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PhaseContrastMicroscopy Secondaryarticle
JeremyBSanderson,JohnRadcliffeHospital,Oxford,UK Article Contents
. Introduction
Phasecontrast microscopy is a method that enables us to see very transparent objects, . HowPhaseContrastWorks
whichareotherwisealmostinvisible by ordinary light microscopy, in clear detail and in . ConstructionofthePhaseContrastMicroscope
goodcontrasttotheir surroundings. This is achieved optically, without altering the . InterpretingthePhaseContrastImage
specimenbystainingorotherprocessing. . TheHaloArtefact
. SettingUpthePhaseContrastMicroscope
Introduction
The human eye perceives only differences in wavelength startedoutinsynchrony,orinphase,arenowoutofphase;
(as colour) and amplitude (as brightness) of the light thisdifferenceisreferredtoasthephasedifferencebetween
reaching it. The eye cannot see differences in the thetwobeams.Allspecimensdiffract,orscatter,light,and
phase relationship between different beams of light. these diffracted beams carry the information about the
Classically, biological specimens have usually been ´
structureoftheobject(Oldfield,1994;PlasekandReischig,
˘
viewed as stained slices of material by bright-field 1998). An image of the object is formed at the primary
transmitted-light microscopy (Figure 1f). Stains are image plane owing to interference between those beams
used to alter both the colour and brightness of the light diffracted by the specimen and the undiffracted (zero
passing through the specimen, and so increase contrast in order) beam. With a stained specimen there is a half-
the image. Cellular function is understood better by wavelength (1l) phase difference between the undiffracted
studying the actual motion, growth, reproduction and 2
beamandthosediffractedbythespecimen.Interferenceof
exchange of cell constituents of living cells than it is by the two sets of beams leads to overall differences in
drawing conclusions from artificially manipulated dead amplitude, which can be detected by the eye as differences
ones. Since cells are generally transparent structures, they in brightness. Coloured objects are merely amplitude
are almost invisible to the eye by bright-field microscopy specimens that selectively absorb light of certain wave-
(Figures 1b,g). lengths within the visible spectrum.
The phase contrast microscope exploits the Thintransparentobjectssuchascellsintroduceaphase
interaction of the illuminating beam of light with the difference between the two beams of only one-quarter
specimen to convert an image of an invisible specimen 1
wavelength( l).Foratheoreticaldiscussionofwhythisis
(or one of very low visibility) into an image that the 4
so, using vector treatment, see Pluta (1989) or Bradbury
eye can detect. Other forms of contrast enhancement, andEvennett(1996).Torenderthesespecimensvisible,itis
such as darkground microscopy, differential interference necessary to have an artificial means of introducing an
contrast microscopy and Hoffmann modulation extra 1l phase difference between the diffracted and
contrast microscopy, have also been developed. For 4 1
undiffracted beams. There would then be 4l phase
furtherdetails,seeBradburyandEvennett(1996).Perhaps difference contributed by the specimen and a further 1l
the most widespread use of phase contrast microscopy in 4
phase difference by the microscope.
biology is for cytology and tissue culture, admirably If the beam illuminating the field of view is constrained
lending itself to quick checking of live cell cultures by an annulus in the first focal plane of the condenser,
Figure 1a).
( it will form an annular image in the back focal plane
of the objective (Figure 2). Suppose that a circular ‘trench’
(or, alternatively, a ridge) matching the image of the
condenser annulus is placed within the back focal plane
HowPhaseContrastWorks of the objective. The optical path traversed by the
undiffracted beam alone can now be selectively advanced
Light can be considered as a wave. When light encounters (or retarded) and the necessary extra 1l phase difference
4
glass, or other optically transparent material denser than between the two beams introduced before they recombine
air,itissloweddown,andthenumberofwavesincreasesin to form the image at the primary image plane of the
proportion to both the density (as determined by the microscope. This ‘trench’ (or ridge) is called the phase
refractive index) and thickness of the material. Consider a ring and it is carried on the phase plate. The phase
second identical beam (from the same source), which ring carries an absorbing layer that reduces the amplitude
moveswhollyinairparalleltothefirstwithoutenteringthe oftheundiffractedzeroorderbeam,reducingitsbrightness
glass. The first beam will have travelled a greater distance to match that of the weaker beams diffracted by the
than the second. Furthermore, the two beams, which specimen.
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PhaseContrastMicroscopy
Figure 1 All parts of this figure show the same field of view of living HeLa cells (a–e) and fixed, embedded HeLa cells in thin section (f–h).
