LSC Article

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MINIREVIEW

Laser-Scanning Cytometry: A New Instrumentation

with Many Applications

Zbigniew Darzynkiewicz,* ,1 Elzbieta Bedner,* , Xun Li,* Wojciech Gorczyca, and Myron R. Melamed

*The Brander Cancer Research Institute and Dep ar tm en t of Pa th ology , N ew Y ork M ed ica l Col lege, Va lh al la , N ew Y ork 10 595;

an d Dep ar tm en t of Pa th ology , Pom era n ia n S chool of M ed ici n e, S zcz ecin , Polan d

T h e l a s e r -s c a n n i n g c y t o m e t e r ( L S C) i s a m i c r o s c o p e -

b a s e d c y t o fl u o r o m e t e r w h i c h h a s a t t r i b u t e s o f b o t h

fl o w a n d i m a g e c y t o m e t r y . La s e r -e x c i t e d fl u o r e s c e n c e

e m i t t e d f r o m fl u o r o c h r o m e d i n d i v i d u a l c e l l s o n a m i -

c r o s c o p e s l i d e i s m e a s u r e d a t m u l t i p l e w a v e l e n g t h sr a p i d l y w i t h h i g h s e n s i t i v i t y a n d a c c u r a c y . T h o u g h

t h e i n s t r u m e n t h a s b e e n a v a i l a b l e c o m m e r c i a l l y f o r

o n l y 3 y e a r s , i t i s a l re a d y u s e d i n a v a r i e t y o f d i f fe r e n t

a p p l i c a t i o n s i n m a n y l a b o r a t o r i e s . T h i s r e v i e w f o c u s e s

o n t h e f o l l o w i n g u n i q u e a n a l y t i c a l c a p a b i l i t i e s o f L S C

w h i c h c o m p l e m e n t t h o s e o f fl o w c y t om e t ry a n d fl u o -

r e s c e n c e i m a g e a n a l y s i s : ( a ) t h e c e l l s a r e p o s i t i o n e d o n

s l i d e s d u r i n g m e a s u r e m e n t s o t h e y m a y b e e x a m i n e d

r e p e a t e d l y o v e r t i m e , a f e a t u r e u s e f u l f o r s t u d i e s o f

e n z y m e k i n e t i c s a n d o t h e r t i m e - r e s o l v e d p r o c e s s e s ; ( b )

s e q u e n t i a l a n a l y s i s o f t h e s a m e c e l l s c a n b e c a r ri e d o u t

u s i n g d i f fe r e n t i m m u n o - o r c y t o c h e m i c a l s t a i n s o r g e -

n e t i c p r o b e s , m e r g i n g i n f o r m a t i o n o n c e l l i m m u n o p h e -

n o t y p e , c e l l f u n c t i o n s , e x p r e s s i o n o f p a r t i c u l a r p r o -

t e in s , D N A p lo i d y a n d c e ll c y c le p o s it io n , a n d /o r

c y t o g e n e t i c p r o fi l e f o r e a c h m e a s u r e d c e l l; ( c ) a n y o f

t h e c e l l s m e a s u r e d c a n b e r e l o ca t e d t o c o r r e l a t e w i t h

v i s u a l e x a m i n a t i o n b y fl u o r e s c e n c e o r b r i g h t fi e l d m i -

c r o s c o p y o r w i t h a n y o t h e r p a r a m e t e r ; ( d ) t o p o g r a p h i c

d i s t ri b u t io n o f fl u o r e s c e n c e m e a s u r e m e n t s w i t h i n t h e

c e l l , i n c y t o p l a s m v s n u c l e u s , p e r m i t s a n a l y s i s o f t h e

t r a n s l o c a t i o n o f r e g u l a t o r y m o l e c u l e s s u c h a s N F B ,

p 5 3 , e t c ., a n d i s e s s e n t i a l f o r FI S H a n a l y s i s ; (e ) h y p e r -

c h r o m i c i t y o f n u c l e a r D N A a s m e a s u r e d b y m a x i m a l

p i x e l fl u o r e s c e n c e i n t e n s i t y a l l o w s o n e t o i d e n t i fy c e l lt y p e s d i f f e r i n g i n d e g r e e o f c h r o m a t i n c o n d e n s a t i o n

s u c h a s m i t o t i c o r a p o p t o t i c c e l l s ; ( f) a n a l y s i s o f t i s s u e

s e c ti o n a r c h i te c t u re a n d o f t h e c o n s ti t u e n t s i n

t r a n s e c t e d c e l l s w i t h i n t i s s u e s e c t i o n s b y r a t i o m e t ri c

a s s a y s n o r m a l i z e d t o D N A c o n t e n t e x t e n d s a p p l i c a -

t i o n s o f L S C i n c l i n i c a l p a t h o l o g y ; ( g ) b e c a u s e c e l l l o s s

d u r i n g s a m p l e p r e p a r a t i o n a n d s t a i n i n g i s m i n i m a l ,

s a m p l e s w i t h a p a u c i t y o f c e l l s c a n b e a n a l y z e d ; a n d

( h ) a n a l y z e d c e l l s c a n b e s t o r e d i n d e fi n i t e l y , e . g . , f o r

a r c h i v a l p r e s e r v a t i o n o r a d d i t i o n a l a n a l y s i s . Po t e n t i a

f u t u r e a p p l i c a t i o n s o f L S C a re d i s c u s s e d . 1999 Academi

P r e s s

INTRODUCTION

Pr ogress in cell biology is being dr iven by t he development of quantitative analytical methods applicableto individual cells or cell organelles. It began in the1950s with the development, by Caspersson and hiscolleagues at the Karolinska Institute in Stockholm, omicrospectrophotometry, followed by microfluorometryand microinterferometry [1, 2]. These methods, whichprovided a means of measuring DNA, RNA, and pro

teins in the cell, initiated the modern era of cell biologybased on quantitative rather than qualitative visuacell analysis. The next methodology significantly contr ibuting t o progress in cell biology was aut oradiography [3]. Applications of autoradiography were wides p r e a d a n d p a r t i cu l a r l y f r u i t fu l i n s t u d y in g ce lreproduction, where they provided a foundation for themodern concept of the cell cycle.

