Microscopy, Micrometry and Cell Counting

D. Banaag[1], T. Justo[1], E.A. Macauyag[2], J, Talavera[1]

DATE PERFORMED !over"#er 1$ an% 2&, 2&1' (!)TR*+TOR,) !AME Maan De -u."an ABSTRACT Thi !"#!$i%!&' () *+&,-*'!, '+ !"#.+$! 'h! 'h!+$i! )&, %!*h)&i % /!hi&, .i0h' %i*$+ *+#1 ) (!.. ) '+ -&,!$ ')&, )&, #!$2+$% '(+ /) i* %!'h+, &)%!.1 Mi*$+%!'$1 )&, C!.. C+-&'i&0 'h)' $!3-i$! i' - )0!4 I& Mi*$+%!'$1, ) S!) U$*hi& E%/$1+5 Si6! i ,!'!$%i&!, '+ /! 78489% (hi*h h) ) :4;;< ,i22!$!&*! 2$+% 'h! .i'!$)'-$! =).-! (hi*h i >? 9% 2+$ )& !%/$1+ )' 'h! ; *!.. *.!)=)0! ')0!4 F+-$ C+$&!$ S3-)$! M!'h+, i !%#.+1!, i& 'h! C!.. C+-&'i&0 P)$' - i&0 ) H!%+*1'+%!'!$4 Th! $-.i&0 )&, 'h! *!.. (h!$! -**! 2-..1 !!& )&, Th! C!.. *+&*!&'$)'i+& +2 '(+ )%#.! +2 HCT@11A, ) '1#! +2 *+.+& *)&*!$ *!.., () ,!'!$%i&!, '+ /! 148B"1?B 2+$ 'h! 2i$ ' +&! )&, >487"1?B 2+$ 'h! !*+&, +&!4 Th! =i)/i.i'1 ) )1 - i&0 T$1#h)& /.-! ) ) ')i& 2+$ &+&@ =i)/.! *!.. () )/.! '+ ,i22!$!&'i)'! =i)/.! *!.. 2$+% &+&@=i)/.! *!.. )&, 'h! P!$*!&' Vi)/.! *!.. +2 'h! )%#.! )$! ,!'!$%i&!, '+ /! 7747< )&, :A4?<, *.) i21i&0 'h!% ) -&,!$0+i&0 'h$+-0h ) 0$+(i&0 #h) ! Introduction T/e angular an% s0at1al resolut1on o2 t/e 3u"an !a4e% eye 1s a#out &.&1 %egrees an% &.&25"" res0ect1vely, 61t/ a co"2orta#le v1e61ng %1stance o2 a#out 1$c". Because o2 t/1s l1"1tat1on, 6e a%/ere to 1nstru"ents 6/1c/ 1"0rove our a#1l1ty to o#serve 7 t/e earl1est one #e1ng t/e 81g/t M1crosco0e. T/1s #rea4t/roug/ 1s a 0ro%uct o2 t/e %1scovery an% a00l1cat1on o2 O0t1cs. )1nce t/en, 1t /as greatly 1"0rove% our un%erstan%1ng o2 t/e #as1c un1t o2 l12e 7 t/e cell. (n t/1s e90er1"ent, t6o #as1c "et/o%s o2 "1crosco0y 1s e90lore%. T/e 21rst one #e1ng M1cro"etry 6/1c/ 1s t/e %eter"1nat1on o2 t/e e9act s1.e o2 an o#:ect un%er a "1crosco0e an% +ell +ount1ng, t/e "anual %eter"1nat1on o2 cell %ens1ty 1n a cell sus0ens1on. Materials and Methods Focusing through a microscope ;/en t/e 0re0are% sl1%e 6as "ounte% onto t/e stage, t/e l1g/t "1crosco0e 6as 2ocuse% us1ng t/e lo6 0o6er o#:ect1ve 61t/ a 1&9 "agn121cat1on. T/1s 6as %one #y turn1ng u0 t/e l1g/t, o0en1ng t/e %1a0/rag", sett1ng t/e con%enser all t/e 6ay u0, an% a%:ust1ng t/e coarse a%:ust"ent 4no# an% 21ne a%:ust"ent 4no# unt1l a clear 1"age 6as v1e6e% t/roug/ t/e eye01ece. Adjusting for phase contrast P/ase contrast 1s a tec/n1<ue use% 1n "1crosco0y to "a91"1.e t/e contrast 6/en t/ere 1s a #r1g/t 21el% so t/e 1"age 0ro%uce% 1s clearer. (n or%er to a%:ust t/e l1g/t "1crosco0e 2or 0/ase contrast, a s0ec1al con%enser 61t/ %122erent annular r1ng %1a0/rag"s 6as use%. Also, t/e eye01ece 6as re0lace% 61t/ a center1ng telesco0e 1n or%er to 0ro0erly see t/e annular r1ng o2 t/e

