Professional Documents
Culture Documents
As you explore the wonder and beauty of these images, take a moment to
read a little about how they were created. Explanations of the techniques
are placed throughout the exhibit and are numbered in ascending order
of complexity. Start here with #1 (Reflected Light Stereomicroscopy) and
work clockwise around the atrium until you reach #11 (2-Photon Confocal).
Each description tends to build a little more on the previous diagrams to
help you understand the technology required to make these images.
NECDPłAH@EI=CAO=NAOAAJ=O?KHKNA@OQ>FA?POKJ=SDEPA>=?GCNKQJ@1DA
?KHKNO=NAPDANAOQHPKB=JEJDANAJP
PN=JOHQ?AJP?KHKN
OQ?D=OPDACNAAJ
OA=SAA@ EJ PDA th LH=?A EI=CA
=J@ PDA NA@ =J@ CNAAJ KB PDA HERANSKNP
OLKNAO=J@AH=PANOEJPDAthLH=?AEI=CA1DA=>OPN=?P?KHKNO=J@L=PPANO
in the 6th LH=?A EI=CA =NA =HOK PDA NAOQHP KB PDA EJDANAJP ?KHKNO KB PDA
?NUOP=HHEVA@OLA?EIAJ
© James E. Hayden, RBC,FBCA
The Wistar Institute
4
!=NGBEAH@
!=NGłAH@IE?NKO?KLUD=O=RANUOEIEH=NREOQ=H?KJłCQN=PEKJPK>NECDPłAH@
AT?ALPPD=PEPD=O=@=NGłAH@OPKLLH=?A@>AHKSPDA?KJ@AJOANHAJO1DA
LQNLKOAKBPDEOOPKLEOPK>HK?G=JU@ENA?PHECDPBNKIL=OOEJCPDNKQCDPDA
OLA?EIAJ=J@KJHU=HHKSHECDPPD=PEO>AJPEJBNKIPDAA@CAOPKHECDPPDA
OQ>FA?P O PDA HECDP ATEPO PDA ?KJ@AJOAN
EP ?NA=PAO = @ACNAA ?KJA
SDE?DCN=VAOL=OPPDAK>FA?PERAHAJO
OKJK@ENA?PHECDPAJPANOPDAHAJOPK
BKNI=JEI=CA%KSARAN
=OPDAHECDPL=OOAOPDNKQCDPDAOLA?EIAJ
OKIA
KBPDAHECDPEONABN=?PA@Ġ>AJP
QLS=N@=J@PDEOEOPDAHECDPPD=PBKNIOPDA
EI=CA1DAłJ=HNAOQHPEOCAJAN=HHU=OLA?EIAJSEPD>NECDPA@CAOOAAJ=C=EJOP
=>H=?G>=?GCNKQJ@
6KQ?=JOAAPDEOSDEPAA@CA@NAOQHPEJPDAOPNQ?PQNAO=NKQJ@PDAKQPOE@A
KBNUVK=JEJPDAthLH=?ASEJJEJCEI=CA4EPDOLA?EIAJOPD=PD=RAOKIA
EJDANAJP?KHKN
HEGAPDANA@EJPDANUVK=JKNPDACNAAJKBPDACN=LAEJPDA
thLH=?AAJPNU
PDA?KHKN?=J=HOK>AOAAJ=C=EJOPPDA>H=?G>=?GCNKQJ@
© James E. Hayden, RBC,FBCA
The Wistar Institute
5
-KH=NEV=PEKJ
-KH=NEV=PEKJIE?NKO?KLUEO=HOKRANUOEIEH=NPKPDA>NECDPłAH@?KJłCQN=PEKJ
AT?ALP EP D=O LKH=NEVEJC łHPANO LH=?A@EJ PDA HECDP L=PDPK?NA=PA OKIA
OLA?E=HABBA?PO1DAłNOPLKH=NEVANEOLH=?A@KRANPDAHECDPOKQN?A
>ABKNA
PDAHECDPNA=?DAOPDAOLA?EIAJ1DAOA?KJ@Ġ?=HHA@PDA=J=HUVAN
EOLH=?A@
=>KRAPDAK>FA?PERA
=BPANPDAHECDPD=OL=OOA@PDNKQCDPDAOLA?EIAJ
-KH=NEVANO=HECJPDAHECDPS=RAOL=OOEJCPDNKQCDPDAIEJKJA@EOPEJ?P@ENA?PEKJ
