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relief phase contrast relief phase contrast a new technique for phase contrast light microscopy jorg piper meduna klinik bad bertrich germany introduction and the background are amplified in the final ...

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                                                                                                                                RELIEF PHASE CONTRAST
              Relief Phase Contrast:A New Technique
              for Phase-Contrast Light Microscopy
              Jörg Piper, Meduna-Klinik, Bad Bertrich, Germany
                                                                    INTRODUCTION                                          and the background are amplified in the final
               BIOGRAPHY                                            Phase contrast is a mode of light microscopy          image, so that minimal differences in refrac-
               Jörg Piper obtained his                              that is widely used for the examination of            tive index are visible even in colourless speci-
               medical degree from the                              transparent and colourless specimens such as          mens with a low contrast and thickness.
               University of Bonn in Ger-                           unstained cells and micro-organisms which               Depending on the configuration and prop-
               many. One field of his sci-                          typically have very low contrast. These objects       erties of the phase ring in the objective, phase
               entific activities is dir-                           do not absorb light, so the amplitude of the          contrast microscopy can be positive or nega-
               ected at light microscopi-                           light waves passing through them remains              tive. In positive phase contrast the specimen is
               cal applications, espe-                              nearly constant. However, they do modify the          visible with medium or dark grey features, sur-
               cially reflection contrast. He has created sev-      phase of transmitted light by around one              rounded by a bright halo; the background is of
               eral mathematical models for the three-              quarter wavelength (l/4) hence we call them           higher intensity than the specimen. In negative
               dimensional quantitative analyses of                 phase objects. Such differences in phase can-         phase contrast the background is darker and
               opaque cellular specimens. Since 1998 he             not be perceived by the eye or by a photogra-         the specimen appears brighter, surrounded by
               has also collaborated with the University of         phy. The Dutch physicist Frits Zernike devel-         adark halo. The bright and dark halos are arti-
               Oradea in Romania as an associate and hon-           oped phase contrast as a new illumination             facts which are one of the major disadvantages
               orary professor. In Germany, he currently            mode to convert phase differences into visible        of phase contrast; they are especially prevalent
               works as senior consultant for internal med-         amplitude differences [10,11] for which he was        in specimens inducing large phase shifts.
               icine, angiology and diabetology.                    awarded the 1953 Nobel prize.                         Recently, advances in the design of objective
               ABSTRACT                                               To achieve phase contrast, two components           phase-ring configurations have led to a new
               Relief phase contrast is a new modification          of brightfield microscopes have to be modi-           technique which reduces halo-effects called
               of conventional phase contrast which leads           fied: 1. The condenser has to be equipped with        apodized phase-contrast microscopy.
               to visible improvements of image quality in          a ring-shaped aperture or mask (the con-                All these modifications used in phase con-
               light microscopy. In particular, the following       denser annulus), which is placed near the con-        trast do not create three-dimensional images
               parameters can be improved: contrast, focal          denser aperture diaphragm. 2. A conjugate             which could be compared with the 3D effects
               depth, sharpness, three dimensionality,              phase plate (or ring) is placed in the back focal     of interference contrast microscopy. Com-
               planeness, and halo artifacts. These effects         plane of the objective. The condenser annulus         pared with brightfield, in phase contrast the
               can be achieved when the ring-shaped                 and the phase ring in the objective have to be        depth of focus is smaller, because the con-
               masks in the condenser are replaced by cres-         optically aligned so that they are conjugate.         denser aperture iris diaphragm is fully open. In
               cent- or punctate-shaped masks. Several              With this arrangement the specimen is illumi-         phase contrast, the intensity of contrast is
               solutions are described which are suitable to        nated by the apex of a cone of light. The light       dependent on the differences of refractive
               create this modification. The achievable             beams which are diffracted by the specimen            indices of the specimen and the surrounding
               improvements of image quality are relevant           pass through the objective lens at various            medium, and the thickness and native contrast
               for all quality levels of objectives. The new        angles which are dependent on the relative            of the specimen. 
               technique can be used for phase contrast             refractive index and the thickness of the spec-         The quality of phase contrast images is
               objectives from different manufacturers, so          imen. The other light components, corre-              strongly determined by the quality of the
               that the usual limitations of compatibility          sponding to the background, pass through the          lenses. Existing chromatic and spherical aber-
               are eliminated.                                      phase ring in the objective which produces an         rations reduce the quality of the resulting
                                                                    additional phase difference. Thus the phase           images more intensively than in brightfield
               KEYWORDS                                             differences between the specimen, its details         microscopy. 
               light microscopy,phase-contrast microscopy,                                                                                  Figure 1: 
               relief phase contrast, phase, interference,                                                                                  (a, b) Optical alignments in conventional
               focal depth, cell relief, halo artifacts, spher-                                                                             phase contrast and relief phase contrast
               ical aberration, image quality                                                                                               microscopy.
