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Multi-view light-sheet microscope for rapid in toto imaging
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We present a multi-view selective-plane illumination microscope (MuVi-SPIM), comprised of two
detection and illumination objective lenses, that allows rapid in toto fluorescence imaging of
biological specimens with subcellular resolution. The fixed geometrical arrangement of the
imaging branches enables multi-view data fusion in real-time. The high speed of MuVi-SPIM
enables faithful tracking of nuclei and cell shape changes, which we demonstrate by in toto
imaging the embryonic development of Drosophila melanogaster.
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3@-"07(S#$S#E($)4."% 6.(@)4.1+% 7(-% ;.$#1:."."% Q1M.1:31?%03/% 2#@:"3;(1?@u$."B% Ann. Phys.%315+%
*TUJ%V*J\5WB%
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:(7.1$(#10@%(70?(1?%#/%70)"#$)#E()%;(#@#?()0@%$E.)(7.1$B%J Microsc%170+%55JTUI%V*JJUWB%
IB% >3($S.1+%<B+%,M#?."+%<B+%G.@%D.1.+%]B+%f(--;"#:-+%<B%m%,-.@'."+%9B%>B%&B%_E-()0@%,.)-(#1(1?%G..E%Y1$(:.%
=(P.%97;"8#$%;8%,.@.)-(P.%X@01.%Y@@37(10-(#1%F()"#$)#E8B%Science%305+%*\\KT*\\J%V5\\ZWB%
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[B% &.@@."+% XB% <B+% ,)47(:-+% QB% GB+% f(--;"#:-+% <B% m% ,-.@'."+% 9B% >B% &B% d.)#1$-"3)-(#1% #/% n.;"0/($4% 90"@8%
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*\B%6"3#1?+%6B%`B+%,3E0--#+%fB+% &##$+%GB%,B+%C4#(+%<B% FB% m% ]"0$."+%,B%9B%G..E%01:% /0$-%@(P.%(70?(1?%M(-4%
-M#HE4#-#1%$)011.:%@(?4-H$4..-%7()"#$)#E8B%Nat Meth%8+%K^KTKI\%V5\**WB%
**B%C0E#3@0:.+% <B+% f0)4$73-4+% FB+% >3/10?.@+% =B% m% &1#E+%FB% l301-(-0-(P.% /@3#".$).1).% (70?(1?% #/%
E"#-.(1%:(//3$(#1%01:%(1-."0)-(#1%(1%@(P(1?%).@@$B%Nat Biotech%29+%[U^T[UJ%V5\**WB%
*5B%C.@@0%n010))4(+%]B%et al.%=(P.H).@@%UG%$3E."H".$#@3-(#1%(70?(1?%(1%-4()S%;(#@#?()0@%$07E@.$B%Nat Meth%
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*UB%,M#?."+%<B+%`."P.."+%XB+%2".?."+%&B+%>3($S.1+%<B%m%,-.@'."+%9B%>B%&B%F3@-(HP(.M%(70?.%/3$(#1%(7E"#P.$%
".$#@3-(#1%(1%-4"..H:(7.1$(#10@%7()"#$)#E8B%Opt. Express%15+%[\5JT[\Z5%V5\\KWB%
*ZB%X".(;($)4+% ,B+% ,00@/.@:+% ,B+% ,)4(1:.@(1+% <B% m% 6#701)0S+% XB% ,#/-M0".%/#"% ;.0:H;0$.:% ".?($-"0-(#1% #/%
$.@.)-(P.%E@01.%(@@37(10-(#1%7()"#$)#E8%:0-0B%Nat Meth%7+%Z*[TZ*J%V5\*\WB%
*^B%&"'()+% !B% F3@-(E@.HP(.M%7()"#$)#E8% M(-4% @(?4-H$4..-% ;0$.:%/@3#".$).1).% 7()"#$)#E.B% X4G% 64.$($+%