The introduction of flow cytometry (FC) initiated athird phase in methods development [46]. AlthoughFC is st ill a relat ively youn g meth odology, it is a lreadyin wide use and ha s found myriad a pplications in basi

and clinical research as it has in the diagnostic clinicalaborat ory [79]. FC offers severa l advan ta ges over th emethods used before. One is the rapidity of cell measurements, allowing one to analyze large populationsof cells, detect rare cells, and distinguish subpopulations of cells according to their different characteristics. Also attractive is the possibility of multiparametric analysis to quantify relationships among severacell constituents in particular cell subpopulations selected by some other feat ur e. Sort ing, another valua blefunction of FC, has been used to select clones of livecells for propagation and even to sort individual chro

1 To wh om cor r esp on d en ce an d r ep r in t r eq u ests sh ou ld b e ad -dressed at Brander Cancer Research Institute, New York Medical

College, 19 Bradhurst Ave., Hawthorne, NY 10532. Fax: 914-347-2804. E -mail: da [email protected].

0014-4827/99 $30.01Copyright 1999 by Academic P res

All rights of reproduction in any form reserved

Experimental Cell Research 249, 112 (1999)

Article ID excr.1999.4477, available online at http://www.idealibrary.com on


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mosomes for development of chr omosomal DN A libra r-es. Flow cytometry with cell sorting is now indispens-

a b l e i n i m m u n ol og y, m o le cu l a r a n d ce ll b i ol og y,cytogenetics, an d the hu man genome pr oject.

There are some limitations of FC, however, whichrestrict its usefulness in certain applications. Amonghese one can list the following:

(a) The individual cells are measured only once.Thus, FC does n ot allow one to a nalyze time-resolvedevents on the same cells, e.g., to measure enzyme ki-netics;

(b) Although, in principle, single cells can be sortedaccording to their measured cell parameters and theirmorphology examined, this procedure is cumbersomeand rarely used. As a corollary, filing the measuredcells for archival preservation is also restricted;

(c) Cell an alysis by FC is at zero spat ial resolution;nitial attempts to resolve cell structure by slit-scanlluminat ion found little acceptan ce. Thus, FC cann ot

provide information on the spatial distribution of fluo-rochromes within the cell, i.e., nuclear vs cytoplasmic

ocalization, un iform ity of distr ibution, localizationwith another fluorochrome, etc.;

(d ) F C d o e s n o t a l l o w o n e t o re s t a i n t h e a l re a d y -measured cells with another probe(s) and merge theresults on a cell-by-cell basis;

(e) Analysis of solid tissue by FC is based on mea-surement of dissociated cells or nuclei, and the isola-ion procedure, whether mechan ical, enzymat ic, or

with detergents, produces a plethora of u ndesirableeffects. Needless to say, the information on tissue to-pography, e.g., relationship of tumor cells to host stro-mal or infiltrating cells, blood vessels, the presence of

islands of proliferating or quiescent cells, etc., is losafter cell dissociation;

(f) Since sample preparation for FC often requiresrepeated centr ifugations, significant cell loss occur sThe loss m ust be compensated by star ting with largnum bers of cells per sample. Therefore, small-sizesamples (e.g., fine needle a spirates; spinal fluid) a reseldom analyzed by FC.

The newly developed, microscope-based laser-scanning cytometer (LSC) offers many of the advantages o

FC and few of the l imitations l isted above [10, 11]Although LSCs, which a re ma nufactured in t he Un itedStat es (CompuCyte Corp., Cambr idge, MA) and in J apan (Olympus Optical Co., Tokyo), became commercially available just a few years ago, numerous reporthave already been published describing their capabilit ies and numerous applications. This review, whilediscussing much of the published data on LSC, is focused on applications that are unique to this instrumentation. An excellent review article describing theinstru ment itself, its ana lytical capabilities, and similarities and differences between LSC vs FC vs auto

mat ed fluorescence ima ge an alysis (FIA) systems warecently published by Kamentsky et al. [11]. It shouldbe noted tha t because of similarity in n ame, th e laserscan ning microscope, a confocal microscope illum inat ed by a scann ing laser beam, and other instr umen tshaving a scanning laser as an illuminat ion source happen to be confused with LSC.

PRINCIPLES OF CELL MEASUREMENT BY LSC

F i gu re 1 p re se n t s a d ia g ra m m a t ic s ch e m e o f t h eLSC. The m icroscope (Olympus Optical Co.) is a n inte

FIG. 1. Scheme representing m ajor components of the LSC. See text for explana tion.

2 DARZYNKIEWICZ E T AL.


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gral part of the instrument and provides essential me-chanical and optical components. The specimen which

s on a glass slide on the stage of the microscope isexcited by a laser beam that rapidly scans the micro-scope slide. In the curr ent inst ru ment s beams from twoasers (argon and helium neon) spatially merged by a

set of dichr oic mirrors a re directed ont o the comput er-contr olled oscillat ing (350 Hz) mirr or which directs t hebeams t hr ough th e epi-illuminat ion port of the micro-scope and images them through the objective lens ontoh e s l i d e . Th e l a s e r b e a m s , t h u s , ra p i d l y s we e p t h e

area of the microscope slide under the lens. Dependingon the lens magnification the beam spot size variesfrom 2.5 (at 40 magnification) to 10.0 m ( a t 1 0

ma gnificat ion). The slide with its position monitored bysensors is positioned on the computer-controlled mo-orized microscope stage and moved, with the stage, at