con%enser. Once v1e6e%, a l1g/t an% %ar4 r1ng 6as seen t/roug/ t/e center1ng telesco0e. T/1s 6as %ue to t/e 1nter0lay o2 l1g/t 0ass1ng t/roug/ t/e con%enser an% t/e retar%at1on o2 l1g/t t/roug/ t/e o#:ect1ve. T/ese t6o r1ngs 6ere cal1#rate% us1ng t/e center1ng scre6s an% 6as "a%e to ecl10se suc/ t/at only a r1ng 6as v1e6e% t/roug/ t/e center1ng telesco0e. ;/en t/1s 6as %one, t/e center1ng telesco0e 6as re0lace% #ac4 61t/ t/e ocular lens. T/e "1crosco0e 6as t/en use% 2or 0/ase contrast. Getting the calibration constant

F1gure 2. For"ula 2or +al1#rat1on +onstant T/e stage "1cro"eter /as 1&& %1v1s1ons an% t/e total lengt/ 1s 1 "1ll1"eter, t/us eac/ %1v1s1on 1s e<u1valent to 1& "1cro"eters or &.&1 "" 0er )MD >Argayosa @ De -u."an, 2&1'?. Cell counting in culture media and viability assay

F1gure 1. Al1gn"ent o2 t/e eye01ece "1cro"eter an% stage "1cro"eter =no61ng t/e cal1#rat1on constant 1s 1"0ortant 2or any eye01ece "1cro"eter use% s1nce s0ec1"ens can #e accurately "easure% 61t/ 1t. -ett1ng t/e cal1#rat1on constant 6as %one 61t/ t/e use o2 t/e eye01ece "1cro"eter >EM? to #e cal1#rate% an% a stage "1cro"eter >)M? use% to cal1#rate t/e EM. T/e )M 6as "ounte% on t/e stage, v1e6e% 61t/ t/e eye01ece, 2ocuse% 61t/ t/e a%:ust"ent 4no#s an% "a%e to al1gn 61t/ t/e EM suc/ t/at at least t6o l1nes co1nc1%e 61t/ eac/ ot/er. T/e v1e6 un%er t/e "1crosco0e loo4e% l14e t/at o2 F1gure 1. A2ter 6/1c/, t/e nu"#er o2 stage "1cro"eter %1v1s1ons >)MD? an% eye01ece "1cro"eter %1v1s1ons >EMD? 6as counte% 2ro" t/e 0o1nt at 6/1c/ t/e l1nes 2ro" #ot/ "1cro"eters co1nc1%e to t/e ot/er 0o1nt 6/ere t/ey al1gn. T/ree tr1als 6ere %one 2or t/1s, us1ng %122erent 0o1nts o2 al1gn"ent 2or eac/ tr1al. A2ter t/e ' )MDs 61t/ t/e1r corres0on%1ng EMDs 6ere o#ta1ne%, t/e cal1#rat1on constant 6as co"0ute% us1ng t/e 2or"ula g1ven #elo6