!1(P."$(-8%#/%>.(:.@;."?%V5\\JW%
*IB%>3($S.1+% <B% m% ,-0(1(."+% GB% oB% dB% 9P.1% /@3#".$).1).% .N)(-0-(#1% ;8% 73@-(:(".)-(#10@% $.@.)-(P.% E@01.%
(@@37(10-(#1%7()"#$)#E8%V7,XYFWB%Opt. Lett.%32+%5I\[T5I*\%V5\\KWB%
*KB%]#.+%`B%9B% m% Q@;."-$+%DB% FB% ,-3:(.$%#/% 13)@.0"% 01:% )8-#E@0$7()%;.40P(#3"%:3"(1?%-4.%/(P.%7(-#-()%
)8)@.$%-40-% E".).:.% ?0$-"3@0-(#1%(1%G"#$#E4(@0%.7;"8#?.1.$($B%Journal of Cell Science%61+%U*TK\%
V*J[UWB%
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E@01.%(@@37(10-(#1B%Nat Meth%8+%Z*KTZ5U%V5\**WB%
5\B%2"(@@+%,B%m%,-.@'."+%9B%>B%&B%F.-4#:%-#%)0@)3@0-.%@0-."0@%01:%0N(0@%?0(1%/0)-#"$%#/%#E-()0@%$.-3E$%M(-4%0%
@0"?.%$#@(:%01?@.B%J. Opt. Soc. Am. A%16+%5I^[T5II^%V*JJJWB%
5*B%l3077.1+% CB% fB+% ].1?+% GB% m% 608@#"% YY+% dB% FB% X."/#"701).% #/% UG% G.)#1P#@3-(#1% Q@?#"(-47$% #1%
F3@-(HC#".%01:%F018HC#".%Q")4(-.)-3".$B%University of North Carolina at Chapel Hill, Department
of Computer Science, Tech. Rep%V5\\JWB%
55B%d()40":$#1+%fB%>B%D08.$(01HD0$.:%Y-."0-(P.%F.-4#:%#/%Y70?.%d.$-#"0-(#1B%J. Opt. Soc. Am.%62+%^^T
^J%V*JK5WB%
5UB%2#1'0@.'+% dB% CB+% f##:$+% dB% 9B% m%9::(1$+% ,B% =B% Digital image processing using MATLABB% VX".1- ().%
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5^B%d0//.@+%FB+%f(@@."-+%CB%9B%m%E.1401$+%<B%Particle image velocimetry: a practical guideB%V,E"(1?."c%
D."@(1R%e.M%o#"S+%*JJ[WB%
List of supplementary items
Supplementary File Title
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,3EE@.7.1-0"8%](?3".%Z% F3`(H,XYF%(70?.%$81-4.$($
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Supplementary!Figure!1!
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Supplementary!Figure!7!
A.@@%-"0)X(1?%(1%-4.%Drosophila%M.1-"0@%.)-#:."7E%=./-%)#@371%$4#U$%70R(737%(1-.1$(-8%
C"#^.)-(#1$%#/%-4.%M.1-"0@%$(:.%#/%0%4($5\MG7A4.""8%Drosophila%.7;"8#%0-%/#3"%:(//.".1-%
-(7.$%O6% g%Z% 4%:./(1.:% 0$%$-0"-% #/%(70?(1?PE% ,(R%13)@.(% 0".%)4#$.1% /#"%-"0)X(1?% O/(M.%
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:.M.@#C7.1-E%F03?4-."%13)@.(%0".%@0;.@@.:%(1%-4.%$07.%)#@#3"%0$%-4.%C0".1-%13)@.3$E%
D(::@.% )#@371% )#"".$C#1:$% -#% -4.% ".?(#1% 4(?4@(?4-.:% ;8% -4.% U4(-.% ;#R% (1% -4.% @./-%
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?"#3C.:%).@@$E%A.@@%70"X."$%0".%).1-".:%#1%-4.%C#$(-(#1%#/%70R(737%(1-.1$(-8%/#"%.0)4%
).@@E%%
%
%
Supplementary!Figure!8!
