33
Virtual Microscopy
• Q u a lity o f th e c yto lo g y slide.
• Im age q u a lity : sharpness, contrast, and color.
• Seam less stitch ing .
It goes w ith o u t saying th a t the m in o r q u a lity o f the o rig ina l
cytology slide cannot be overcom e b y sup erior slide acq uisition
technology. For educational purposes, it is therefore advisable to
prepare m u ltip le slides i f possible and select the best fo r im m e -
diate scanning before c o lo r fading sets in .
M o st VSASs are designed to reach reasonable diagnostic q ual-
ity, a llo w in g the p atholog ist to m ake a reliab le diagnosis.4,6,10-12
D epending on the ap p lication, th is approach is ab solutely
adequate since it m in im ize s storage space, scanning tim e, and
expenditure fo r optics. W ith the advent o f im p roved te ch no lo -
gies, m axim a l q u a lity w ill sooner o r late r be standard even
th ou g h m in im a l diagnostic q u a lity w ill s till be good enough in
the fu tu re .
Im age q u a lity is dictated u ltim a te ly b y the laws o f optics.1 In
m ost VSASs, c onventional m icroscope technique is applied fo r
im age acquisition. T he reso lu tio n o f the v irtu a l slide is therefore
dependent on the physical properties o f optical devices used in
tra d itio n a l microscopy. M ost scanning systems use a x 40 dry
objective. In tra d itio n a l settings, x 40 objectives w ith a num eric
aperture (N A ) o f 0.75 are used fo r diagnostic purposes. A x 40
p lan ap ochrom at objective (n o rm a lly used to take images) has
an N A up to 0.95, p rovid in g hig h er lateral reso lu tio n w h ile
depth o f fie ld is reduced. A x 20 p lan ap ochrom at objective w ith
an N A o f 0.75 results in the same lateral re s o lu tio n as a stand-
ard x 40 objective used fo r diagnostics. Som e suppliers argue
th a t by using a x 20 plan ap ochrom at instead o f a x 40 objective,
the scanning tim e w o u ld be reduced b y a factor o f fo u r w ith o u t
c om p rom ising im age quality. Yet th is claim is o n ly true w h en
com paring tw o objectives w ith the same N A. A c q u isitio n speed
is tig h tly b o un d to image quality, w ith hig h er acq uisition speed
o fte n achieved at the cost o f quality, as m en tio n ed in the previ-
ous section.
As a ru le o f th um b , a p p roxim ately 1 -1 .5 m egapixels are
needed w h e n taking pictures w ith a x 40 objective and a stand-
ard camera to m atch the optophysical re s o lu tio n o f a standard
microscope. Increasing the re s o lu tio n o f the camera above this
value increases im age size w ith o u t y ie ld in g m ore in fo rm a tio n
and sho uld therefore be avoided.
O p tim a l c olor in fo rm a tio n is achieved b y collecting the red,
green, and b lue com ponents o f lig h t at each pixel, resulting in
2 4 -b it c olor data. However, m an y C C D chips use a so-called Bayer
m ask, w h ic h measures o n ly 8 bits o f one c olor at a pixel. The
2 4 -b it c olor data are th en created b y in te rp o la tin g c olor in fo r-
m a tio n between 8 -b it data m easured at nonadjacent pixels. Line
scanners m o s tly w o rk w ith in d ivid u a l red, green, and b lue detec-
tors th a t sam ple 2 4 -b it c olor data at each d ig ital slide pixel.
Seam lessly stitched
images
are
especially im p o rta n t in
cytopathology. M eanw hile, m ost c om m ercially available VSASs
provide a good im age a lig n m e n t and relevant overlap between
im age tiles is n o long er a p rob lem , b u t it sho uld nevertheless be
considered w h e n rating the q u a lity o f a v irtu a l slide (Fig. 3 3.2) .
Focus Planes
D iffe re n t strategies are used to a tta in focused images. The m ost
accurate m eth od w o u ld be to autofocus at every im age p osition.
However, th is can lead to alig ning artifacts i f the z-p o sitio n o f
tw o adjacent images differs m ore th a n the depth o f sharpness.
F ig . 3 3 .2 E x a m p le o f im p re c is e a lig n m e n t o f im a g e tile s . The b lue line
m arks th e jo in in g o f tw o im age tiles.
This is especially a p rob lem in uneven and th ic k samples. Fur-
therm ore, autofocusing each image is the m ost tim e-consum ing .
For fla t samples, a so-called focus m ap can be constructed by
autofocusing a lim ite d n um b er o f images at predefined in te r-
vals. T he z-p o sitio n o f the rem a in in g images is interpolated.
W h e n scanning at x 40 (sm all depth o f sharpness), focus maps
sho uld be applied o n ly fo r ab solutely fla t samples i f the req uire-
m ents fo r im age q u a lity are high.
A m a jo r drawback o f m ost v irtu a l slides is the lack o f m u l-
tip le focus planes th a t a llo w the user to slig h tly change focus
w ith in the z-axis. C ytocentrifuge preparations o r collection o f
m aterial in liq u id m edia resulting in "m o n o la y e r" preparations
u su a lly pose few focusing problem s, as the level o f focus is s im i-
la r at each slide loc a tio n (Fig. 33.3A ). Therefore m ost vendors
o f VSASs show examples o f liquid-based cytology preparations
on th e ir homepages o r solely h isto lo g y slides, w h ic h are less
p rob lem atic in th is regard. M ultila ye red cytology smears, u n lik e
single dispersed cells, result in d iffe re n t planes o f v is io n fo r cell
clusters and therefore require greater depth o f focus in regions
w ith dense cell groups. T his fact represents the m a in obstacle in
the p rod u c tio n o f v irtu a l cytology slides. V iew ed w ith a tra d i-
tio n a l microscope, dense clusters o f cells m ay s till be inte rp ret-
able by ad justm ent o f focus in the z-axis. T his does n o t h o ld
true fo r a v irtu a l m icroscope, w h ic h o n ly provides an im age o f
a single focus level. VSASs use autom atic focus adjustm ents o r
predefined focus maps to com pensate fo r subtle changes in the
to p olog y o f a specim en. T he focus in an im age tilin g system is
lim ite d to one focus p o sitio n fo r each in d ivid u a l im age tile. It
fo llo w s th a t image tiles o f th ic k cell groups o r uneven histolog ic
sections cannot y ie ld the same visual in fo rm a tio n as a tra d i-
tio n a l m icroscope (Fig. 3 3.3B ), as som e regions o f the image
w ill always be o u t o f focus (Fig. 33.4A ). To avoid th is drawback,
tw o d iffe re n t approaches, b o th scanning the slide at m u ltip le
focus planes, have been developed.
1. z-stack: im ages fro m p red e fine d slid e areas o r fro m th e
w h o le slid e are acq uired a t m u ltip le focus p lanes (u p to
a b o u t 4 0 ). T h e server d elivers th e a d d itio n a l p lanes to
th e c lie n t o n req uest ( F ig . 3 3 .4 B ).
1015
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