F1gure '. A /e"atocyto"eter T/e cell concentrat1on o2 3+TA115, a ty0e o2 colon cancer cell, 6as %eter"1ne% us1ng a /e"atocyto"eter, a ty0e o2 count1ng c/a"#er 61%ely use% 2or cell count1ng. T/e /e"atocyto"eter conta1ns gr1%s an% <ua%rants "a41ng 1t eas1er to count cells 6/en 1t 1s "ounte% un%er t/e l1g/t "1crosco0e. 8ess t/an 1$Bl o2 t/e cell sa"0le 6as ta4en an% "ounte% onto t/e /e"atocyto"eter us1ng ca01llary act1on 61t/ a cover sl10 0lace% on to0 o2 1t. T/e "et/o% use% 1n count1ng t/e cells 6as t/e 2our corner s<uare "et/o%. T/1s 6as %one #y count1ng t/e nu"#er o2 cells 0resent 61t/1n 1n eac/ o2 t/e 2our <ua%rants o2 t/e /e"atocyto"eter seen 1n F1gure 2. T/e cells C$D a#ove 61t/1n t/e #oun%ary o2 t/e <ua%rant 6as counte%. A2ter o#ta1n1ng t/e nu"#er o2 cells 2or eac/ <ua%rant, t/e average value 6as co"0ute% an% use% 2or t/e 2or"ula #elo6 >to co"0ute 2or t/e cell concentrat1on 7 cellE"l?

F1gure F. For"ula 2or +ell +oncentrat1on T/e %1lut1on 2actor >DF? 6as co"0ute% #y gett1ng t/e 21nal volu"e o2 %1lute% solut1on over t/e al1<uot ta4en 2ro" t/e stoc4 solut1on. T/1s 1s use% only 12 t/e stoc4 solut1on 6as %1lute%, 12 not, t/en DF G 1. For t/e v1a#1l1ty assay, t/e cell sa"0le use% 6as BAJCCFA.1, a ty0e o2 "ur1ne "acro0/age. T/e %ye e9clus1on "et/o% 6as e"0loye% 6/ere1n try0to0/an #lue 6as use% to %eter"1ne nonAv1a#le cells 6/1c/ 6ere sta1ne% #lue. T/e sus0ens1on 6as 0re0are% 61t/ a 1 2 rat1o 2or try0to0/an #lue an% t/e cell sa"0le res0ect1vely. A00ro91"ately, 5&Bl o2 try0to0/an #lue 6as a%%e% to 12&Bl o2 t/e cell sa"0le, result1ng 1n a DF e<ual to 1.$. T/e "et/o% e"0loye% 2or cell count1ng 1s t/e sa"e as t/e 0rev1ous one "ent1one%. 3o6ever, t/e count1ng o2 cells 6as se0arate% 1nto t6o grou0s, v1a#le an% nonA v1a#le. A2ter6ar%s, t/e overall cell concentrat1on 6as co"0ute%, along 61t/ D v1a#le cellsE"l an% D nonAv1a#le cellsE"l. T/e gro6t/ curve stage 6as also recor%e% 1n t/e results. (t 1s 0$+(i&0 12 t/e 0ercentage o2 v1a#le cells 1s /1g/er t/an t/e 0ercentage o2 nonAv1a#le cells, ,1i&0 12 1t 1s t/e o00os1te an% ')'i+&)$1 12 t/e t6o values are e<ual. RESULTS Microscopy O#:ect1ves use% 1n t/e "agn121cat1on o2 sa"0le s0ec1"ens are 1nscr1#e% 61t/ a ser1es o2 letters an% nu"#ers 6/1c/ 1n%1cate t/e #as1c 0ro0ert1es o2 t/e lens >Dav1%son, 2&1'?. T/ese 1nscr10t1ons 1nclu%e% P/2 !EOF8*AR F&E&.C$ 15&E&.1C P/2 F&E&5$ 15&E&.1C P8A! 15E&.'$ 15&E&.1C P8A! 1&&E1.2$ Oel 15&EA

T/ese 1nscr10t1ons 0rov1%e t/e #as1c c/aracter1st1cs o2 t/e o#:ect1ve to #e use% accor%1ng to t/e nee%s o2 t/e "1crosco01c tec/n1<ue an% t/e "1crosco0e 1tsel2. Annular r1ng %1a0/rag"s >F1g.1? o2 t/e P/ase contrast "1crosco0e s1"1larly 1n%1cate t/e s0ec121c 0ur0oses an% 2unct1on o2 eac/ o#:ect1ve.