9//.)-$% #/% 01($#-"#C()% C#(1-G$C".0:% /31)-(#1% #1% -4.% (70?(1?% #/% ).@@% 7.7;"01.$% (1%
Drosophila%.7;"8#E%(a)%L#(1-G$C".0:%/31)-(#1%OL,KP%#/%-4.%D3Q(G,LND%($%.@#1?0-.:%0@#1?%
-4.%7()"#$)#C.h$% :.-.)-(#1%0R($E%(b)% N1%01% .0"@8% Drosophila%.7;"8#+%7#$-% ).@@$%0/-."%
).@@3@0"($0-(#1%@(.%)@#$.%-#%-4.%.7;"8#h$%$3"/0).i%-4.%7.7;"01.$%;.-U..1%-4.%).@@$%0".%
-43$% 1.0"@8% C."C.1:()3@0"% -#% -4.% .7;"8#h$% $3"/0).E% 64.% (@@3$-"0-(#1% $4#U$% 0% )"#$$G
$.)-(#1%-4"#3?4%0%Drosophila%.7;"8#%C."C.1:()3@0"%-#%-4.%.7;"8#h$%@#1?%0R.$E%=./-%-U#%
)#@371$%$4#U% -4.%#"(.1-0-(#1% #/%-4.%L,K% ".@0-(M.%-#% -4.%.7;"8#+% U(-4% -4.%.7;"8#h$%
01?3@0"% C#$(-(#1% #/% Zj% 01:% IZj+% ".$C.)-(M.@8E% 64.% /3$.:% (70?.% O"(?4-% )#@371P% ($%
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0".%.M."8U4.".%)@#$.%-#%C0"0@@.@E%(c)%k"(.1-0-(#1%#/%-4.%7()"#$)#C.h$%L,K%".@0-(M.%-#%-4.%
#"(.1-0-(#1%#/%-4.%/@3#".$).1-% 7.7;"01.$%:.-."7(1.$%(70?.%]30@(-8%O.7;"8#%$3"/0).%
U0$%l31"#@@.:l%0$%:.$)"(;.:%(1%,3CC@.7.1-0"8%1#-.%WPE%N1%0%$(1?@.%D3Q(G,LND%(70?.%$.-%
U(-4#3-%$C.)(7.1%"#-0-(#1%O@./-%-U#%)#@371$P+%7.7;"01.$%U(@@%;.%;.$-%".$#@M.:%U4.".%
-4.8% 0".% 1.0"@8% C0"0@@.@% -#% -4.% 7()"#$)#C.h$% L,K+% U4(@.% (1% -4.% 0".0$% U4.".% -4.%
7.7;"01.$%0".% 1.0"@8% C."C.1:()3@0"% -#% -4.% L,K+% (70?.% )#1-"0$-% 01:%".$#@3-(#1%0".%
$(?1(/()01-@8%".:3).:E%N1%0%/3$.:%(70?.%C"#:3).:%/"#7%-U#%"#-0-.:%D3Q(G,LND%:0-0$.-$%
O"(?4-%)#@371P+%-4.%L,K% ($%1.M."%C."C.1:()3@0"%-#% -4.%).@@% 7.7;"01.$i%-4.% ".$3@-(1?%
(70?.%)#1-"0$-%01:%".$#@3-(#1%0".%-4."./#".%$3//()(.1-%-#%".$#@M.%(1:(M(:30@%).@@$%0@7#$-%
018U4.".%#1%-4.%.7;"8#h$%$3"/0).%O,..%,3CC@.7.1-0"8%Q(:.#%*ZPE%
Supplementary!Figure!9!
F.-0(@.:%M(.U%#/%-4.%?0CVSG7A4.""8%Drosophila%.7;"8#%$3"/0).E%(a)",.?7.1-0-(#1%#/%-4.%
7.7;"01.$%(1%01% .7;"8#%:3"(1?%.0"@8% )8)@.%*V%O.R)@3:(1?% C#@0"%".?(#1$PE%2".8$)0@.%
:0-0%($%-4.%/3$.:%01:%31"#@@.:%(70?.%U(-4%".:%@(1.$%:.1#-(1?%$.?7.1-.:%).@@%;#":."$E%(b)"
A@#$.G3C%#/%-4.%4(?4@(?4-.:%".?(#1%(1%O0PE%(c)"6(7.G@0C$.%M(.U%#/%).@@%7#M.7.1-$%01:%
$40C.$% :3"(1?% .0"@8% Drosophila!7#"C4#?.1.$($E% 64.% @./-% $)4.70-()% :.$)"(;.$% -4.%
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(1%O)P+%).@@%$C..:$%)01%;.%)0@)3@0-.:E%6(7.%($%:./(1.:%/"#7%-4.%/("$-%(70?.%#/%-4.%-(7.G
@0C$.%O/#3"%7(13-.$%;./#".%#1$.-%#/%M.1-"0@%/3""#U%/#"70-(#1PE%\""#U$%@0;.@@.:%O*GSP%
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!
Supplementary!Figure!10!