0.5-m s t ep s p er l a se r s ca n , p erp e n dicu l a rly t o t h es c a n . Li g h t s c a t t e re d b y t h e c e l l s i s i m a g e d b y t h econdenser lens and recorded by scatter sensors. Fluo-rescence emitted by the specimen is collected by theobjective lens partially directed to a CCD camera formaging. Another part of fluorescence light is directedh rou g h t h e s ca n l en s t o t h e s ca n n i n g m i rror. Up on

reflection, it passes t hr ough a ser ies of dichr oic mirr orsand optical interference filters to reach one of the four

photomultipliers. Each photomultiplier records fluorescence at a specific wavelength range, defined by the

combination of filters and dichroic mirrors. A lighsource, additional to the lasers, provides transmittedillumination which is used to visualize the objectsthr ough a n eyepiece or t he CCD camer a. The measu rement of cell fluorescence (or light scatter) is computercontrolled and triggered by setting a threshold contourfor the cell above background (Fig. 2). For each measured object th e following par amet ers ar e r ecorded byLSC:

(a) Integrated fluorescence intensity over the integration contour which can be adjusted t o a desiredwidth with r espect t o the th reshold cont our , represent

ing the sum of intensities of all pixels within the area(Fig. 2);

(b) The value of maximal pixel within this area, socalled peak or max pixel value;

(c) The area within the integration contour, r epresenting the number of pixels within the contour area

(d) The perimeter of the contour (in micrometers);(e) Fluorescence inten sity integra ted over t he ar ea o

a t oru s of desired width defined by the periphera l contour located around (outside of) the primary integration contour. Thus, if the integration contour is set forthe nucleus based on, e.g., red fluorescence (DNA

FIG. 2. Different settings for analysis of nuclear, total, and/or cytoplasmic fluorescence. When nuclear DNA is stained with the red

fluorescing dye (e.g., PI), the threshold contour (T) is set on red signal to detect the nucleus, e.g., as in a. The integration contour (I) is thenet a few pixels outside of T to ensure that all nuclear fluorescence is measured and integrated (a). However, when cytoplasmic fluorescences also measured, I is set far away from T to ensur e th at fluorescence emitted from cytoplasm is int egrated a s well (b). It is also possible t

eparat ely measure n uclear and cytoplasmic fluorescence as shown in c. The peripheral contours (P) are then set a t the desired n umber opixels outside of I and the fluorescence intensities emitted from both areas, within the I boundary and within the P torus, are separately

measu r ed a n d sep ar ately in teg r ated . I n each case th e b ack g r ou n d con to u r is au to matically set ou tsid e th e cell a n d th e b ack g r ou n dfluorescence is subtr acted from nuclear, cytoplasmic, or total cell fluorescence. The actual cell contours, a s they a ppear on the monitor, a r

hown in d.

3LASER-SCANNING CYTOMETRY


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stained by propidium), th en th e integrated (or maximalpixel) green fluorescence of FITC-stained cytoplasmcan be measured separat ely, within the integrationcontour (i.e., over t he n ucleus) and within the periph-eral contour, i.e., over the rim of cytoplasm of desiredwidth outside the nucleus. It sh ould be noted tha t allabove values of fluorescence (a, b, an d d) are aut omat -cally corrected for background which is measured lo-

cally outside the cell, within the background contour

(Fig. 2);(f) The slide position on X a n d Y coordinates of the

maximal pixel; an d(g) The computer clock time at the moment of mea-

surement.Ratios of the r espective para meters are easily preset

as a n ew parameter, and the rat iometric data a re thencollected or calculated during data analysis. The spec-rum overlap measured by individual photodetectors

can be electronically compensated during data analy-sis.

There are many similarities between LSC and FC

(Ta bl e 1 ). Th e m e a su re m en t s b y LS C a r e ra p id a n dwith optimal cell density up to 100 cells can be mea-sured per second. The accur acy and sensitivity of cellfluorescence measu rement s by LSC ar e compar able tohe most advanced flow cytometers [10, 11]. Other fea-ures that can be measured, such as integrated fluo-

rescence intensity of the cells, time of measurement,and forward light scatter also are identical for bothnstru ments. However, r ight an gle (side) light scatt er,

common to FC, cannot be measured by LSC. LSC, onh e ot h e r h a n d , m e a su r e s in d iv id u a l p ix el va l u es ,

which cannot be measured by F C. This pa rameter re-

flects inhomogeneity of the fluorochr ome distributionwit h t h e a n a l yz ed ob je ct , a n d t h e p ea k p ix el v a lu erepresents t he ma ximal concent ra tion per a rea imagedon a single pixel. In contrast, peak fluorescence measured by FC represents the peak value of the analogelectronic signal from fluorescence integration of thecellular fluorescence. The possibility of different iaanalysis of fluorescence emitted from nucleus vs cytoplasm is another feature of LSC absent in FC.

The most characteristic feature of LSC distinguishing it from FC is that cell analysis is done on a slideThis offers th e possibility of visua l cell examina tion t oassess morphology and corr elat e it with th e measu redparameters. It also allows cell image capture, analysisand/or display. Furthermore, additional cytofluorometric ana lysis of th e sam e cells is possible usin g new setsof mar kers or oth er cont our ing thr esholds. The resultsof the sequent ial measuremen ts can th en be int egra tedin list m ode fashion, u sing th e mer ge capability of thei n st r u m e n t . Ap p li ca t i on s t h a t d es ce n d fr om t h e s eunique featu res of LSC ar e discussed below.

CELL MORPHO METRY

As mentioned, in contrast to measurements by FCwhich are at zero spatial resolution, LSC offers thep os s i b il i t y of a n a l y zi n g fl u o roch r o m e l oca l i za t i onwithin the cell and relating it to cellular structuresApplications of LSC exploiting this feature can be categorized in three groups.