F1gure $. Annular r1ng %1a0/rag"s o2 t/e 0/ase contrst "1crosco0e. >A? >B? >+? Micrometry T/ree tr1als 6ere %one 1n t/e co"0utat1on o2 t/e cal1#rat1on constant o2 t/e "1cro"eter eye01ece. T/e average cal1#rat1on constant 1s &.&15C M1l1"eters 0er Eye P1ece M1cro"eter D1v1s1on >""E e"%? . (n t/1s l1g/t, #e1ng o#serve% o2 /av1ng a %1a"eter o2 $e"%, un%er t/e "1crosco0e, t/e co"0ute% cell %1a"eter 6as &.&H''"" >H'.'I"?. Assu"1ng t/at an 1"age o2 t/e cell /av1ng a %1a"eter o2 $c" 1s seen 2ro" t/e "1crosco0e, t/e "agn121cat1on o2 t/e s0ec1"en 6as 5&&9. Hemocytometry and ia!ility Assay +olon cancer cells 6ere use% 1n t/e /e"ocyto"etry count. T6o cultures o2 "ur1ne "acro0/ages >JCCFA.1? la#ele% as 2las4s B an% + 6ere teste% 2or t/e1r total cell concentrat1on an% 0ercentage o2 v1a#le cells. Ta#le 1. Results o2 +ell +ount1ng Flas4 B Flas4 + +oncentrat1on 1.'$91&$ J.'H91&$ >cellsEMl? Percentage o2 HH.HD C5.&D K1a#le +ells

-ro6t/ curve -ro61ng )tage )tage

-ro61ng )tage

(n #ot/ 2las4s, t/e 0ercentage o2 v1a#le cells 1s /1g/er t/an t/e nonAv1a#le cells. T/1s 1n%1cates t/at #ot/ o2 t/e sa"0les are 1n t/e -ro61ng stage #ecause "ore cells are al1ve t/an %ea%. "ISCUSSI#$ Microscopy M1crosco0es use a t6oAlens syste" t/e o#:ect1ve lens syste" >3PO, 8PO an% O(O? /av1ng a "agn121cat1on o2 F&9, FA1&9 an% 1&&9 res0ect1vely an% t/e ocular lens syste" /av1ng a "agn121cat1on o2 a#out 1&9. T/e total "agn121cat1on o2 t/e 1"age 1n t/e "1crosco0e 1s o#ta1ne% 2ro" t/e 0ro%uct o2 #ot/. T/e 1"age as 1t 1s enlarge% 1s 1nverte%. T/1s 1s #ecause t/e "1crosco0e, s0ec121cally t/e o#:ect1ve 2or"s a real 1"age t/roug/ a conve9 lens. T/1s 1s s/o6n 1n t/e 21gure #elo6.

