PART THREE
Special Techniques in Cytology
m icroscopy and th e m u ltitu d e o f techniques based on its
p rinciples are exhaustively described in the m onographs o f Tay-
lo r and associates3-5 and b y Thaer and Sernetz.6 Phase-contrast
m icroscopy is treated b y B ennet and associates.7 W o lte r p ro -
vides a superb and exhaustive discussion o f the m icroscopy o f
phase structures.8 For the lig h t optical m icroscopy o f b iolog ic
m aterials u nd er p olarized lig h t, a n u m b e r o f specialized texts
are available: the w e ll-k n o w n text b y B ennett9 and a p articu-
la rly w e ll-w ritte n chapter b y S c hm id t.10 A p p lic a tio n o f p olariza-
tio n optical m ethod s to h isto p a th o lo g y is the exclusive subject
o f a text b y Scheuner and H utschenreiter.11 In ou e discusses
very h ig h -se n sitivity p o la rizin g m icroscopy.12 For interference
m icroscopy, th e m ost com prehensive discussion o f lig h t o p ti-
cal in s tru m e n ta tio n is given b y Krug and colleagues.13 There is
also the text b y H a le 14 and the rig orous discussion o f the optics
involved b y Francon.15 Video-enhanced m icroscopy is discussed
b y In o u e 1
and b y A lle n and cow orkers.16-18 C onfocal m icros-
copy is the subject o f a text b y W ils o n and Sheppard19 and o f the
excellent m onog rap h edited b y Pawley.20 D ig ita l im age analysis
in the cytop atholog ic and h isto p atho lo g ic la b o ra to ry is the sub-
ject o f texts b y Baak and O o rt,21 O b erholzer,22 and Burger and
associates.23
It is the goal o f th is chapter to present those principles o f
lig h t optical m icroscopy the und erstanding o f w h ic h is essential
to atta in effective use o f the instru m e nt.
Image Formation
In a ll im aging m od alities, it is the perform ance o f the objective
th a t determ ines the in fo rm a tio n contents o f the image. It m ay
therefore be h e lp fu l to begin w ith a discussion o f im age fo rm a -
tio n in a lig h t optical microscope. To facilitate an und erstanding
o f the processes involved in image fo rm a tio n , and o f the fac-
tors u ltim a te ly lim itin g perform ance, idealized m od el objects
are useful. For th e im aging o f an object p o in t in to an image
p o in t, we consider a very sm all, round , b a ck-illu m in ate d object,
Optic axis
Fig. 32.1 Object point imaged into image point: geometric optics
representation.
such as a tin y p in h o le in a m etallized slide. For the discussion o f
image reso lu tio n and d iffra c tio n -lim ite d im aging, we consider
m odel objects such as gratings (i.e. arrays o f parallel, opaque,
and fu lly transparent stripes), each grating w ith a d iffe re n t spac-
ing o f the stripes.
The im aging o f an object p o in t in to an im age p o in t b y an
objective is show n in Fig. 3 2 .1 . T he process is illustra ted by
draw ing straight lines, o r rays, to show the resulting m agnifica-
tio n and h o w the rays em anating fro m the object p o in t com e to
a sharp focus at the image p o in t. T his representation by geom et-
ric optics is e m in e n tly useful fo r the design o f optical systems.
Lig ht paths are indicated b y straight lines th a t bend at sharply
defined angles w h e n refracted by an optical elem ent and th a t
intersect to fo rm a m ath em a tic ally fin e im age p oint.
G eom etric optics is, however, a sim p lifie d abstraction, w h ic h
provides valid predictions o n ly u nd er con d itio ns in w h ic h lig h t
interacts w ith relative ly large objects. G eom etric optics ignores
the wave nature o f lig ht.
In reality, lig h t propagates in the fo rm o f electrom agnetic
waves. T he w avefield has a d istin ct structure, defined b y the
frequency o f the ra d ia tio n , the a m p litud e o f the electrom ag-
netic field , and the divergence o f the w avefronts. The im aging
o f a lu m in o u s object p o in t in to an im age p o in t is therefore
better represented b y th e process show n in Fig. 3 2.2 , in w h ic h
the focus is seen as the p o in t o f greatest c on stric tio n o f the
wavefield.
G eom etric optics, w ith its rays and extrem ely sharp focus,
ignores the in h e re n t structure o f the im aging m ed ium . It
assumes the w avelength to be zero. Thus all objects are large
w h en com pared w ith th is in h e re n t structure, and re so lu tio n is
in fin ite in geom etric optics. In th e real w o rld , lig h t waves in te r-
act w ith objects. W h e n the objects are com parable in d im e nsion
w ith the w avelength o f lig h t, the predictions derived fro m geo-
m etric optics are no lon g er valid. To describe the inte rac tion o f
lig h t w ith sm all objects, one has to consider d iffra c tio n theory.
M ost certainly th is is the case w h en one discusses the lim it o f
reso lu tio n o f a lig h t optical microscope.
Fig. 32.2 Object point imaged into image point: converging wavefronts
shown.
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