanning electron microscopy of metals using light emission. Proc. 5th Europ. congr. on Electron Microsc. 502. Karnovsky, M. J. (1961) A formaldehyde-glutaraldehydefixative of high osmolarity for use in electron microscopy. J . Cell. Biol. 27, 137A. Soni, S.L., Kalnins, V.I. & Haggis, G.H. (1975) Localization of caps on mouse B lymphocytes by scanning electron microscopy. Nature, 255, 717. Spurr, A.R. (1969) A low-viscosity epoxy resin embedding medium for electron microscopy. J . Ultrastruct. Res. 26, 31.
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