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Light Microscopy – Secondaryarticle
Brightfield and Darkfield Article Contents
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. Brightfield(Kohler)Illumination – Theory
¨
Illumination . Brightfield(Kohler)Illumination – Practice
. DarkfieldIllumination – Theory
RonaldJOldfield,MacquarieUniversity,Sydney,Australia . DarkfieldIllumination – DryandImmersionSystems
. RheinbergIllumination
Themicroscopeimage,especiallyits contrast, is profoundly influenced by the way that . Practical Applications of Darkfield Microscopy
light is directed on to the specimen. The essential controls required are of intensity, . Filters in Light Microscopy
evenness, area of illumination and angle of illumination.
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Brightfield (Kohler) Illumination – considerably to the cost of a microscope, but extends its
Theory performance and efficiency.
An illumination system for professional microscopy
Thecondenser mustsatisfy the following:
The article ‘Light Microscopy’ describes the role of the . The specimen field should be fully and evenly illumi-
condenser and gives definitions of terms used in this nated.
section. Withhistheoriesofmicroscopelensperformance, . Theintensity of the illuminated field should be variable
Ernst Abbe determined the requirements of specimen within wide limits.
illumination. Essentially, the light source should comple- . Thediameteroftheilluminatedfieldshouldbevariable
tely fill the back focal plane (bfp) of the objective. This is to match the fields of view of the more commonly used
achieved by having a condenser for each objective, objectives, from about 0.2mm to 4mm.
matching the objective numerical aperture (NA). The . The angular aperture (NA) of the condenser should be
microscope illuminator will include a lens or lenses that variable to match the range of objective NAs, from
may act as part of the condenser. This extends the about 0.1 to 1.3.
complexity of the practical use of the condenser; manu- . Selection of wavelengths (colour) may be desirable for
facturers have devised many variations of condensers and someaspectsofspecialized microscopy.
illuminators, the choice of which depends on quality and
price that can be afforded.
Kohler illumination employs a collector lens, close to the
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Source-focusedillumination filament, to form an image of the light source in the
condenserlens‘aperture’,i.e.theplaneofthecondenseriris
Earlier instruments focused an image of the light source diaphragm (better called the aperture diaphragm). The
itself, such as the broad flame of an oil lamp, on to the filament image should completely fill the diameter of the
specimen. Obviously, focusing the filament of a modern aperture diaphragm. This enables an image of the light
electric lamp on to the specimen will give very uneven source to be formed in the bfp of the objective; the light
lighting, and such a technique is not favoured today. In source image will fill that plane, a requirement for the
most microscopes of teaching laboratories, some modifi- illuminationofthefullNAoftheobjective.Thefunctionof
cationofsource-focusedilluminationisemployedbecause the aperture iris is to change the illuminating NA, and to
of its cheapness. At its simplest, a ground glass filter is matchittotheobjective in use.
interposed between the light source and the condenser to Adjacenttothecollectorlensisthefield(iris)diaphragm.
present a more evenly illuminated specimen. Thecondenserlensfocusesanimageofthefielddiaphragm
ontotheplaneofthespecimen.Asthefieldirisis opened
andclosed,thediameteroftheilluminatedspecimenfieldis
¨ changed. To maximize contrast, no more of the specimen
Kohlerillumination
should be illuminated than is actually observed (or
August Kohler (1866–1948) devised an illumination photographed).
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system specifically for photography through the micro- All textbooks will have diagrams attempting to explain
Figure1) but they are quite intimidat-
scope. It is the system now acknowledged by all Kohlerillumination(
¨
manufacturers as being preferred for simple observation ing until one understands that two different ray paths are
as well as for recording microscope images. It adds beingconsideredintheonefigure.Inaddition,eachofthe
ENCYCLOPEDIA OF LIFE SCIENCES © 2002, John Wiley & Sons, Ltd. www.els.net 1
Light Microscopy – Brightfield and Darkfield Illumination
Retina looking down a microscope? How can I superimpose an
image of a measuring device on the specimen?) may be
answeredwithreferencetoKohlerilluminationdiagrams.