               ACKNOWLEDGEMENTS                                                                                                             (a) Conventional phase contrast. Condenser
               The author thanks Mrs Ingrid Kerner and Mr                                                                                   annulus (bright) and phase-ring (dark) are
               Robert de Herrera for their helpful assis-                                                                                   properly aligned, concentric and conjugate. 
               tance in preparing the manuscript.                                                                                           (b) Relief phase-contrast. The annulus is
                                                                                                                                            replaced by a modified arc-shaped mask
               AUTHOR DETAILS                                                                                                               (bright), which when properly aligned over-
               Prof. Dr med. Jörg Piper,                                                                                                    laps with the phase ring (dark).
               Clinic Meduna, Clara-Viebig-Str. 4,                                                                                          (c) Simplified optical pathway for relief-phase
               D-56864 Bad Bertrich, Germany                                                                                                contrast microscopy (modified from [5]).
               Tel: +49 (0) 2674 182 3184,                                                                                                  Key: 1 = light source; 2 = modified mask; 3 =
               Email: webmaster@prof-piper.com,                                                                                             condenser; 4 = specimen; 5 = background
               Website: www.prof-piper.com                                                                                                  light; 6 = light bent by the specimen; 7 =
                                                                                                                                            phase ring; 8 = eyepiece with intermediate
                                                                                                                                            image; 9 = eye.
               Microscopyand Analysis21(4):9-12 (UK),2007
                                                                                                             MICROSCOPY AND ANALYSIS JULY 2007                               9
                                                                                                                                         Figure 2:  
                Furthermore, phase contrast can only be
              achieved when the phase rings in the objec-                                                                                Implementation of relief phase contrast using non-
              tives and the condenser annuli are specifically                                                                            transparent slides with ring-shaped apertures. Images of
              adjusted for each other. Normally, a particular                                                                            the condenser aperture taken with a phase telescope. 
              ring-shaped mask can be used for one or two                                                                                (a) The aperture diaphragm is partially closed and then
              special objectives within a well-defined range                                                                             the margin of the slide is moved in from the right (shad-
              of magnification. For example, one condenser                                                                               owed area of aperture). The darker ring is the phase ring
              annulus could used with objectives for 103or                                                                               of the objective.
              163 magnification, a second mask for 253                                                                                   (b) Final alignment of the margin of the slide and the
              and 403, and a third mask for 1003 magnifi-                                                                                aperture diaphragm. 
              ing oil-immersion objectives. When objectives                                                                              (c) An annular ring for phase contrast (small diameter,
              and condensers are used from different manu-                                                                               bright) is shifted from the right, overlapping the phase-
              facturers, a misalignment can result. There-                                                                               ring (dark).
              fore, the objectives and condenser should                                                                                  (d) Final alignment with partially closed aperture
              preferably be from the same manufacturer.                                                                                  diaphragm, visible on the right (dark).
                The principles of conventional phase con-                                                                                (e) An annular ring for darkfield (large diameter, bright)
              trast and its beam path were discussed in a                                                                                is overlapping the phase ring (dark) on the left side. The
              recent article in Microscopy and Analysis [2].                                                                             aperture diaphragm (dark) is partially closed, its edges
              Moreover, several very instructive internet-                                                                               visible on the left.
              based interactive tutorials are available which                                                                            (f) Final alignment with partially closed aperture
              demonstrate the effects of positive, negative                                                                              diaphragm.