to %1verge 2ro" a 0o1nt on t/e sa"e s1%e o2 t/e lens as t/e o#:ect >t/1s 1s t/e locat1on o2 t/e v1rtual 1"age?. T/e a#1l1ty o2 t/e "1crosco0e to 0rov1%e a large an% %eta1le% 1"age o2 a sa"0le 1s #ase% on t/e conce0ts o2 "agn121cat1on an% resolut1on. Magn121cat1on 1s t/e a#1l1ty o2 t/e "1crosco0e to enlarge t/e 1"age o2 t/e s0ec1"en 6/1le resolut1on re2ers to t/e 1ts a#1l1ty to %1st1ngu1s/ t6o 0o1nts as %1st1nct o#:ects >Talaro, 2&&H?. T/e l1"1t o2 resolut1on %eter"1nes t/e resolv1ng 0o6er o2 t/e "1crosco0e an% 1s calculate% us1ng t/e A##e e<uat1on 8.R G &.51L E !.A 6/ere L 0erta1ns to t/e 6avelengt/ o2 l1g/t 0ass1ng t/roug/ t/e s0ec1"en 1n nano"eters an% !.A. or t/e nu"er1cal a0erture. T/e nu"er1cal a0erture "easures t/e a#1l1ty an% e221c1ency o2 t/e lens to #en% l1g/t rays. (t 1s e<ual to ns1n>a? 6/ere n 1s re2ract1ve 1n%e9 >nGcEv? an% a as M o2 t/e angle o2 1nco"1ng l1g/t. T/e value o2 s1n>a? never #eco"es larger t/an 1. T/ere2ore to 1ncrease resolut1on, t/e "ater1al #et6een t/e sa"0le an% t/e o#:ect1ve "ust #e c/ange% as to 1ncrease t/e re2ract1ve 1n%e9. T/1s 1s t/e reason 6/y o1l 1s a00l1e% to O1l (""ers1on O#:ect1ves an% co0e 61t/ t/e "agn121cat1on #roug/t #y 1t. Manu2acturers o2 "1crosco0es 0rov1%e t/e c/aracter1st1cs o2 t/e o#:ect1ves nee%e% 1n e90er1"ents #y "eans o2 1nscr1#1ng a ser1es o2 letters an% nu"#ers on t/e sur2ace >Dav1%son, 2&1'?. T/ese 1n2or"at1on a1%s t/e stu%ents an% sc1ent1sts al14e 1n c/oos1ng t/e a00ro0r1ate o#:ect1ves 2or t/e1r stu%y. Ty01cally, t/e na"e o2 t/e "anu2acturer 0rece%es all ot/er 1n2or"at1on >at t/e to0"ost?. O0t1cal correct1ons are also 1n%1cate%. F1el% curvature correct1ons "ay #e re0resente% #y P8A! >Flat 21el% o0t1cal correct1on? or !EOF8*AR >Fluor1te a#errat1on correct1on? >Dav1%son, 2&1'?. T/e ty0e o2 "1crosco0e t/e o#:ect1ves are a00ro0r1ate 2or are 1n%1cate% #y a##rev1at1ons as 6ell >e.g D 2or Dar421el%?. T/e 1nscr10t1on P/2 re2ers to P/ase

F1gure 5. For"at1on o2 Real (nverte% ("age #y t/e O#:ect1ve lens T/e eye01ece lens t/en acts as a "agn12y1ng glass an% "agn121es t/e 1"age 2or"e%. A real 1"age 1s 2or"e% 6/en l1g/t rays or1g1nat1ng 2ro" a 0o1nt on one s1%e o2 a lens >1.e. t/e o#:ect? are re2racte% #y t/e lens so t/at t/ey 2ocus >co"e toget/er? to a 0o1nt on t/e ot/er s1%e o2 t/e lens at t/e 1"age locat1on 6/1le a v1rtual 1"age 1s 2or"e% 6/en l1g/t rays or1g1nat1ng 2ro" a 0o1nt on one s1%e o2 a lens >1.e. t/e o#:ect? are re2racte% #y t/e lens so t/at t/ey %1verge >"ove a0art 2ro" eac/ ot/er? on t/e ot/er s1%e o2 t/e lens. ;/en t/ese rays are trace% #ac4 1n a stra1g/t l1ne >1gnor1ng t/at t/ey 6ere actually #ent #y t/e lens? t/en a00ear