=(?4-G$4..-%0@(?17.1-%3$(1?%0%/@3#".$).1-%7.:(37E%(a)%f4.1%1#%.7($$(#1%/(@-."%($%3$.:%
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)(")3@0"%$40C.$%(1%-4.%(70?.E%,('.%#/%-4.%)(")@.$%($%"#3?4@8%C"#C#"-(#10@%-#%-4.%:($-01).%#/%
-4.%C0"-()@.%O01:% -4."./#".%/"#7%-4.% C0"X.:%@0$."%;.07P% /"#7%-4.%/#)0@%C@01.%#/%-4.%
:.-.)-(#1%0"7E%=0"?.%M0"(0-(#1%(1% -4.%)(")@.$`% $('.$%(7C@(.$% 0R(0@%7($0@(?17.1-%#/% -4.%
(@@37(10-(#1%#;^.)-(M.%@.1$+%U4(@.%31(/#"7@8%$('.:%)(")@.$%O0$%($%-4.%)0$.%(1%-4.%/(?3".P%
C#(1-%-#%0R(0@%7($0@(?17.1-%#/%-4.%:.-.)-(#1%#;^.)-(M.%@.1$E%(b!c)%k1).%01%.7($$(#1%/(@-."%
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?.1."0-.$%01%(70?.%#/%-4.%;.07%U(-4%(-$%U0($-%1.0"%-4.%).1-".%#/%-4.%(70?.E%64()X1.$$%#/%
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3$./3@%-#%:($C@0).%-4.%;#-4%@(?4-G$4..-$+%7#M(1?%.0)4%U0($-%/#"%0%/"0)-(#1%#/%-4.%@(?4-G
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-#%:#3;@.%@(?4-G$4..-`$%3$0;@.%@.1?-4%U(-4#3-%0//.)-(1?%(-$%-4()X1.$$E%N1%-4($%.R07C@.+%-U#%
@(?4-G$4..-$%U(-4%3$0;@.%@.1?-4$%O:./(1.:%0$%-U().%-4.%c08@.(?4%"01?.1P%#/%*SSe7%0".%
:($C@0).:%/#"%0CC"#R(70-.@8%*ZZe7+%".$3@-(1?%(1%01%.//.)-(M.%@(?4-G$4..-%U(-4%-4()X1.$$%
;.-U..1%5ESe7% 01:%SE5e7% #M."%-4.% U4#@.%Drosophila%.7;"8#E%(f)%k1).%-4.%,LND%($%
0@(?1.:+% ?0@M01#7.-"()% $)011."% ($% 3$.:% -#% $)01% -4.% ;.07% #M."% -4.% /(.@:% #/% M(.U+%
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%
Supplementary!Note!1!
Light!sheet"properties"
L"#C."-(.$%#/%-4.%@(?4-G$4..-%)01%;.%.0$(@8%#;-0(1.:%/"#7%-4.%(70?.%?.1."0-.:%;8%0%U.@@%
/#)3$.:% C0"X.:% ;.07% (@@37(10-(1?% 0% /@3#".$).1-% 7.:(37% O$..% 0@$#% ,3CC@.7.1-0"8%
L"#-#)#@% *% 01:% ,3CC@.7.1-0"8% K(?E% *Z:PE% N1% #3"% )0$.+% -4.% ;.07% U0($-% "0:(3$% U0$%
7.0$3".:%-#%;.%mno5ES%pq%Orst%"0:(3$*P+%U4()4%)#"".$C#1:$%-#%:.C-4%#/%/#)3$%O-U().%
-4.%c08@.(?4%"01?.P%#/%5uno *SS%pqE%\/-."%-4.%U0($-$%#/%-4.%;#-4%;.07$%0".%:($C@0).:%
/#"%"#3?4@8%*ZZ%e7%O,3CC@.7.1-0"8%K(?E%*Z.P+%.//.)-(M.%@(?4-G$4..-%-4()X1.$$%($%;.-U..1%
5ES%e7%01:%SE5%e7%#M."%-4.%U4#@.%Drosophila%.7;"8#%O0"#31:%5ZZe7%(1%:(07.-."PE%,('.%
#/%-4.%@(?4-G$4..-%(1%-4.%:(".)-(#1%C."C.1:()3@0"%-#%-4.%(@@37(10-(#1%0R($%($%:./(1.:%;8%-4.%