              and apodized phase contrast based on realis-
              tic animations [1,3]. The advantages of phase
              contrast in comparison with other illumination          of several annular rings, usually existing in           MATERIAL AND METHODS:
              modes have been described [4,5,8,9].                    Zernike phase condensers, can be used for               Relief phase contrast was developed on two
                In this article a new modification of phase           relief phase contrast, if the turret is rotated         Leica (Leitz) microscopes, a Dialux and an HM-
              contrast is presented which can improve the             into an abnormal position, so that annular              Lux III. These microscopes are both able to
              quality of the conventional phase contrast              ring and phase ring overlap slightly. The con-          achieve positive phase contrast. The Dialux
              images by higher contrast, enlarged focal               denser aperture diaphragm is closed as                  was equipped with a Zernike phase contrast
              depth, reduced haloing and less-visible spher-          described above.                                        universal condenser, the HM-Lux III with a kit
              ical aberration. This method, called relief-              The individual position of the phase ring in          of separate slides for phase contrast and dark
              phase contrast, can also be used when existing          the objectives is not important for realising           field which can be shifted into an existing
              phase-contrast objectives are built by differ-          relief phase contrast, because the position of          brightfield condenser. In this way, the several
              ent manufacturers. Moreover, in most cases              the illuminating light sector can be adjusted to        varieties of technical realisation described
              this technique requires only one light-modu-            the phase ring with a high degree of variabil-          above could be evaluated.
              lating element in the condenser, which is suit-         ity. Thus, objectives made by different manu-             The images of the phase ring and annular
              able for all existing phase-contrast objectives.        facturers can be used simultaneously. More-             ring constellations, controlled by a phase tele-
                                                                      over, the condenser annuli can be directly              scope, were taken using Canon Powershot A
              PRINCIPLES OF RELIEF PHASE                              modified as when they are covered by an                 620 and Casio Exilim EX-Z 110 cameras. The
              CONTRAST                                                opaque plate with a small aperture for the              microscopical images were taken using Olym-
              In conventional phase contrast, the condenser           transmitted light. In this case it is not necessary     pus Camedia C 7070 and Canon EOS 350D/20D
              annulus is completely transparent, so that the          to close the condenser aperture diaphragm.              cameras operated with remote switches. 
              passing light beams create a light cone which           Alternatively, the annulus could also be                  When slides for phase contrast are used (Fig-
              illuminates the specimen in a concentric circu-         replaced by other constructions, suitable to            ure 3a), the margin of the illuminating sector
              lar manner (360°). When the condenser annu-             achieve circumscribed small light beams,                can be built by the edge of the slide and the
              lus and the objective phase ring are correctly          which can be adjusted to the phase rings in             aperture diaphragm (Figure 2 a,b) or by the
              aligned, both illuminating elements are                 the objective (see Discussion).                         annular ring itself and the aperture
              exactly conjugate; the alignment is usually               As in normal phase contrast, the quality of           diaphragm (Figure 2 c,d). Annular rings for
              controlled using a centering telescope (phase           the resulting images can be optimized also in           darkfield microscopy (Figure 3c), which are
              telescope) as shown in Figure 1a.                       relief phase contrast by closing the field              characterized by much higher diameters, can
                To achieve relief phase contrast, the trans-          diaphragm so that it is just seen at the edges of       also be used (Figure 2 e,f). Moreover, a slide
              parent annulus has to be replaced by a smaller          the field of view (as in Köhler illumination).          with an annular ring can directly be prepared
              transparent sector, which can be crescent-
              shaped or a small round aperture. This light
              sector has to be aligned with the phase ring in
              the objective in the usual manner, so that it is
              conjugate (Figure 1b). With this modification,
              the specimen is only illuminated from one
              defined direction by oblique light beams (Fig-
              ure 1c). Typically, the resulting images have
              more contrast, focal depth and three dimen-
              sionality and less visible spherical aberration
              compared with conventional phase contrast.
                Crescent- or circular-shaped sectoral masks
              can be achieved in several ways, controlled
              using a phase telescope: 1. Sliding components                       a                 bdc                                                        e
              with an annular ring can be moved into the
              path of light and the condenser aperture
              diaphragm can be partially closed so that just          Figure 3:  
              a small part of the annular ring remains trans-         (a-c) Standard condenser sliders for conventional phase contrast (a), relief phase contrast with a special mask (b), and darkfield (c) microscopy. 
              parent for light. 2. Condenser turrets with a kit       (d,e) Prototype condenser sliders with semicircular (d) and circular (e) shaped masks. 