4DAJłHPANO=NAPQNJA@=PNECDP=JCHAOPKA=?DKPDAN
PDAHECDPL=OOEJC
PDNKQCDPDAłNOPEO?KILHAPAHU>HK?GA@>UPDAOA?KJ@=J@JKHECDP?=J>A
OAAJĠPNUPDEOSEPDPSKL=ENKB-KH=NKE@OQJCH=OOAO
%KSARAN
EB=OLA?EIAJ
LH=?A@>APSAAJPDAPSKATDE>EPO=?ANP=EJLNKLANPU?=HHA@>ENABNEJCAJ?A
EPSEHH>AJ@OKIAKBPDAHECDPBNKIPDAłNOPLKH=NEVANB=NAJKQCDPKL=OO
PDNKQCDPDAOA?KJ@1DEOEOKBPAJOAAJEJ?NUOP=HOHEGAPDAN@LNEVAAJPNUKB
3EP=IEJ
PDAPDLNEVAAJPNUKBOQ??EJE?=?E@=J@QNA=
=J@PDAthLNEVA
AJPNU KB LKP=OOEQI ?DHKN=PA 1DA KRAN=HH ABBA?P EO = OLA?PNQI KB ?KHKNO
@ALAJ@EJCKJPDAOLA?EIAJOOLA?Eł?LNKLANPEAO=J@KNEAJP=PEKJ
© James E. Hayden, RBC,FBCA
The Wistar Institute
6
-KH=NEV=PEKJ
SEPD/DAEJ>ANC#EHPAN
The lighting technique shown in the 19th place winning entry of a bird
feather is a simple expansion of the polarization described in the previous
(#5) technique. In this example, standard cross polarization creates colors
and contrast in the birefringent specimen, but additional color is added by
inserting a red/blue quadrant Rheinberg filter below the specimen. This
filter adds out of focus colored light to the background and also attenuates
the colors created by the cross polarization.
© James E. Hayden, RBC,FBCA
The Wistar Institute
7
-KH=NEVA@
Reflected Light
1DEOPA?DJEMQA?KI>EJAOPDAE@A=OKBNAŃA?PA@HECDPIE?NKO?KLUĠPA?DJEMQA
=J@LKH=NEV=PEKJIE?NKO?KLUĠPA?DJEMQA
>U=@@EJC=BAS=@@EPEKJ=H
D=N@S=NAIK@Eł?=PEKJOPKPDAQLNECDPIE?NKO?KLA#ENOP
PDAEHHQIEJ=PEKJ
H=ILDKQOAEOIKQJPA@=>KRAPDAOLA?EIAJ=J@=OLA?E=HD=HBIENNKNNAŃA?PO
HECDP@KSJPDNKQCDPDAK>FA?PERAPKB=HHKJPDA?NUOP=HHEJAOQ>FA?PEJPDEO
thLH=?AAJPNU@@EJC=SNEJGHAPKPDAOAPQL
=LKH=NEVANEOLH=?A@EJPDA
HECDPL=PD>ABKNAPDAIENNKN
=HECJEJCPDAHECDPS=RAOEJ=@EOPEJ?PKNEAJP=PEKJ
>ABKNA PDAU NA=?D PDA OLA?EIAJ O PDA HECDP NAŃA?PO KBB PDA OQ>FA?P
EP
?D=JCAOKNEAJP=PEKJ
=??KN@EJCPKPDA?D=N=?PANEOPE?OKBPDA?NUOP=HO1DA
EI=CABKNIEJCHECDPEO?KHHA?PA@EJPDAK>FA?PERA=J@?KJPEJQAOQLPDNKQCD
PDAD=HBIENNKN
=J@=J@LKH=NEVEJCłHPANĠ=J=HUVAN
KJEPOS=UPKPDA?=IAN=
BKNEI=CA?=LPQNA1DA=J=HUVAN>HK?GO=JUHECDPPD=POP=UOEJPDAKNECEJ=H
KNEAJP=PEKJ
=J@KJHUL=OOAOPDAHECDPPD=PEONAKNEAJPA@>UPDAOLA?EIAJ
© James E. Hayden, RBC,FBCA
The Wistar Institute
8
!EBBANAJPE=H&JPANBANAJ?A
KJPN=OPÎ!&
!& I=GAOQOAKB=HHPDAAHAIAJPOKBPDAOP=J@=N@LKH=NEVA@HECDPIE?NKO?KLA
ĠPA?DJEMQA
>QP=@@O=@@EPEKJ=HLNEOIOPD=P=HPANPDALD=OAKBPDAHECDP