con%enser annulus an% Oel "eans O1l 1""ers1on >Dav1%son, 2&1'?. T/e nu"#ers #elo6 t/e "ent1one% 1nscr10t1ons re0resent 2urt/er "ec/an1cal c/aracter1st1cs an% %eta1ls. T/e 21rst nu"#ers %1v1%e% #y a slas/ re0resent t/e lateral "agn121cat1on >r1g/t? an% nu"er1cal a0erture >le2t?. T/at 1s, an 1nscr10t1on 1&&E1.2$ "eans t/at t/e o#:ect1ve /as a lateral "agn121cat1on o2 1&&9 an% a nu"er1cal a0erture o2 1.2$. T/e ne9t set o2 nu"#ers s1"1larly %1v1%e% #y a slas/ re0resent t/e tu#e lengt/ >r1g/t? an% t/e 6or41ng %1stance >le2t? >Dav1%son, 2&1'?.A0art 2ro" t/e tec/1n1cal 0ro0ert1es o2 t/e o#:ect1ve lenses, lenses "ay 0resent certa1n a#errat1ons 6/1c/ "ay #e e1t/er c/ro"at1c or s0/er1cal ones. +/ro"at1c a#errat1ons result 2ro" t/e lenses t/at act as 0r1s"s an% /ence result to 1"ages 61t/ color 2r1nges aroun%. )0/er1cal a#errat1ons 0ro%uce 1"ages t/at a00ear curve% an% 2u..y 1nstea% o2 #e1ng l1near or 2lat >Breg"an, 1JJ5?. ("ages seen un%er t/e "1crosco0e are not al6ays 61t/ t/e1r #est <ual1ty 0oss1#le. +r1t1cal 1llu"1nat1on 1s l1"1te% #y t/e 2or"at1on o2 an 1"age o2 t/e l1g/t source 1n t/e s0ec1"en 1"age 0lane. T/1s 1s a%%resse% #y =N/ler 1llu"1nat1on #y ensur1ng t/e 1"age o2 t/e l1g/t source 1s 0er2ectly %e2ocuse% 1n t/e sa"0le 0lane an% 1ts con:ugate 1"age 0lanes. =N/ler 1llu"1nat1on re<u1res several o0t1cal co"0onents to 2unct1on +ollector lens an%Eor 21el% lens, F1el% %1a0/rag", +on%enser %1a0/rag" an% +on%enser lens. T/ese co"0onents l1e 1n t/1s or%er #et6een t/e l1g/t source an% t/e s0ec1"en an% control t/e 1llu"1nat1on o2 t/e s0ec1"en. T/e collectorE21el% lenses act to collect l1g/t 2ro" t/e l1g/t source an% 2ocus 1t at t/e 0lane o2 t/e con%enser %1a0/rag". T/e con%enser lens acts to 0ro:ect t/1s l1g/t, 61t/out 2ocus1ng 1t, t/roug/ t/e sa"0le. T/e 2ocus an% central c/aracter1st1cs o2 t/e 1"ages 1n l1g/t "1crosco0y "ay #e o2 t6o ty0es 0ar2ocal or 0arcentral >Rac46oo%@3arr1s >e%?, 1JJ5?. Par2ocal 1n%1cates t/at u0on c/ang1ng 2ro" one o#:ect1ve to anot/er, t/e 2ocus %oes not

c/ange an% /ence sl1g/t tun1ng o2 t/e 21ne a%:ust"ent 1s only nee%e%. On t/e ot/er /an%, 1n Parcentral, 1t 1s t/e center o2 t/e 21el% t/at %oes not c/ange 6/en t/e o#:ect1ves are c/ange% >Breg"an, 1JJ5?. M1crosco0es use% 2or 0/ase contrast "1crosco0y are e<u100e% 61t/ con%enser annulus an% 0/ase 0late. T/e con%enser annulus 2ocuses t/e l1g/t 1nto t/e s0ec1"en. Due to t/e result1ng /ollo6 l1g/t 2ro" t/e con%enser annulus, t/e 6avelengt/ o2 %122racte% an% un%122racte% l1g/t 1s %122erent1ate%. D122racte% l1g/t 1s slo6er t/an t/e un%122racte% l1g/t. T/e /u"an eye can not %etect suc/ %122erence 1n 6avelengt/. T/e 0/ase 0late t/en "agn121es t/e %122erence 1n l1g/t 6avelengt/ an% 1s %etecte% #y t/e eye as a contraste% 1"age. T/e 0/ase 0late /as s1lver coat1ng an% glass lens t/1nner t/an t/e o#:ect1ve lens t/at allo6s t/e un%122racte% l1g/t to 0ass 2aster t/an t/e %122racte% l1g/t. >Tr1#e et al., 1JC$? T/e con%enser annulus an% 0/ase 0late 6or4s toget/er to 1ncrease t/e %122erence 1n 6avelengt/ o2 %122racte% an% un%122racte% l1g/t t/at 1s %etecte% as a contrast 1"age #y t/e eye. T/e o0t1cal 0at/ 2or #r1g/t 21el% , %ar4 21el% an% 0/ase contrast "1crosco0y are s/o6n #elo6