(a)LivingHeLacellsinculturebyphasecontrast.(b)Thesamecellsbytransmitted-lightbright-fieldmicroscopy.(c)Inbright-fieldmode,withoutphase
contrast, closing the condenser diaphragm will enhance contrast to some degree, but at the expense of resolution in the image. This method is to be
avoided.(d)Sameimageas(a),buttheimagehasbeentakenwiththeannulusandphaseplateoutofalignment(seealsoFigures3e,f).(e)Theuseofa
greenfilterimprovesthequalityofthephasecontrastimage.(f)StainedHeLacells,togetherwiththebright-fieldimage(g)forcomparisonwiththephase
contrast image (h).
Parts(h)and(i)areincludedforcomparisonofphasecontrastimagesoflivingcellswiththosethathavebeenfixed,embeddedandsectionedthinly.
Themannerinwhichcellsandtissuesarefixed(ifatall)andpreparedwillinfluencetheresultingphasecontrastimage.Thelivingcells(h)exhibithigh
contrast,wherethereisarelativelyhighdifferenceofrefractiveindexbetweenthecellsandthewaterymediumtheyarecontainedin.Thesectionsofcells
embeddedinresinin(i)exhibitlowercontrast.This is because there is a smaller difference of refractive index between the cell constituents and the
backgroundresin. Likewise, cells fixed in methanol, an extracting fixative, exhibit a higher contrast image than those fixed in paraformaldehyde, a
crosslinking fixative that retains more of the cytoplasm.
Figures(a)–(e)weretakenusingaZeissAxiovert25,invertedmicroscopefortissuecultureusinga32NA0.5longworkingdistanceobjective.Figures
(f)–(h) were taken using a Zeiss Axiophot microscope equipped with a Plan Neofluar 40NA 1.30 oil immersion phase contrast objective.
ConstructionofthePhaseContrast Interpreting the Phase Contrast Image
Microscope Providedthattheundiffractedanddiffractedbeamsareout
of phase with one another by 1l overall, they will interfere
A special set of objectives, fitted with phase plates, 2
is normally needed for phase contrast microscopy. toformavisibleimage,anditdoesnotmatterwhetherthe
diffracted beams are retarded or advanced by 1l with
Manufacturers generally provide several different sizes of 4
annuli in the condenser to match objectives of differing respect to the undiffracted beam. Two forms of phase
magnification and numerical aperture (Figure 3d). These contrast microscopy are therefore possible; these are
annuli can normally be rotated within the condenser referredtoaspositiveandnegativephasecontrast.Positive
housing, and brought onto the optical axis of the phasecontrastreferstothemostwidelyusedsystemwhere
microscope as required (Figures 3a,b). Provision is usually the phase plate is constructed with a ‘trench’, so that the
madeforcentringeachannuluswithrespecttotheoptical diffracted beams (passing outside the phase ring) travel
axis of the condenser. one-quarter of a wavelength further than the zero order
beams.Structureswitharefractiveindexhigherthantheir
2 ENCYCLOPEDIAOFLIFESCIENCES/&2002MacmillanPublishersLtd,NaturePublishingGroup/www.els.net
PhaseContrastMicroscopy
Primary image plane appreciable width and some diffracted rays will inevitably
(phase contrast) pass through it, causing the haloes that are a familiar part
ofphasecontrastimages.Inpositivephasecontrastobjects
of refractive indexhigher than the background form an
image in which these dark structures are surrounded by a
bright halo, and lined internally with a darker halo. In
negative phase contrast, the situation is reversed. Phase
contrast is not suited for making precise linear measure-
ments:itisdifficulttoassessaccuratelythepreciseposition
Objective back focal of an edge in the image owing to the halo artefact.
plane and phase plate
SettingUpthePhaseContrast
Objective Microscope
Specimen Set the microscope up, in proper adjustment for Kohler
(phase object) ¨
illumination for bright-field microscopy, using a well-
stained specimen. Ensure that the condenser is set at the
Condenser correctheight,andiscentred.Ifindoubt,refertoBradbury
andBracegirdle(1998)orOldfield(1994).Withoutaltering
the focus, replace the stained specimen with the transpar-
entone.Openthecondenseraperturefully.Swinginalow-
Condenser power (10 or 20) phase contrast objective; the
front focal plane specimen will probably not be visible. Insert the correct
annulus; an indication of the appropriate annulus is
usually marked on the barrel of the objective in green
Annular diaphragm script (e.g. Ph3).