M axi m al pi xel (fl u ores cen ce peak ) an aly si s. In thifirst group are applications that util ize the maximapixel measurement as a feature discriminating the cell

TABLE 1

Differences an d Similarities between Cell Ana lysis by F C a nd LSC

F C LSC

Cell st a in in g a n d m ea su r em en t In su spen sion On a slide

Correlation of cell measurement with analysis of cell morphology(image ana lysis)

On ly a ft er sor t in g P ossible

Analysis of differences in intracellular fluorochrome localization

(e.g., nucleus vs cytoplasm)

P ra ct ica lly n ot possible P ossible

An alys is of h igh est loca l in ten sit y of flu or och rom e in a cell N ot possible P ossible (m axim al pixel)

Nu m ber of m ea su r em en t s of a given cell On e Sever a lSequ en t ia l m ea su r em en t s of a cell in t im e (k in et ics) Not possible P ossible

Sequ en t ia l a n a lyses of a cell wit h differ en t pr obes Not possible P ossibleCell loss du rin g st ain in g a nd m ea su rem en t Sign ifica nt , depen ds on n um ber of

centrifugations

Minimal (5%)

Ar ch iva l st or a ge of t h e m ea su r ed cells Not possible P ossible

An a lysis of t issu e sect ion s Not possible P ossibleAnalysis of neighboring cell-to-cell interactions; tissue

architecture analysis

N ot possible P ossible

Sem ia u t om a t ic F ISH a n a lysis P r a ct ica lly n ot possible P ossible

An a lysis of cells by ligh t sca t ter in g In for wa r d a n d side dir ect ion s On ly in for wa r d dir ect ionCell/ch r om osom e sor t in g P ossible Not possible

Speed of cell m ea su r em en t Up t o 10,000 cells per secon d U p t o 100 cells per secon dMu lt ipa r a m et er a n a lysis P ossible P ossible



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Maximal p ixel DNA-associated fluorescence is a sensi-ive mar ker of chr omat in condensat ion. N amely, DNAn condensed chromatin, e.g., in mitotic or apoptotic

cells, shows increased stainability (per unit area ofchr omatin image) with most fluorochr omes. Thu s, evenf th e integr at ed fluorescence of th e an alyzed cells (rep-

resenting th eir DNA cont ent) is the sam e, the ma ximalpixel values of the cell with condensed chromatin is

great er compa red t o th e cell with m ore diffuse chr oma-i n s t ru ct u re . DNA h y p erch rom i cit y wa s u s ed a s a

mar ker t o distinguish m itotic and immediately postmi-otic G 1 cells from interphase cells [12, 13]. Although

mitotic cells can be recognized by FC u sing a var iety ofmarkers [reviewed in 14], the advantage of this ap-proach by LSC is t hat a single fluorochr ome is u sed todiscriminate between G 1 vs S vs G 2 vs M phase cells.Ther efore, additional color dye(s) can be u sed t o detectother cell constituents, e.g., cyclins, cytokeratin, cyto-k i n es , o r i m m u n op h e n ot y p e m a r k e rs , on t h e s a m eprepar ation. By a pplying t his principle, pulse labeling

of DNA-replicat ing cells with Br dU (detected with an ti-BrdU mAb) was combined with identification of mitoticcells by LSC to study the cell cycle kinetics by thefraction of labeled mitoses (FLM) meth od [15]. TheFLM m eth od, originally developed for au tora diogra phy[16], provides a wealth of inform ation on cell cyclekinetics but is cumbersome and time consuming andherefore rar ely used. Its adaptation to LSC drama ti-

cally simplifies the procedure and shortens t ime ofan alysis [15].

Similar to mitosis, chromatin condensation duringapoptosis also ma nifests by DN A hyperchr omicity. Ap-

optotic cells, therefore, can also be identified by theirhigh value of ma ximal pixel of DNA-associated fluores-cence, and their identification can be combined withanalysis of the cell cycle distribution [Fig. 3; Refs. 17and 18]. H owever, because both mitotic and apoptoticcells are characterized by high maximal pixel value,heir distinction from each other is not possible by this

method. This l imitation is of par ticular importa ncewhen a poptosis is indu ced by mitotic inhibitors su ch a sTaxol or vincristine a nd th erefore th e sam ple conta insarge proportions of both mitotic and apoptotic cells.

It should be noted, however, that several other meth-

ods of identification of apoptotic cells, including theirrecognition by th e presence of DNA st rand breaks,decreased mitochondrial transmembrane potential, orfractional DNA cont ent [reviewed in 19], h ave beensuccessfully adapted to LSC [18, 2022]. The possibil-ty t o subject th e measur ed cells t o morphological ex-

amination provided by LSC is particularly importantn s t u d i e s o f a p o p t o s i s . Th i s i s d u e t o t h e fa c t t h a t

apoptosis was originally defined by morphological cri-eria [23] and cell morphology still remains the gold

sta nda rd t o ident ify this m ode of cell deat h. Using LSC,for example, it was possible to discriminate between

the genuine apoptotic cells and false-positive cells inperiph era l blood and bone mar row of leukemic patientundergoing chemotherapy [18]. The latter cells weremonocytes/ma crophages containing apoptotic bodie(probably ingested from the disintegrating apoptoticcells) in t heir cytoplasm. While both th e genuine apoptotic cells and the false-positive cells contained num e rou s DNA s t ra n d b rea k s a n d were i n dis t in g u is h

able by FC, analysis of their morphology by LSC madepossible th eir positive identificat ion [18]. Based on th iobservation and other findings it was concluded thatLSC is th e inst ru men t of choice in a na lysis of apoptosi[18, 24].