$)011(1?%"01?.%#/%-4.%?0@M01#7.-"()%$)011."$%01:%U0$%$.-%0-%0"#31:%WWZe7%/#"%(70?(1?%
#/%Drosophila%.7;"8#$%O0"#31:%WZZ%e7%(1%@.1?-4PE%
=0-."0@% ".$#@3-(#1% OU(-4(1% -4.% /#)0@% C@01.P% #/% -4.% 7()"#$)#C.% ($% :.-."7(1.:% ;8% -4.%
137."()0@%0C."-3".%#/%-4.%:.-.)-(#1%#;^.)-(M.%@.1$%01:%U(@@%;.%0"#31:%ZEVS%e72%(1%)0$.%#/%
-4.% VZRvZEYf% @.1$% 01:% H*Z% 17% .7($$(#1% O0$% 3$.:% /#"% Drosophila%(70?(1?PE%\R(0@%
".$#@3-(#1%O0@#1?%-4.%:.-.)-(#1%0R($P%U(@@%;.%:"(M.1%C"(70"(@8%;8%-4.%0R(0@%".$#@3-(#1%#/%
-4.%:.-.)-(#1%@.1$%O5E5Y%e7%U(-4%VZRvZEYfP+%0@-4#3?4%0%7(1#"%(7C"#M.7.1-%O0"#31:%
*Zw%0-%-4.%U0($-%#/%.0)4%@(?4-G$4..-P%($%.RC.)-.:%:3.%-#%-4.%@(?4-G$4..-%(@@37(10-(#13E%
!
Supplementary!Note!2!
Microscope"instrument"control"architecture"
64.%7()"#$)#C.%)#1-"#@%.@.)-"#1()$%($%;3(@:%0"#31:%-U#%4(?4G$C..:%010@#?%#3-C3-%)0":$%
O[N%LANGHJSS%01:%LAN.GH5WI+%[0-(#10@%N1$-"37.1-$%A#"CEPE%\10@#?%#3-C3-%)4011.@$%0".%
3$.:%-#% :(".)-@8%$.-% -4.%@0$."% (1-.1$(-(.$%O3$(1?%01% 0)#3$-#G#C-()0@%-310;@.% /(@-."+%$..%
kC-()0@% $.-3C"(1% D.-4#:$P+% C#$(-(#1$% #/% abGL(.'#% $-0?.% 01:% ?0@M01#7.-"()% $)011."$%
O-4"#3?4%D(1(,0R%B#0":$+%A07;"(:?.%6.)41#@#?8%N1)EPE%6#%.1$3".%7()"#$.)#1:%C".)($.%
-(7(1?+%M#@-0?.%-"0).$%/#"%-4.$.%:.M().$%0".%C".G)#7C3-.:%01:%-4.1%3C@#0:.:%#1-#%-4.%
)0":$%-#?.-4."%U(-4%)#"".$C#1:(1?%:(?(-0@%-"0).$%-#%-"(??."%)07."0+%\k6K%;@01X(1?%01:%
(@@37(10-(#1%$-.."(1?%/@(CG7(""#"E%K(@-."%U4..@+%$C.)(7.1%"#-0-(#1%01:%$C.)(7.1%M."-()0@%
-"01$@0-(#1%0".%)#1-"#@@.:%#M."%c,5S5%C#"-$+%0$%-4.("%#C."0-(#1%($%1#-%-(7.%)"(-()0@E%
\1%(7C#"-01-%C0"-%#/%D3Q(G,LND%($%-4.%.//()(.1-%01:%/0$-%401:@(1?%#/%-4.%@0"?.%07#31-$%#/%
:0-0E%64.%/3@@%(70?.%0)]3($(-(#1%01:%C"#).$$(1?%C(C.@(1.%)#7C"($.$%#/%/#3"%)#7C3-."$_%0%
7()"#$)#C.%)#1-"#@%)#7C3-."%ON1-.@%A#".5F3#%"311(1?%0-% SE*H%2>'+%V%2B%c\DPi%-U#%
)07."0%)#7C3-."$%O.0)4%/.0-3"(1?%-U#%]30:G)#".%5EV2>'%N1-.@%a.#1%AL!$+%5V%2B%#/%c\D%
01:%\".)0%\cA%*YYZN%c\NF%)#1-"#@@."%U(-4%/#3"%5%6B%40":%:"(M.$Pi%01:%0%:0-0%010@8$($%
)#7C3-."%O$..%N70?.%/3$(#1"(1%D.-4#:$PE%\1%YZ%6B%c\NFH%;0$.:%$-#"0?.%$8$-.7%O3$(1?%0%
Sf0".%IJWZGY(%c\NF%)#1-"#@@."P%($%3$.:%-#%$-#".%-4.%@0"?.%07#31-$%#/%:0-0%C"#:3).:%;8%