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Eyepiece Theseparate functions of the two iris diaphragms, field
EYE exit pupil and aperture, are not appreciated by many microscope
users, and is a frequent source of serious error in
microscope manipulation.
Intermediate
image EYEPIECE
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Brightfield (Kohler) Illumination –
Practice
There is a complexity to the instructional diagrams and
written instructions for Kohler illumination. In addition,
Objective ¨
back focal plane eachbrandofmicroscope–eachmodel–islikelytohavea
different set of instructions. But there are three sequential
OBJECTIVE steps commontoall systems:
Specimen
1. An image of the filament is focused on to the
(condenser) aperture diaphragm. In older micro-
CONDENSOR scopes, the lamp collector lens was focused to form a
Aperture
iris diaphragm sharp image of the filament on the aperture dia-
phragm. Modern systems are said to have their light
sources prefocused and precentred, and include
groundglass filters to make it impossible to check this
step.
2. Animageofthefieldirisdiaphragmisfocusedonthe
specimen. The condenser is raised or lowered for this
step, and lamp-centring facilities will have to be
engaged. The field iris is then opened or closed until
its image matches the size of the observed specimen
field.
3. The condenser NA is matched to the objective NA.
Field The objective bfp is examined (after removing an
iris diaphragm
LAMP eyepiece) to check the ‘seven-eighths’ position of the
COLLECTOR aperture iris (see the article ‘Light Microscopy’).
LENS Imperfect specimens may demand further adjustment
oftheapertureiristoenhancecontrastand/ordepthof
Filament field.
Figure 1 Kohler illumination. The diagram on the left highlights the
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image-formingraypaththatincorporatesthefour conjugateplanes Witheachchangeofobjective, the NA and field diameter
associated with the specimen plane. On the right, the illuminating or also change; strict Kohler illumination therefore requires
aperture ray path is emphasized – four conjugate planes that incorporate ¨
the filament and the lens ‘apertures’. re-adjustment of steps 2 and 3.
AcondenserwithNAgreaterthan1.0isdesignedtobe
‘immersed’ with a drop of immersion oil between the
two ray paths has four conjugate planes, planes that are condenser top lens and the slide undersurface. Although,
images of each other, images on each other of preceding because it is messy, this is rarely done in routine
planes. Familiarity with these two sets of four planes microscopy, it is quite appropriate for those instances
greatly facilitates understanding of the microscope and is when the very highest level of microscope performance is
assumed knowledge in the instructions for more elegant sought.
techniques such as differential interference contrast and Intensity control is by a variable transformer or
fluorescence. Rather simple questions with quite difficult rheostat; the aperture iris should not be used for this
answers (such as Where should the eye be placed when purpose.
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Light Microscopy – Brightfield and Darkfield Illumination
DarkfieldIllumination – Theory DarkfieldIllumination – Dry and
It greatly limits the potential of the microscope to accept ImmersionSystems
that the role of the instrument is merely to produce an
image that is an exact, enlarged copy of the specimen. The simplest darkfield attachment is a homemade patch-
Especially with living material, contrast (i.e. variations in stop(Figure3), a clear disc with an opaque centre. The disc
theimageofcolourorintensity)maybeverypoor,andthe is inserted in the normal brightfield Abbe condenser, close
imagepractically invisible. It is a common experience that tothepositionofthecondenseraperturediaphragm;older
smalldustparticlesintheairareeasiertodiscerniflightis microscopeshadafilterholderconvenientforthispurpose.
directed not into the eye, but across the observer’s line of Theaperturediaphragmitselfmustbeleftfullyopen.The
vision.Althoughthedustistoosmalltobe‘resolved’bythe opaque centre of the disc prevents ‘direct’ illumination
eye, particulate matter is seen, detected or made visible, from entering the objective; the diameter of the opaque
even to the extent that measurement could be made of region is not particularly critical, roughly one-half to two-
particle movement. The same principle is used in micro- thirds the diameterofthecleardisc.Thetransparentouter
scopybypreventingdirectilluminatingraysfromentering margins of the patchstop transmit a hollow cone of light
theobjective. Darkfieldmicroscopy(manyprefertheterm towards, but missing, the objective. Only if a specimen is
‘darkground’) renders the object as bright against a dark present will diffracted or reflected rays be redirected and
background, considerably enhancing the contrast and accepted by the objective. In spite of its rather amateur
visibility of small objects (Figure 2). status,thepatchstopisaveryusefulmicroscopeaccessory,
Figure 2 Plasmodiumvivaxinahumanbloodsmear;brightfield(left),darkfield (right). The bar represents 10 mm.