Early during apoptosis the proapoptotic regulatoryprotein Bax undergoes translocation into mitochondria[25], where, most likely, it is involved in facilitatingrelease of cytochrome c and dissipation of t he mitochondrial t ran smembra ne potential [26]. Int erestinglythe mitochondrial tran slocation of Bax, which is reflected by th e increase in its local densit y (as a resu lt oaccum ulat ion in mitochondr ia), can be detected by LSCa l s o b y m a x im a l p ix el of B a x i m m u n o flu ore s ce n ceana lysis (Fig. 4). Likewise, t he tr an slocation of cytochrome c from mitochondria into cytosol during apoptosis can be detected by a decrease in maximal pixel oits imm unofluorescence (man uscript in prepar ation)The a na lysis of ma ximal pixel to detect tr an slocat ion omacromolecules (when the translocation is associatedwith chan ge in th eir local density) may find ma ny oth erapplicat ions, e.g., to monitor a ctivat ion or dea ctivat ionof th e signal tr ansdu ction molecules, receptor cluster

ing, etc. (Table 2).The maximal pixel value was also useful as a markerdiscriminat ing lymphocytes, m onocytes, a nd granu locytes [27]. These cell types differ between themselvesb y t h e d e gr e e of ch r o m a t i n con d e n s a t i on . C on s equently, stainability of their DNA with propidium, reflected by m aximal pixel value, is also different [27]Another par ameter measur ed by LSC that is corr elated(inversely) with chromatin condensation is fluorescence ar ea. This para meter reflects n uclear size an d ialso different for lymphocytes, monocytes, and granulocytes [27]. Single-color an alysis by LSC, t her efore

yields differential count of white blood cells similar totha t pr ovided by FC based on simultan eous a nalysis oforwar d a nd side light scatt er [79].

N u clear vs cytopla sm ic fl u orescen ce. The secondgroup of applications of LSC is associated with i tability to spatially resolve fluorescence within the cela n d ca n b e a p p li ed t o a n a l yze t ra n s i t of i n div id u aproteins, detected im mu nocytochem ically, between differen t cell compar tm ent s, nota bly between nu cleus andcytoplasm. Tran slocation of individual proteins fromcytoplasm to nucleus often reflects activation, and aclassical example of such a protein is nuclear factor



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kappa B (NF-B). This ubiquitous factor is involved inregulation of diverse immune and inflamma tory re-sponses and also plays a role in control of cell growth

and apoptosis [28]. In its inactive form NF-B remains

in th e cytoplasm sequestered t hr ough int eraction withIB protein. Rapid translocation of NF-B from cytoplasm to nucleus occurs in response to extracellular

signals or DNA dam age an d is considered to be a hall

TABLE 2

Major Applications of LSC

Applica t ion s Adva n t a ges, exa m ples

m m u n oph en ot ypin g Min im a l cell loss (specia l u t ilit y for h ypocellu la r sa m ples), sa vin gs on

reagents [38, 39]Cell cycle a n a lysis Det ect ion of m it ot ic a n d post m it ot ic cells by a n a lysis of m a xim a l pixel

of DNA-related fluorescence [12, 13, 15, 17, 20, 30]Det ect ion of a popt osis E xa min at ion of cell m or ph ology, det ect ion of a popt ot ic cells by a na lysis

of maximal pixel of DNA-related fluorescence [17, 18, 48]

Analysis of enzyme kinetics, dru g upt ake, or ligandbinding

True kinetics (or binding affinity) measu red on the same individualcells (receptors) [37]

Analysis of activation or deactivation of macromolecules by

their tra nslocations

Cytoplasmic/nuclear translocations (e.g., activation of NF-B or p53)

[29], mitochondrial translocations (e.g., Bax, detected by maximalpixel an alysis, see Fig. 4)

F ISH a n a lysis Ra pid, sem ia u t om a t ic, u n bia sed select ion of cells [11, 32]Correlation of cell function with morphology and other cell

attr ib u tes

Unique t o this instru menta tion, offers a possibility to probe functions o

live cells and directly correlate functional events with cell morphologyand/or the changes tha t can be detected only on fixed cells

S tu dies of cell-t o-cell in ter act ion s P os sible by a na lys is of t he n eigh bor in g cells gr owin g on t he s lid e(identified by their recorded XY position)

Ap p li ca t i on s i n p a t h ol og y, t i ss u e s e ct i on a n a l ys is R a pi d a n a l ys is of e xp r e ss ion of ce ll p r ol ife r a t ion a n t i ge n s , h or m on ereceptors, etc., a s prognostic markers, ana lysis of tissue (tumor)

architecture [4247]

FIG. 3. Ident ificat ion of apoptotic cells by LSC based on h igh values of ma ximal pixel detecting r ed fluorescence or fra ctiona l DNA cont en

of propidium iodide (PI)-stained cells. Exponentially growing HL-60 cells, untreated (A) or treated with 0.15 M DNA topoisomerase nhibitor camptothecin (CPT) for 3 (B) or 4 h (C) to induce apoptosis, were stained with PI in the presence of 100 g/ml of RNase A [21, 22]

The contour maps represent bivariat e distributions of cells with respect to their integrated red fluorescence (DNA content) vs ma ximal redfluorescence pixel value. Only mitotic cells (M) have high ma ximal pixel values in t he un tr eat ed cultur e. Apoptotic cells (Ap) tha t a re pr esen

n the CPT-treated cultures are characterized either by increased red fluorescence maximal pixel value or by a low (fractional sub-G 1) DNAcontent. The relocation feature of LSC allows one to observe morphology of the cells selected from particular regions of the bivariate

distributions. The cells with h igh maximal pixel value or with fractional DNA content show chromat in condensat ion and nuclear fragmenation, typical of apoptosis (Ap; bottom four panels).