        10      MICROSCOPY AND ANALYSIS JULY 2007
                                                                                                                                     RELIEF PHASE CONTRAST
              for relief phase contrast when it is covered by          ab
              an opaque black mask (Figure 3b); in this case,
              the condenser aperture diaphragm has to be
              wide open. The resulting control image, which
              is visible with the phase telescope, is illus-
              trated in Figure 1b.
                 The turret of a Zernike condensor can be
              rotated into several positions to achieve mar-
              ginal overlappings of annulus and phase ring.
              The condenser aperture diaphragm has to be
              used for additional limitation of the illumi-
              nating field. Annular rings with various diam-
              eters can be used so that the configuration of
              the resulting illuminating field can be vari-            cd
              able, corresponding to Figure 2 d,f.
                 A binocular viewing tube should preferably
              be used to align the illuminating elements. In
              this way, one eyepiece can be removed and
              the phase-telescope can be inserted in its
              place. Thus, the effects of manipulations can
              be controlled simultaneously by the remain-
              ing eyepiece. When an optimal alignment is
              found, the centering telescope can be
              replaced by the second eyepiece, so that nor-
              mal binocular examination can then be made. 
                                                                      Figure 4:  
              RESULTS                                                 Buccal epithelial cells, 40x objectives HFW = 100 µm. (a) Conventional phase contrast with a basic corrected objective (Olympus A 40x Plan 0.65NA).
              When phase-contrast objectives that were not            (b) Relief phase contrast using the same objective. (c) Conventional phase contrast with a more highly corrected objective (Leica Phaco Plan 40x 0.65NA).
              highly corrected were used, the quality of the          (d) Relief phase contrast with the Leica objective.
              images could be dramatically improved. Relief
              phase contrast achieved a higher contrast               denser; alternatively a brightfield condenser           diameter of the lenses, the lower the width of
              with enlarged focal depth and often                     can be modified for phase contrast by placing           the aperture diaphragm and the remaining
              improved sharpness. The relief of the speci-            special slides into the light path. In these cases,     resolving power, when relief-phase-contrast is
              men mostly appeared more three dimen-                   the condenser aperture diaphragm has to be              achieved. When ring-shaped masks are
              sional, similar to interference contrast images.        closed to achieve correct alignment of the illu-        directly modified to crescent- or punctate-
              The planarity of the microscopical image was            minating mask in the condenser and the phase            shaped masks, the condenser aperture iris
              improved, because the effects of spherical              ring in the objective. Closing the aperture             diaphragm can be fully open. The specific fea-
              aberration were lower. Halo artifacts were              diaphragm can improve the focal depth and               tures of the specimen and the individual rela-
              often reduced (Figure 4 a,b).                           sharpness in the same manner as is usual in             tive diameter of the existing phase ring deter-
                 Even when highly corrected phase-contrast            brightfield. In conventional phase contrast,            mines which option (large or small aperture
              objectives were used (e.g. planachromatic or            the quality of the microscopical images is not          diaphragm) leads to better optical results.
              planapochromatic lenses), the images result-            influenced by the aperture diaphragm. The                 Relief phase contrast can be regarded as a
              ing from relief phase contrast had more con-            resulting three-dimensional appearance can              complementary method which can improve
              trast, enlarged focal depth and more appar-             contribute to the gobal quality of relief phase         the quality of conventional phase contrast
              ent three-dimensional aspects (Figure 4 c,d).           contrast images as well as the diminution of            images.
                 Compared with interference contrast, relief          existing spherical aberrations. It can be                 Manufacturers of microscopes could imple-
              phase contrast often produced images with               regarded as an additional advantage of these            ment relief phase contrast if existing con-
              higher or complementary information of spec-            modifications that objectives made by differ-           denser annuli in conventional phase contrast
              imen details (Figure 5 a,b, Figure 6 a-c).              ent manufacturers can be used simultaneously            systems were replaced by crescent- or punc-
                 The brightness of the microscopical image            also in cases of misalignments in conventional          tate-shaped masks. In this case, relief phase
              was lower than that using conventional phase            phase contrast caused by non-compatibility.             contrast could be achieved without closing the
              contrast, because the area of the illuminating            The resolving power is reduced when the               aperture diaphragm. Moreover, manufactur-
              light beams was more reduced (about -2.0 or             aperture diaphragm is closed. The lower the             ers could create modified turrets equipped
              -3.0 EV). Therefore, higher light intensities           diameter of the phase ring in relation to the           with unconventionally pre-aligned annular
              have been necessary.