S=RAO=OPDAUL=OOPDNKQCD)EGAPDALKH=NEVANO
PDA!& LNEOIO=NAHK?=PA@
KJAEPDANOE@AKBPDAOLA?EIAJ1DAłNOPLNEOIOAL=N=PAOA=?DHECDPS=RAEJPK
HECDP S=RAO SEPD = OLA?Eł? LD=OA =HECJIAJP >ABKNA PDAU EJPAN=?P SEPD PDA
OLA?EIAJ1DA?D=N=?PANEOPE?OKBPDAOLA?EIAJPDAJ=HPANPDALD=OA=OPDAHECDP
L=OOAOPDNKQCD
?NA=PEJCEJPANBANAJ?A1DAOA?KJ@!& LNEOI>NEJCOPDAHECDP
S=RAO>=?GPKCAPDAN
>QPPDAEJPANBANAJ?ABNKIPDAOLA?EIAJ?NA=PAO=?D=JCA
EJPDAOD=LAKBPDAHECDPS=RA1DALD=OAKBPDAHECDPEO=HPANA@IKOPHU>UPDA
@AJOEPUKBPDAOLA?EIAJ=OEPL=OOAOPDNKQCD
OKPDACNA=PAOPABBA?PO=NAOAAJ
=PPDA>KQJ@=NEAOKB@AJOAN=NA=O
HEGAPDAA@CAKBPDAOLA?EIAJ
1DALKH=NEVANOEJPDEOPA?DJEMQAAJD=J?APDALD=OAABBA?PO=J@=HOKLNKRE@A
=J=@@EPEKJ=HOLA?PNQIKB?KHKNEJOKIAOLA?EIAJOPD=PATDE>EP>ENABNEJCAJ?A
1DA ?NUOP=HHEJA OPNQ?PQNAO EJ PDA th LH=?A AJPNU =NA J=PQN=HHU PN=JOL=NAJP
=J@PDA?KJPN=OP=J@?KHKNOSANA?NA=PA@KJHU>A?=QOAKBPDAKLPE?=HABBA?PO
KBPDA!& PA?DJEMQA4EPD>EKHKCE?=HOLA?EIAJOHEGAPDA@E=PKIOEJPDAth
LH=?AAJPNU
!& EOCAJAN=HHUQOA@PKLNKRE@A=OD=@KSA@
@EIAJOEKJ=HHKKG
1DA=@@EPEKJ=H?KHKNOAAJEJOE@APDAOAOLA?EIAJOEOPDAEJDANAJP?KHKNKBPDA
OPNQ?PQNAOPDAIOAHRAO
© James E. Hayden, RBC,FBCA
The Wistar Institute
9
#HQKNAO?AJ?A
#HQKNAO?AJ?AIE?NKO?KLUKB>EKHKCE?=HPEOOQAONAHEAOKJOLA?E=HŃQKNAO?AJP@UAO
KN EJDANAJP LNKPAEJO LNK@Q?A@ >U ?AHHO PK H=>AH OLA?Eł? L=NPO KB PDA ?AHHQH=N
OPNQ?PQNA 4DAJ PDAOA O=ILHAO =NA K>OANRA@ PDNKQCD = OP=J@=N@ >NECDPłAH@
IE?NKO?KLAĠPA?DJEMQA
JKPDEJC?=J>AOAAJQPSDAJPDAOAOLA?EIAJO=NA
AT=IEJA@EJPDA@=NG=J@EHHQIEJ=PA@SEPD=>NECDPHECDPKB=OLA?Eł??KHKN
PDAU
CHKS>NECDPHUEJ=JKPDAN
1DA GAU PK PDEO PA?DJEMQA HEAO EJ = OLA?E=H łHPAN >HK?G PD=P KJHU HAPO ?ANP=EJ
?KHKNOL=OOEJ?ANP=EJ@ENA?PEKJOOKEP?=JOAL=N=PAKQPPDAOLA?Eł??KHKNO
RANU>NECDP
SDEPAHECDPBNKI=IAN?QNUH=ILLNKRE@AOPDAEHHQIEJ=PEKJOKQN?A
4DAJPDEOHECDPEO@ENA?PA@EJPKPDAłHPAN>HK?G
EPL=OOAOPDNKQCD=łHPANKB=
OLA?Eł??KHKN
?=HHA@PDAAT?EP=PEKJłHPAN&JKQN@E=CN=IAT=ILHA
PDEOLNKRE@AO
PDA>HQAHECDP1DAHECDPEONAŃA?PA@KBB=OLA?E=HIENNKN
?=HHA@=@E?DNKE?=J@EO
@ENA?PA@@KSJ=PPDAOLA?EIAJ
PDNKQCDPDAK>FA?PERA
BNKIPDAPKL
1DA >HQA AT?EP=PEKJ HECDP OPEIQH=PAO PDA ŃQKNAO?AJP I=NGANO EJ PDA ?AHH =J@
?=QOAOPDAIPKŃQKNAO?A
KNCHKS
EJ=>NECDPCNAAJ?KHKN1DEOAIEPPA@CNAAJ
HECDPĠ=OSAHH=ONAŃA?PA@>HQAHECDP
PDAJPN=RAHO>=?GQLPDAK>FA?PERAPKBKNI