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mark of activation [28]. NF-B was detected immuno-cytochemically in severa l leukemic cell l ines with

FITC-tagged ant ibody, and its presence in the nu cleusvis-a-vis cytoplasm was monitored by LSC measure-ments of green fluorescence (FITC) integrated over thenucleus vs over the cytoplasm, respectively [29]. Acti-vation led to a rapid increase in NF -B-associated flu-orescence mea sur ed over th e nu cleus concomita nt witha decrease in fluorescence over the cytoplasm, whichwa s re fle ct e d b y a l a rge in cre a s e i n t h e n u cl ea r t ocytoplasmic fluorescence ratio. One of the virtues ofhis assay is that NF-B activat ion could be corr elated

with cell m orphology, immu nophenotype, or cell cycleposition [29]. This applicat ion of LSC can be extend ed

o monitor other factors that upon activation accumu-ate in cytoplasm and/or undergo translocation to the

n u cl eu s , s u ch a s t u m o r s u p p res s or p 5 3 a n d s ig n a lransduction or cell cycle regulatory molecules.

Since LSC allows one to int egrat e (mer ge) th e resu ltsof two or more measurements, it is possible to measurehe same cells twice, once with the contour setting to

measure only nucleus and subsequently with a settingha t m easures both nu cleus and cytoplasm. Such ana l-

ysis revealed nu clear expression of cyclin B1 whichcould be compar ed with tota l cellular expression of th isprotein [30, 31].

Fluorescence in situ hybridization (FIS H). FISHana lysis represents the third type of LSC application

t h a t a re b a s e d o n t h e c a p a c i t y o f t h i s i n s t ru m e n t t ospatially resolve the distribution of fluorescent regionswithin the cell [11, 32]. The software developed for thisapplication allows one to establish, within a primarycontour representing nu cleus stained with a part iculardye (e.g., propidium), a second set of contours representing an oth er color (e.g., FITC) fluorescence. Fivesecondary features are then measured in addition tothe major featur es listed ear lier, nam ely (a) num ber osecondary contours (i.e., FISH spots); (b) distance between t he nea rest s pots; (c) int egrat ed and (d) ma ximapixel fluorescence; and (e) fluorescence ar ea. The th ree

last pa ram eters (c e) are measur ed for each secondar ycontour (Fig. 2).

An obvious advan ta ge of LSC over visua l an alysis oFISH is the unbiased selection of the measured cellsa n d s em i a u t om a t e d , r a p i d m e a s u r em e n t . F u r t h e rmore, ana lysis of the integrated fluorescence int ensityof the secondary contours may yield information pertaining to th e degree of amplificat ion of part icular genome sections. However, as emph asized by Kam ent skyet al. [11, 32], semiautomated FISH measurements byLSC are subject to potential traps and require highquality technical pr eparat ions.

FIG. 4. Detection of Bax translocation from cytosol to mitochondria by analysis of the increase of maximal pixel of Bax immunofluores-

cence. MCF-7 cells were induced to apoptosis by their exposure to 0.15 M camptothecin (CPT). Apoptosis of MCF-7 cells is observed witha delay (2472 h) and is preferential to S-phase cells (52). Bax was detected in these cells immunocytochemically, with the FITC-conjugated

Ab; DNA was counterstained with PI. Accumulation of Bax in mitochondria prior to and in early stages of apoptosis was revealed by thencreased m aximal pixel value; the int egrated F ITC fluorescence was also increased but to a lesser degree th an the maximal pixel value (no

hown). The DNA content frequency histogram s of the gated su bpopulations of the cells with low a nd high ma ximal Bax pixel values (ahown in 72-h CPT-treated culture) reveal th at the latter consist of predominan tly S-phase cells (manuscript in prepara tion).



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ANALYSIS OF TIME-RESOLVED EVENTS

Current methods assaying cellular enzyme activitysuffer limitations. Biochemical assays of cell extracts,cell fractions, or isolated proteins in bulk provide no

nformation on individual cells needed to assess thentercellular variability of cell populations, detect rare

cells or cell subpopulations with distinct features, orrelate the measur ed par ameter s to normal or abnorma lcells, etc. Although individual cells are rapidly mea-sured by FC, each cell is mea sured only once when inflow. Time-resolved events, therefore, cannot be mea-sured on a cell by cell basis. The kinetics of enzymaticreactions [e.g., 33], change in pH [34], bursts of ionizedntracellular calcium [35], or oxidative product forma-i on [3 4] m e a su re d b y F C a re b a s ed on s eq u en t i a l

measurements of single cells over t ime but not the

same single cells.LSC provides the means to measure kinetic reac-

ions within individual cells in large cell populations(Fig. 5). Thus, r epeated measurement of a group ofcells within a selected area of the slide allows one torecord a ll fluorescence par am eter s as a function of time[37]. Using t he fluorogenic subst ra te di-(leucyl)-rhoda-mine 110, the kinetic activity ofL-aminopeptidase wasmeas ur ed in severa l cell types by LSC [37]. The ra te offluorescein diacetate (FDA) hydrolysis by esterases aswell as the ra te of upt ake of th e lysosomotrophic fluo-rochrome acridine orange (AO) was also assayed [37].

S ev era l h u n d re d ce lls p er s a m p le ca n b e m e a su re dwith a t ime resolution of 10 to 60 s. Since the t imeresolution is inversely proportional to the number omeas ur ed cells per sam ple, fewer cells can be an alyzedi f t h e t i m e i n t e rv a l b e t we e n m e a s u re m e n t s m u s t b e

short. The kinetic curves constru cted for individuacells can be matched with the respective cells, thelatter identified by their position on the slide or classified by their fluorescence image or by light microscopy a fter staining with conventional absorption dye[37].