                 As specimens were illuminated from one                a                                                      b
              direction by oblique light beams, the back-
              ground of relief phase-contrast images some-
              times appeared with variable brightness,
              especially when objectives with low magnifi-
              cations were used. Similar effects are also
              known from interference contrast. 
                 All the relevant findings are presented in
              Table 1 which compares the major similarities
              and differences of conventional phase con-
              trast with relief phase contrast.
              DISCUSSION:
              When a conventional phase-contrast micro-               Figure 5:  
              scope is available, relief phase contrast can be        Buccal epithelial cells in a very thin layer of saliva with Newton´s rings. 40x objectives. HFW = 70 µm. Exposure by electronic flash.
              achieved quite simply using a Zernike con-              (a) Relief phase contrast using Leica Phaco Plan 40x 0.65NA objective. (b) Interference contrast using Leica NPL Fluotar 40x 0.65 ICT.
                                                                                                                 MICROSCOPY AND ANALYSIS JULY 2007                                 11
                                                                                                                                                                             Figure 6:
                                                                                                                                                                             Thin-layer crystallization of a water soluble pigment. Cov-
                                                                                                                                                                             erslip preparation with Newton´s rings. HFW = 240 µm.
                                                                                                                                                                             (a) Conventional phase contrast using Leica Phaco Plan
                                                                                                                                                                             40x 0.65NA. (b) Relief phase contrast using same lens as
                                                                                                                                                                             (a). (c) Interference contrast using Leica NPL Fluotar 40x
                                                                                                                                                                             0.65NA ICT.
                                                                                                          Key Features                             Conventional                                   Relief 
                  rings which could overlap the phase rings tan-                                                                                  Phase Contrast                            Phase Contrast
                  gentially when they are rotated into their                              use objectives from all manufacturers                            no                                        yes
                  fixed position. Thus, the aperture diaphragm
                  could be closed to improve focal depth and                              illuminating light beams                                    concentric                                 eccentric
                  sharpness.                                                              condenser aperture diaphragm                                   open                                open or smaller
                     Alternatively, circular or semicircular shaped                                                                                                                     (various modifications)
                  masks could be built (Figure 3 d,e) which could                         3D images / relief effects                                       no                                        yes
                  be shifted excentrically into the light path to
                  overlap phase rings tangentially. Thus, bright-                         contrast                                                        good                                     higher
                  field mode could be used when the aperture                              sharpness                                                      good                              potentially higher 
                  diaphragm is opened, and relief phase con-                                                                                                                        (depending on the specimen)
                  trast could result when the aperture                                    depth of focus                                                narrow                                     higher
                  diaphragm is much smaller. Universal con-                                                                                                                       sometimes lower (when aperture
                  densers could be equipped with several                                  resolving power                                                 high                           diaphragm is smaller)
                  removable turrets to achieve all varieties of                                                                                                                             sometimes lower 
                  relief phase conrast as well as conventional                            halo artifacts                                                  high                      (depending on the specimen)
                  phase contrast.                                                         influence of spherical aberration                               high                                     lower
                       Moreover, special condensers for relief
                  phase contrast could be created, equipped                               influence of chromatic aberration                               high                              sometimes lower
                  with various technical modifications to                                 brightness of the microscopical image                           high                                     lower
                  achieve small, circumscribed illuminating light
                  beams with a variable position, length, width                           homogeneity of background                                       high                sometimes lower (when objectives with
                  and shape. Thus, two separate iris diaphragms                                                                                                                      low magnification are used)
                  could be superimposed on each other, eccen-                            Table 1:  A comparison of the major characteristics of conventional phase-contrast and relief phase-contrast microscopy.