D3Q(G,LNDE%\%C"(M0-.% *Z%?(?0;(-%9-4."1.-% #C-()0@%1.-U#"X% 0@@#U$%/#"%/0$-% -"01$/."%#/%
(70?.$%;.-U..1%-4.%7()"#$)#C.+%$-#"0?.%$8$-.7%01:%(70?.%C"#).$$(1?%)#7C3-."+%01:%
)#7731()0-(#1%;.-U..1%-4.%7()"#$)#C.%)#7C3-."$E%
%
64.%7()"#$)#C.%($%)#1-"#@@.:%;8%0%)3$-#7GU"(--.1%C"#?"07%)#:.:%(1%=0;Q(.U%O[0-(#10@%
N1$-"37.1-$%A#"CEP%-40-%401:@.$%-4.%)#7731()0-(#1%U(-4%-4.%.@.)-"#1()$%01:%-4.%#-4."%
)#7C3-."$E%
!
Supplementary!Note!3!
Assessment"of"imaging"time"
\@@G0"#31:%(70?(1?%U(-4%D3Q(G,LND%($%/0$-."%-401%U(-4%0%".?3@0"%,LND4%#"%7,LND5%/#"%
-U#%:($-(1)-%".0$#1$E%64.%/("$-%($$3.%)#1)."1$%-4.%-(7.%-4.%$C.)(7.1%($%0)-30@@8%(70?.:%
O.R)@3:(1?% -4.% -(7.% ".]3(".:% /#"% "#-0-(#1P% 0-% .0)4% -(7.% C#(1-E% D3Q(G,LND% C"#:3).$%
M("-30@@8% ".?($-.".:% (70?.$+% U4(@.% "#-0-(#1% ;0$.:% 73@-(GM(.U% (70?(1?% U(-4% ,LND% #"%
7,LND% ".]3(".$% (70?.G;0$.:% (70?.% ".?($-"0-(#1E% 64.% @0--."% 1.).$$(-0-.$% $3//()(.1-%
#M."@0C%;.-U..1%-4.%:(//.".1-%M(.U$+%U4()4%(1.M(-0;@8%@.0:$%-#%$#7.%0".0$%;.(1?%(70?.:%
73@-(C@.%-(7.$E%64.%.R0)-%$('.%#/%-4.%1.).$$0"8%#M."@0C%:.C.1:$%#1%-4.%C"#C."-(.$%#/%-4.%
$C.)(7.1%O$C.)(7.1%U(-4%@#U% C.1.-"0-(#1% :.C-4% 01:v#"% @0)X(1?% $40"C% /.0-3".$% U(@@%
?.1."0@@8%".]3(".%@0"?."%#M."@0C%;.-U..1%-4.%M(.U$PE%N1%-4.#"8+%-4($%#M."@0C%)#3@:%;.%
".:3).:%-#% 0% /"0)-(#1% O0$% @(--@.% 0$% 5ZwP% #/% -4.% -#-0@% $C.)(7.1% $('.+% 4#U.M."% 0$% -4.%
.//.)-(M.1.$$%#/%-4.% ".?($-"0-(#1%)011#-% ;.%0$$.$$.:% 31-(@%(-%($% 0)-30@@8%.R.)3-.:+% @#U%
#M."@0C% C3-$% ".$3@-$% #/% -4.% .1-(".% ".)#":(1?% 0-% "($XE% D#".#M."+% $(1).% ?.#7.-"()0@%
".@0-(#1$%;.-U..1%-4.% M(.U$%0".%1#-% X1#U1%;./#".401:+%(-% ($%:(//()3@-%-#% .1$3".%-40-%
.M."8%C0"-%#/%-4.%$C.)(7.1%($%)#M.".:%;8%0-%@.0$-%#1.%M(.U%(/%-4.%#M."@0C%($%-##%@#UE%N1%
C"0)-().+%-#% 0M#(:% -4.% C"#;@.7$% #/%(1$3//()(.1-%#M."@0C+% -4.% U4#@.% $C.)(7.1%($% #/-.1%
)4#$.1% -#% ;.% (70?.:% 0/-."% .M."8% "#-0-(#1+% .//.)-(M.@8% :#3;@(1?% O(1% 7,LNDP% #"%
]30:"3C@(1?%O(1%".?3@0"%,LNDP%-4.%-(7.%".]3(".:%/#"%(70?(1?E%
,.)#1:@8+%-4.%$C..:%U(-4%U4()4%0%?.@G4.@:%$C.)(7.1%)01%C".)($.@8%"#-0-.%($%@(7(-.:E%64.%
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Supplementary!Note!4!