F1gure C. >Fro" le2t to r1g/t? 81g/t Pat/s o2 Br1g/t F1el%, Dar4 F1el% an% P/ase +ontrast M1crosco0e

Micrometry

A sea urc/1n e"#ryo 1n t/e FAstage cleavage 1s a00ro91"ately J&I" 1n %1a"eter >-1l#ert, 2&&&?. T/1s value 1s close to t/e "easure"ent calculate% 1n t/e e90er1"ent >H'.' I"? 6/1c/ "ay re2lect an accurate cal1#rat1on o2 t/e o#:ect1ves. M1cro"etry serves to #e a s1gn121cant an% 1n%1s0ensa#le tool 1n t/e c/aracter1.at1on o2 cells an% ot/er l1v1ng "1croorgan1s"s. Hemocytometry and %ia!ility assay T/e <uant121cat1on o2 cells 1n a cell sus0ens1on can #e %one %1rectly 1n a "1crosco0e t/roug/ t/e use o2 count1ng c/a"#ers l14e t/e /e"ocyto"eter. T/e /e"ocyto"eter /as a count1ng area "ar4e% 61t/ gr1% an% o2 4no6n %1"ens1ons >-allag/er an% ;1ley, 2&&$?. +oversl10s use% 2or "ount1ng on /e"ocyto"eters are es0ec1ally "a%e to #e t/1c4er t/an t/e convent1onal "1crosco0y coversl10s #ecause t/ey "ust #e a#le to overco"e t/e sur2ace tens1on o2 a %ro0 o2 l1<u1% >-r1goryev, 2&&'?. )a"0les "ove to t/e count1ng c/a"#er t/oug/ ca01llary act1on. De0en%1ng on t/e s1.e o2 t/e target organ1s", %eter"1nat1on o2 t/e nu"#er o2 cells 2ollo6 t/e centralAs<uare rule 2or s"all cells 6/1le t/e 2ourAcorner s<uare "et/o% 1s e"0loye% 1n largeAs1.e% cells l14e t/e "ur1ne "acro0/ages. Total cell count, as 0er2or"e% 1n t/e colon cancer cells, 0rov1%es t/e nu"#er o2 all cells 61t/out %1st1nct1on #et6een v1a#le an% nonAv1a#le cells. >3ogg, 2&&$? To %1st1ngu1s/ l1ve 2ro" %ea% cells, a v1a#1l1ty assay 6as 0er2or"e% us1ng try0an #lue as an e9clus1on %ye. Try0an #lue 1s e9clus1on test 1s a ra01%, s1"0le an% 1se% 1ne90ens1ve "et/o%. T/e co"0ro"1se% "e"#rane 1ntegr1ty o2 nonAv1a#le cells allo6 t/e u0ta4e o2 t/e %ye t/us %ea% cells a00ear as #lue. K1a#le cells a00ear clear or unsta1ne%. >8ongoA)or#elloet al., 2&&5? T/e 0ercentage o2 v1a#le an% nonAv1a#le cells %eter"1ne% t/e gro6t/ curve stage o2 t/e cells. T/e /1g/er 0ercentage o2 t/e v1a#le cells versus t/e nonAv1a#le cells suggests t/at t/e cells are 1n t/e log 0/ase o2 t/e curve gro6t/.