Repeat ed scanning of the same cells causes fluorescence fading. The fading, which ma y be extensive whentime intervals between scanning are short, unfortunately imposes a limitation on time resolution of thekinetic measur ement. H owever, the fading ra te a s welas the fluorescence recovery rate can be measured in

the same cells by LSC [37] and results corrected appropriately.

CELL IMMUN OPH ENOTYPING

The usefulness of FC for immunophenotyping, especially in analysis of hematological malignancies, is indisputable. LSC can also be adapted to carry out routine immu nophenotyping. Mu lticha mber microscopslides were developed which can be used to au tomatically screen the cells against up to 36 a nt ibodies on asingle slide by LSC [38, 39]. The chambers are filled

FIG. 5. Analysis of enzyme kinetics by LSC. L-Aminopeptidase activity was measured in white blood cells from human peripheral bloodusing a fluorogenic substrate, di-(leucyl)-rhodamine 110, and recording increase in fluorescence intensity of individual cells with time [37]

The slides were then stained with Giemsa and examined by light m icroscopy. Individual lymphocytes, m onocytes, and granulocytes werdentified (their image recorded in the cell galleries; right pa nels) and mat ched with t heir r espective kinetic plots [37].



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with cell sus pension by capillary a ction. In t he a bsenceof serum or other proteins in t he su spension, th e cellsstrongly att ach t o the floor of th e chambers by electr o-static interactions [37, 38]. Various antibody combina-ions are then introduced into the chambers, the cells

are incubated in their presence for 3060 min, and,following th e rin se, their fluorescence is measu red. Therat e of ana lysis is relatively fast, a s it tak es an overall

20 min to screen the cells distributed in 12 chambersabeled with a panel of 36 ant ibodies, measu ring 3000o 5000 cells per chamber [38].

Although th e ra te of measuremen t by LSC is slowerh a n F C , a n d t h e l a c k o f s i d e l i g h t s c a t t e r a n a l y s i smpedes discriminat ion of lymphocytes from mono-

cytes an d gran ulocytes, cert ain a dvan ta ges of LSC mayout weigh th ese deficiencies. Thus, LSC is preferr ed forhypocellular samples which cannot tolerate repeatedce n t r ifu g a t ion s t h a t le a d t o ce ll los s . I t m u s t b estressed that loss of cells during centrifugations, asrequired for FC analysis, is not random but preferen-

ial to different cell types [27]. LSC is also economical:because of small sample size in LSC, the cost of thereagents (mAbs) is reduced by more than 80% com-pared to FC [38]. Fur th ermore, LSC provides th e pos-sibility to relocate immunophenotyped cells for addi-ional analysis or archival preservation. This feature is

discussed later in the article.

APPLICATIONS OF LSC IN PATHOLOGY

Cytometry still plays only a minor role in anatomicpathology. In the two most common types of prepara-

ions, histologic sections and fine-needle a spirates(FNA), diagnosis is greatly dependent on the judgmentand experience of the examiner a nd is likely to remainso. H owever, by quan tifying key a ttr ibutes of selectedcells in a specimen of known diagnosis, cytometry cancon t r i bu t e u s e fu l p rog n os t i c i n form a t i on a n d h e l pguide therapy. LSC is par ticularly suitable for thisask . FNA sa mples provide adequa te n um bers of cells

for analysis by LSC and no significant cell loss occursduring the staining and measurement [40, 41]. In his-ologic sections, a reas of interest th at may be a minor

component of the whole section can be selected to ex-

clude extraneous tissues from measurement. As al-ready noted, the slides can be destained an d rest ainedo measure additional attributes of the same cells; the

relocat ion feat ur e of LSC allows one to pr ecisely iden-ify each cell by its location on the slide. Several pub-ications do account for the usefulness of LSC in anal-

ysis of tissue sections or FNA sa mples [40 46].One of the dra wbacks inher ent in m easur ing const it-

uents of the cells in histologic sections is that most ofhe cells are transected at different levels. Thus, be-

cause only a fraction of a cell or nucleus, u nkn own insize, is assa yed, such measur ement provides no infor-

mat ion about qua nt ity of the measur ed constitu ent percell. However, a ratiometric analysis, relating the quantity of th e measu red n uclear const ituent per u nit of DNAnormalizes the data and makes them comparable between sections of different thickness. Such normalization is easily accomplished by LSC, where contouringcan be done on the DNA-associated fluorescence andanother color fluorescence, representing th e m easur ed

n u cle a r con s t it u e n t a n d i n t egra t e d wit h i n t h e s a m econtour, is expressed as a rat io per DNA-associatedfluorescence. This method of normalization was applied to a study of estrogen and progesterone receptorsin human breast cancer [46].

LSC also offers advant ages over cur rent meth ods oimage analysis that with few exceptions (e.g., Feulgenstaining) rely entirely on light-absorbing dyes an d a renot qua nt itat ive. The basic approach of image an alysisto classify cells by their light microscopic morphologyfails to take advantage of important functional information inherent in the immuno- and cytochemical as

says of LSC and FC.Still to be worked out ar e th e comput er-assisted a n

alytical met hods th at will be needed to fully exploit t heinformation in histologic sections. In the case of solidtumors this includes the relationship between tumocells and reactive host cells, stroma, proliferating vessels, etc. and the distribution of proliferating vs apoptotic cells within the tumor, the expression of growthfactor receptors in tumor cells according to location andin relation to host cells and blood vessels, and the effectof drug therapies on the functional measurements othe cells. The nu mber of measur able featu res is grow

ing, providing new tools to characterize and monitorhuman tumors in ways not possible by conventionalight microscopy.

The possibility of sequential cell measurements asoffered by LSC ha s alr eady been explored in an alysis othe effect of infectious agents, such as human granulocytic ehr lichiosis or a denoviru s, det ected immu nocytochemically within the cell, on proliferation and expression of the proliferat ion- or apoptosis-associatedmarkers such as tumor suppressor p53 protein, cyclinsinhibitors of cyclin-dependent kinases, proapoptotiBax, or anti-apoptotic Bcl-2 proteins, by the infected

cells [47, 48].