Calculation"of"transformation"parameters"
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Supplementary!Note!5!
Equirectangular"projection"of"Drosophila"embryo"surface"""
f(-4%0%$(1?@.%IZ#%"#-0-(#1%-4.".%0".%.(?4-%M(.U$%#/%-4.%.7;"8#+%,3CC@.7.1-0"8%K(?E%WE%
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Supplementary!Note!6!
Further"details"on"analysis"of"Drosophila"embryo"images"
Early!Drosophila!development!
Drosophila!melanogaster!.7;"8#?.1.$($%-0X.$%C@0).%(1$(:.%01%.??%U(-4%)#1$-01-%$40C.%
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Supplementary!Protocol!2!
Diagnostic"specimen"preparation"
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Supplementary!Protocol!3!
Drosophila"embryo!preparation!and!mounting!!
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References!
*E% ,0@.4+%BE%9E%\E%³%6.()4+%DE%AE%Fundamentals!of!PhotonicsE%Of(@.8%³%,#1$_%5ZZJPE%
5E% 2"(@@+%,E%³%,-.@'."+%9E%>E%&E%D.-4#:%-#%)0@)3@0-.%@0-."0@%01:%0R(0@%?0(1%/0)-#"$%#/%#C-()0@%
$.-3C$%U(-4%0%@0"?.%$#@(:%01?@.E%J.!Opt.!Soc.!Am.!A%16+%5HWY´5HHW%O*IIIPE%
SE% 91?.@;".)4-+%AE% <E%³% ,-.@'."+%9E% >E% c.$#@3-(#1%.1401).7.1-% (1%0% @(?4-G$4..-G;0$.:%
7()"#$)#C.%O,LNDPE%Opt.!Lett%31+%*VJJ´*VJI%O5ZZHPE%
VE% >3($X.1+%<E+%,U#?."+%<E+%F.@%B.1.+%KE+%f(--;"#:-+%<E%³%,-.@'."+%9E%>E%&E%kC-()0@%,.)-(#1(1?%
F..C%N1$(:.%=(M.%97;"8#$%;8%,.@.)-(M.%L@01.%N@@37(10-(#1%D()"#$)#C8E%Science%305+%
*ZZJ´*ZZI%O5ZZVPE%
WE% >3($X.1+% <E% ³% ,-0(1(."+% FE% bE% cE% 9M.1% /@3#".$).1).% .R)(-0-(#1% ;8% 73@-(:(".)-(#10@%
$.@.)-(M.%C@01.%(@@37(10-(#1%7()"#$)#C8%O7,LNDPE%Opt.!Lett.%32+%5HZY´5H*Z%O5ZZJPE%
HE% ,U#?."+%<E+%Q."M.."+%LE+%2".?."+%&E+%>3($X.1+%<E%³%,-.@'."+%9E%>E%&E%D3@-(GM(.U%(70?.%
/3$(#1%(7C"#M.$%".$#@3-(#1%(1%-4"..G:(7.1$(#10@%7()"#$)#C8E%Opt.!Express%15+%YZ5I´
YZV5%O5ZZJPE%
JE% &.@@."+%LE%<E%et!al.%K0$-+%4(?4G)#1-"0$-%(70?(1?%#/%01(70@%:.M.@#C7.1-%U(-4%$)011.:%
@(?4-%$4..-G;0$.:%$-"3)-3".:G(@@37(10-(#1%7()"#$)#C8E%Nat!Meth%7+%HSJ´HV5%O5Z*ZPE%
YE% L".$$+%fE%>E+%6.3X#@$X8+%,E%\E+%Q.--."@(1?+%fE%6E%³%K@011."8+%BE%LE%Numerical!Recipes:!
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