(n a%%1t1on to /e"ocyto"etry, ot/er "et/o%s 2or t/e %eter"1nat1on o2 cell %ens1ty 1nclu%e tur#1%1"etr1c "et/o%, 2lo6 cyto"etry an% 0la<ue assay "et/o%. (n tur#1%1"etr1c "et/o%, tur#1%1ty 1s "easure% t/roug/ t/e use o2 s0ectro0/oto"eter or tur#1%1"eter. +ell concentrat1on 1s t/en %eter"1ne% #y correlat1ng t/e o#serve% tur#1%1ty an% cell nu"#er. A "ore tur#1% sa"0les "eans /1g/er cell %ens1ty. Anot/er "et/o% 2or cell %ens1ty %etect1on 1s 2lo6 cyto"etry. T/e e"1tte% 2luoresence an% l1g/t scatter 1s %etecte% as t/e cells 2lo6 1n a s1ngleA21le "anner t/roug/ a 2ocuse% laser l1g/t. T/e recor%e% l1g/t 0ulse 1s converte% to an electr1cal s1gnal #y a 0/oto%1o%e or 0/oto"ult10l1er. An est1"ate o2 t/e total nu"#er o2 cells 1s correlate% 61t/ t/e cell count o#ta1ne% a2ter a 219e% t1"e 0er1o% 2or 2lo6. 8astly, t/e 0la<ue assay "et/o% ut1l1.es ser1al %1lut1on o2 cell sus0ens1on , usually v1ral, gro6n 1n a "onoloyer o2 /ost cell culture. Eac/ un1t o2 t/e sus0ens1on "a4es one 0la<ue. Pla<ues are local1.e% 1n2ecte% an% %ea% cells e9a"1ne% an% enu"erate% #y sta1n1ng t/e cell "onolayers. >-ol%"an an% -reen, 2&&J? RE&ERE$CES [1] Dav1%son M;. 2&1'. M1crosco0e o#:ect1ve s0ec121cat1ons. MicroscopyU' Source o( Microscopy Education. Retr1eve% 2ro" /tt0 EE666."1crosco0yu.co"Eart1clesEo0t1cs Eo#:ect1ves0ecs./t"l 2$ !ove"#er 2&1'. [2] -allag/er )R an% ;1ley EA. 2&&H. Current )rotocols Essential La!oratry Techni*ues. Jo/n ;1ley @ )ons, (nc., Englan%. 0.F.2.J ['] -1l#ert )F. 2&&&. "e%elopmental Biology, 5t/ e%. )un%erlan% >MA? )1nauer Assoc1ates [F] -ol%"an E an% -reen 83. 2&&J. )ractical Hand!oo+ in Micro!iology. 2n% e%1t1on. +R+ Press. *)A. 0. 1C, 2&A21

[$] -r1goryev O. 2&1'. +ell +ount1ng 61t/ a 3e"ocyto"eter Easy as 1, 2, '. B1tes1.eB1o Bra1n2oo% 2or B1olog1sts. Retr1eve% 2ro"/tt0 EEcellculture.#1tes1.e#1o.co"Eart1cle sEcellAcount1ngA61t/AaA/e"ocyto"eterAeasyA asA1A2A'E 2$ !ove"#er 2&1' [5] 3ogg ). 2&&$. Essential Micro!iology. Jo/n ;1ley @ )ons 8t%. Englan%.0. J2 8ongoA)or#ello -)A, )ay%a" -, Baner:ee D an% Bert1no JR. 2&&5. +ytoto91c1ty an% +ell -ro6t/ Assay. Cell and Tissue Culture Assorted Techni*ues. Elsev1er )c1ence. *)A 0.'1H Ru.1n, ).E. 1JJJ. Plant "1crotec/n1<ue an% "1crosco0y. O92or% *n1vers1ty Press, O92or%, !e6 Oor4 Tr1#e MA, Eraut MR an% )noo4 R=. 1JC$. Light Microscopy, +a"#r1%ge *n1vers1ty Press. s-reat Br1ta1n 0F'

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