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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
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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.
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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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