Remove an eyepiece and insert a centring-telescope
Figure 2 Raydiagramofthephasecontrastmethod.Theheavylines (sometimescalleda‘phasetelescope’),orinsertaBertrand
representtheundiffractedbeams,whilethediffractedbeamsareshownby lens system into the optical path to image the back focal
´˘
dashedlines. Adapted with permission from Plasek and Reischig (1998).
plane of the objective through the eyepieces. Whichever
deviceisused,focusonthephaseplatewithintheobjective.
surroundingsgiverisetodiffractedbeamsretardedbyone- The image of the annulus in the condenser (which is
quarterwavelength,andthesemorehighlyrefractingareas conjugate with the objective’s phase plate) will also be in
will thus appear darker in the final image, against a lighter focus.
background (Figures 1a,h). Positive phase contrast is Usingthecentringadjustmentsprovidedfortheannuli,
responsible for the commonly recognized appearance of and without disturbing the normal centre position of the
a cell, with the nucleus, lysosomal compartments and the condenser itself, superimpose the image of the condenser
cell membrane appearing darker than their surroundings. annulus precisely over that of the objective phase ring
Thephasecontrasteffectismaximalatregionsofsudden Figure 3e). The centring screws used for this superimposi-
(
change in optical path difference (‘edges’), and is less tion (usually set at 908 or 1208 on the condenser housing)
pronounced where the change in optical path difference arenotthoseusedforKohlerillumination.Theyareeither
¨
between adjacent areas is not so abrupt (‘wedges’), a captive on the condenser (Figure 3d), or may be recessed
phenomenon known as ‘shading-off’. As a consequence, hexagonalscrewsattherearofthecondenser,requiringan
the centre of one structure may appear the same shade of Allenkeyforadjustment.Ifindoubtonthispoint,referto
grey as that of another of quite different refractive index. themanufacturer’sinstructions.Onceadjusted,theannuli
Phase contrast is better suited to structures with an ‘edge’ in the condenser should remain centred over a lengthy
rather than structures with ‘wedge’ boundaries. period;itshouldnotbenecessarytorecentreeachtimethe
microscope is used. Remove the centring-telescope and
replace the eyepiece, or remove the Bertrand lens. For an
invertedmicroscopethealignmentprocedureisusuallythe
TheHaloArtefact same.
Although in practice the phase contrast system works
Most beams diffracted by the specimen will not pass over the full spectrum of white light, it must necessarily
through the phase ring. However, the phase ring has an be manufactured for illumination of one wavelength,
ENCYCLOPEDIAOFLIFESCIENCES/&2002MacmillanPublishersLtd,NaturePublishingGroup/www.els.net 3
PhaseContrastMicroscopy
Figure3 (a)and(b)showthetopviewofdifferenttypesofphasecontrastcondenser,inwhichthevariousannuliarecontainedwithinahousing.
This permits them to be changed quickly and efficiently as required. (c) The commonly encountered green inscription engraved on the barrel of a
phasecontrastobjective.Thecorrectannulustouseisdenoted,shownherebythedesignationPh3.(d)Theundersideofthecondenserin(b),revealingthe
separatecontrolsforcentringthecondenserontotheopticalaxisduringalignmentofthemicroscope,andthoseforindependentlyaligningtheannulus
withthephasering.Thedifferentsizesofannulicanalsobeseen.(e)and(f)showtheeffectsonthephasecontrastimageofnothavingtheannulusand
phaseringinabsolutealignment.
generallyselectedas550nm.Thisischosenbecausetheeye Bradbury S and Evennett PJ (1996) Contrast Techniques in Light
is most sensitive to green light and objectives are best Microscopy. Oxford: Bios Scientific Publishers.
corrected for spherical aberration at this wavelength. Oldfield R (1994) Light Microscopy: An Illustrated Guide. London:
Hence,foroptimumcontrast,agreenfiltershouldbeused Wolfe.
´
PlasekJandReischigJ(1998)Transmitted-lightmicroscopyforbiology:
in the illuminating light path (Figure 1e). If a satisfactory ˘
phase contrast image is not obtained (e.g. Figure 1d), first a physicist’s point of view, part 2. Proceedings of the Royal
Microscopical Society 33: 196–205.
check that the microscope is correctly set up for Kohler
¨ Pluta M (1989) Phase contrast microscopy. In: Advanced Light
illumination, and then that the condenser is correctly Microscopy, vol. 2: Specialized Methods, chap. 5, pp. 1–90. Oxford:
centredandsetattherightheight.Ifthisfailstoremedythe Elsevier.
situation, check that the image of the annulus is of the
correctsizeanditsimageispreciselysuperimposedoverthe FurtherReading
phase ring.
Beck R (1989) The Development of the Phasecontrast Technique for
Microscopy. In memoriam Fritz Zernike 1888–1966. Scientific and
References Technical Information. Vol. IX, No. 5, June 1989. Wild Leitz.
Ross KFA (1988) Phase contrast and interference microscopy. Micro-
BradburySandBracegirdleB(1998)Introduction to Light Microscopy. scopy 36: 97–123.
Oxford: Bios Scientific Publishers. ZernikeF(1955)HowIdiscoveredphasecontrast.Science121:345–349.
4 ENCYCLOPEDIAOFLIFESCIENCES/&2002MacmillanPublishersLtd,NaturePublishingGroup/www.els.net
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