Figure 3 Equipmentfordarkfieldmicroscopy.Fromtheleft:apatchstop,a drydarkfieldcondenser,animmersiondarkfieldcondenser,andanoil-
immersionobjectivewithiris diaphragm.
3
Light Microscopy – Brightfield and Darkfield Illumination
capable of serving objectives of NA less than about 0.7. If worktheobjectiveirisisclosed(just)sufficientlytoexclude
the microscope is equipped with a phase contrast direct rays.
condenser, the 100 or 40 annuli make excellent,
centrabledarkfieldpatchstopsforthe(phaseornon-phase)
4,10 and 20 objectives. If the condenser NA is RheinbergIllumination
greater than 1.0, immersing the condenser to the slide
(adding one or more drops of immersion oil between the
condensertoplensandthebottomoftheslide)mayprove Replacing the opaque area of the conventional patchstop
helpful by increasing the effective NA of the condenser. by a deep blue transparent filter will change the black
Since the 1980s, dry darkfield condensers (Figure 3) background of a darkfield image into a blue background.
havebecomeavailable; they are called ‘dry’ to distinguish Similarly, if a red filter replaces the clear area of the
them from the immersion systems more fully discussed patchstop,thespecimenwillappearred,ratherthanwhite.
below. The immersion darkfield condensers were quite Acolourlessobjectnowappearsbrightredonadeepblue
difficult to use, mainly because they were so messy; there background. Beautiful optical staining effects, loved by
is no such problem with dry systems. In addition, dry magazine editors, can be obtained by using combinations
darkfield condensers do not have the centring pro- ofcolouredfiltersinthisway–suggestedfirstbyRheinberg
blems sometimes associated with the patchstop. These in 1896.
darkfield condensers function by having reflective curved
surfaces to give highly oblique lighting on the specimen
Figure 2).
( Practical Applications of Darkfield
Immersiondarkfieldcondensers(Figure3) Microscopy
Thesearenotsocommonlyusednowthatdrysystemsare At low magnifications, darkfield is extremely helpful to
commercially available. However, if an oil-immersion findandfocusnear-transparentpreparations.Thematerial
objective with an NA of 1.2 or more must be used for the doesnotevenhavetobeinfocus,abrightblurshowingits
observation, then the darkfield condenser must have an presence.
even higher NA, making oil-immersion condensers essen- An exciting prospect of darkfield microscopy is that
tial. These are made with mirrored surfaces so that the particles below the resolving power of the objective might
illuminating cone NAs are approximately 1.2 to 1.4, even be revealed; it is suggested that the minimum visible
moreobliquethanwiththedrycondensers.Itisessentialto diameterofawhitepointonablackbackgroundislimited
immersethecondensertotheslide,otherwisetotalinternal bytheintensity of illumination, not the NA of the optical
reflection occurs at the condenser glass/air boundary and system. More practically, the high contrasts generated by
no light gets to the slide. Immersed objectives and darkfield permit low magnifications, i.e. wider fields, for
immersed condensers make for troublesome microscopy. investigating the presence or absence of very small
Oil-immersion objectives intended for darkfield work pathogens,protozoa,bacteria,etc.(Figure4).Somecaution
should have an iris diaphragm in the objective bfp. For may need to be exercised in interpreting the image.
brightfieldworktheobjectiveirisisleftopen;fordarkfield Although, in the recent past, darkfield has been of prime
Figure 4 AbloodsmearwithTrypanosomasp.:brightfield(left), darkfield (right). The bar represents 10 mm.
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