                  trically aligned, which could then be shifted
                  and turned eccentrically. Alternatively, two
                  excentrically rotating disks could be created,                         long distances and thick glass slides. In this                        REFERENCES
                  one disk-shaped as a transparent light mask,                           case, the illuminating beams will first pass                          1.   Abramowitz M. et. al. Phase contrast microscopy. Olympus
                  the other disk as a non-transparent overlap-                           through the coverslip instead of the object                                Microscopy Resource Center: www.olympusmicro.com
                  ping element, eccentrically superimposed on                            slide; the surface of the specimen will be illu-                      2.   Horn E. and Zantl R. Phase-contrast-light microscopy of
                  the light mask. Two non-transparent slides                             minated from the top instead of from the bot-                              living cells cultured in small volumes. Microscopy and
                  could also be used, overlapping marginally                             tom and shadow figures might occur on the                                  Analysis 101:15-17 (EU), 2006
                  and suitably shaped, so that a small light beam                        surface of the object slide comparable with                           3.   Murphy D. W. et. al. Phase contrast microscopy. Nikon
                  could result. In all cases, small sectoral illumi-                     the shadow effects obtained by reflexion con-                              MicroscopyU: www.microscopyu.com 
                  nating light beams could be achieved by vari-                          trast with oblique illuminating beams [6,7].                          4.   Murphy D. Phase contrast microscopy. In: Fundamentals of
                  able transparent gaps, resulting from the posi-                          When expensive high-end lenses are not                                   light microscopy and electronic imaging. Wiley-Liss, New
                  tion of the double iris diaphragm, the double-                         available, relief phase contrast can still lead to                         York, 97-112, 2001. 
                  disk or double-slide system.                                           excellent image quality when only less well                           5.   The phase contrast microscope. Nobel Foundation:
                     For high-end motorized microscopes,                                 corrected objectives are used. These improve-                              http://nobelprize.org/physics/educational/microscopes/
                  motorized condensers for relief phase contrast                         ments in quality are visible as well in live                          6.   Pera F. et al. Morphometrische Untersuchungen der
                  could be built, equipped with several freely                           microscopy, photographic images or movies.                                 Formänderung von Blutzellen während der Ausstrich-
                  programmable function buttons. In this way,                                                                                                       präparation. Verh. Anat. Ges. 76:147-148, 1982
                  illuminating light beams with optimized align-                         CONCLUSIONS                                                           7.   Piper J. Dreidimensionale Zytometrie und Rekonstruktion
                  ment could be achieved and reproduced with                             The quality of the images produced by con-                                 des Reliefs roter Blutzellen im Reflexionskontrast.
                  a high precision according to the existing kit of                      ventional phase contrast microscopy can be                                 http://www.prof-piper.com/Forschung/Zytologie/
                  phase-contrast objectives.                                             significantly improved when the usual annular                              Zytometrie/zytometrie_0.html
                     When the microscope is equipped with a                              masks in the phase-contrast condenser are                             8.   Robertson D. The phase contrast microscope. In: The world
                  rotary stage, the position of the specimens can                        excentrically aligned with the phase rings in                              beneath the microscope. Weidenfeld and Nicolson, London,
                  be changed according to their individual                               the objectives or replaced by other elements.                              UK. pp. 26-30, 1970.
                  three-dimensional texture and the direction of                           Several ways for the technical implementa-                          9.   Slagter E. and Slagter H. Imaging of phase objects. In: Light
                  the illuminating light beams. Thus, in special                         tion of relief phase contrast exist; these are                             and electron microscopy. Camb. U. Press, pp149-167, 1992.
                  cases, the effects of 3D imaging could be opti-                        dependent or independent of the position of                           10.  Zernike F. Phase-contrast, a new method for microscopic
                  mized or intensified. Alternatively, this opti-                        the condenser aperture diaphragm. Thus, the                                observation of transparent objects. Part I. Physica 9:686-698,
                  mizing effect could also be achieved when the                          various parameters which are influenced by                                 1942.
                  condenser is pivoted, so that it can rotate                            the illuminating aperture can be adapted to                           11. Zernike F. Phase-contrast, a new method for microscopic
                  around the optical axis.                                               the individual features of the specimen. In                                observation of transparent objects. Part II. Physica 9:974-
                     More intensified three-dimensional images                           contrast to conventional phase contrast,                                   986, 1942.
                  could also be achievable when an inverted                              objectives from different manufacturers can                                        ©2007 John Wiley & Sons,Ltd
                  microscope is used for relief phase contrast,                          be used simultaneously, so that their compati-
                  combined with phase-contrast objectives for                            bility is improved.  
           12       MICROSCOPY AND ANALYSIS JULY 2007
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