FUTURE APPLICATIONS

Although only a few years have passed since LSCha s become comm ercially available, num erous pu blications ha ve alrea dy appear ed describing a plethora of itsapplicat ions (Table 2). This is an in dicat ion t ha t LSC ia versatile, multitask instrumentation that immediately has found utili ty in many different fields. Theun ique capa bilities, as discussed ear lier, ma ke LSC theinstru ment of choice in a variety of stu dies.



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The major virtue of LSC, which will be the drivingforce for its fut ur e applications, is t he m erge capability,he possibility it offers to relocate the once measured

cells for further analysis. As mentioned, this feature isessential in studies of the time-resolved events such asenzyme kinetics. It will be used to study metabolicchanges, transmembrane transport rates of drugs, me-a bolites, etc., as well as oth er cell functions th at can be

probed by changes in time. Likewise, association con-stants of the fluorochrome-conjugated ligands with therespective receptors can easily be assessed for individ-ual cells by LSC by r epeatedly measur ing ligand bind-ng to the same cells as a function of increasing ligand

concentrations. The relocation feature also allows sec-ondary measurements of the once probed cells, usingoth er mar kers. It also makes th ese cells accessible forvisual examination and image analysis. Furthermore,heir additional analysis in the futur e, after archival

preservation, is feasible. Individual cells thus can bemmun ophenotyped and, when still alive, subjected to

functional assays, e.g., for a particular organelle, oxi-dat ive meta bolism, pH , enzyme k inetics, etc. Followingfixation (95% cells in itially at ta ched electr ostat icallyremain attached after fixation), the very same cells canb e p rob ed for DNA con t e n t (t o a s s es s DNA p l oi dyand/or cell cycle distribution) or DNA replication (afterprelabeling with BrdU), as well as for content of anyntra cellular constitu ent tha t can be detected immu no-

cytochem ically. To obtain th eir cytogenetic profile, th esame cells ma y th en be probed by F ISH. Conventionalstaining with absorption dyes followed by microscopycan identify t he measured cells a nd correlate their

morphology with any of the measured parameters. If desired, a more sophisticated image analysis of t heselected cells can follow: A simp le linka ge of LSC t o th e

m a ge a n a l y s is s y st e m (K on t r on K S 1 0 0 s y s t e m )hrough standard connections has recently been de-

scribed [49]. The slide may be stored indefinitely, andh en wh en a n ew probe is developed, this pr obe may be

applied to the sa me cells an d th e results from early andate studies integrated. Large cell populations can be

stu died along th e scheme described above to detect cellheterogeneity and identify cells with rare features orcell subpopulations with different featu res. Clear ly,

he relocation feature offered by LSC opens an in-finite nu mber of applications of th is instr umen t in cellbiology.

The factor th at may limit sequential an alyses of th esame cells is the necessity of removal of the fluores-cence from the earlier analyses prior to the next mea-surement. Currently available means of enzymatic orchem ical r emoval of th e fluorochr ome, or its bleaching,may n ot always be effective and n ew methods mu st bedeveloped. However, addition of uv light laser to LSCwill enha nce the possibility of sequential measure-ments with different color probes, without the neces-

sity for fluorochr ome removal. F ur ther more, a combination of fluorescence and time-delayed luminescenceprobes which ar e both color an d t ime resolved [50] an dwhich can be adapted to LSC can double the analyticacapability of this instr umen t.

The capa bility of spa tial localization of fluorochr omewithin the cell (nucleus vs cytoplasm, its highest locaconcentration by maximal pixel analysis, spatial dis

tribution of FISH probes) is another feature of LSCthat will attract new applications. One of the obvioususes of LSC will be in analysis of micronucleation, e.g.in muta genicity or environmental studies [51]. Thesoftware that is already available is adequate to automat ically identify and coun t individual n uclei and micronuclei within the cell. LSC is also expected to become useful for mea sur ement tr an slocat ion of differentfactors, such as NF-B [30], p53, components of thesignal transduction pathway, etc., from cytoplasm tonucleus.

Spatial resolution of the fluorochrome and the possibility of localization of the measured cells on slideoffer an opportu nity for an alysis of cell-to-cell int era ctions. Signa l tr an sfer between th e cells, cell-to-celtr ansport of t he fluorochr ome-tagged molecules, an dlocal differen ces in cell prolifera tive potent ial or apoptosis all can be studied on the cells attached to slidesbefore and after their fixation. Likewise, th e role ocytokines or other growth factors released from individual cells on proliferation or apoptosis of the neighboring cells, whether in cell monolayers or in tissuesections, also can be studied by LSC. This can be ac

comp lished by a na lyzing localization of th e cells probedwith imm unocytochemical m ar kers detecting t he pr esence of these factors vis-a-vis th e localization of t heaffected cells probed with markers of proliferation orapoptosis.

LSC also has a potent ial to become a n indispensabletool in the laboratory of every pathologist, providingquan titat ive da ta on FNA, tissue sections, or cytologysmears. As new diagnostic and prognostic mar kers arerapidly being developed and their clinical utility becomes m ore and more evident, th e n eed for quantitative assa ys of these ma rk ers a lso becomes evident . Thecapabilities of LSC predetermine it to serve t his fun ction.

This study was supported by NCI Grant CA 28704, Chemotherapy

Foundation, and Robert A. Welke Cancer Research F oundation. Dr

E. B ed n er was a r ecip ien t o f th e Alf r ed Ju r zy k o wsk i Fo u n d atio n

Award.

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Received J anu ary 8, 1999

Revised version received March 3, 1999


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