PDA EI=CA 1DA @E?DNKE? IENNKN HAPO PDA CNAAJ HECDP L=OO NECDP PDNKQCD
>QP
PDA>HQAHECDPEOIKOPHUNAŃA?PA@=S=UJU>HQAHECDPPD=PI=GAOEPPDNKQCDEO
?KILHAPAHUOPKLLA@>U=>=NNEANłHPAN=PPDAPKLKBPDAłHPAN>HK?GPD=POPKLO
=HH>HQAHECDPBNKIL=OOEJC
>QPHAPOPDACNAAJPDNKQCD
!EBBANAJPłHPAN>HK?GO=NAJAA@A@BKN@EBBANAJPŃQKNAO?AJPI=NGANO
OKPDAOA
PULA KB EI=CAO =NA CAJAN=HHU P=GAJ KJA ?KHKN =P = PEIA =J@ PDAJ ?KI>EJA@
EJPKKJALE?PQNA=BPANS=N@1DAIKOP?KIIKJ?KHKN?KI>EJ=PEKJO=NA>HQA
CNAAJ=J@NA@
=OODKSJEJPDAł>NK>H=OPOKBPDAthLH=?AAJPNU
@@EPEKJ=H LKOPLNK?AOOEJC KB PDA EI=CAO =HOK LNKRE@AO AJD=J?A@ @AP=EH
JKP KPDANSEOA OAAJ &J PDA th LH=?A AJPNU KB @ERE@EJC 5AJKLQO ?AHHO
I=JU
LE?PQNAOSANAP=GAJKBPDAOQ>FA?P=OPDAIE?NKO?KLAS=OBK?QOA@PDNKQCDPDA
OLA?EIAJ
?NA=PEJC=ļOP=?GKBEI=CAOA?=QOAKBPDAPDE?GJAOOKBPDA?AHHO
KJHUOKIAKBPDAEI=CAEOEJOD=NLBK?QOEJA=?DļOHE?AOLA?E=H=HCKNEPDI
?=HHA@@A?KJRKHQPEKJS=OPDAJQOA@PKATPN=?PKJHUPDAOD=NLAOP=NA=OKBA=?D
EI=CAEJPDAOP=?G=J@PDAJ?KI>EJAPDAOAPK?NA=PA=OEJCHAEI=CASEPDCNA=PAN
@ALPD=OD=NLJAOOPD=JSKQH@KPDANSEOA>ALKOOE>HA
© James E. Hayden, RBC,FBCA
The Wistar Institute
10
Confocal and Polarization
%QPHQECNOKETQUEQR[KUCPCFXCPEGFHQTOQHƀWQTGUEGPEGOKETQUEQR[YJKEJRTQXKFGU
more detail, less blur and the ability to visually “section” through a specimen. The
DCUKEEQPEGRVUQHƀWQTGUEGPEG
VGEJPKSWGCNNCRRN[DWVVJGTGCTGCHGYOCLQT
differences. The light source for confocal needs to be very bright, so lasers are most
QHVGPWUGF$GECWUGNCUGTUCTGOQPQEJTQOCVKENKIJVUQWTEGU
QPGEQNQTVJGTGKUPQ
PGGFHQTCſNVGTDNQEMYKVJCPGZEKVCVKQPſNVGT6JGNCUGTNKIJVKUVTCPUOKVVGFVJTQWIJ
a very small pinhole, providing a high resolution spot of light to illuminate the
URGEKOGP6JKUURQVQHNKIJVKUTGƀGEVGFQHHCFKEJTQKEOKTTQTCPFUECPPGFCETQUUVJG
ſGNFQHKPVGTGUVKPCXCTKGV[QHYC[UETGCVKPIVJGGOKVVGFƀWQTGUEGPVNKIJVCVGXGT[
RQKPVKVRCUUGU#UYKVJVJGƀWQTGUEGPEGFKCITCO
VGEJPKSWGVJGGZEKVCVKQPNKIJV
KPQWTKNNWUVTCVKQPKUDNWGCPFVJGGOKVVGFƀWQTGUEGPEGKUITGGP
6JGGOKVVGFNKIJVCUYGNNCUUQOGQHVJGGZEKVCVKQPNKIJVRCUUGUDCEMVJTQWIJVJG
objective, the dichroic mirror, and then through a special, adjustable pinhole which
has the effect of blocking most of the out-of-focus light coming from parts of the
specimen above and below the plane of focus. The image formed by this light is
HWTVJGT OCIPKſGF CPF ECRVWTGF D[ C URGEKCN FGXKEG ECNNGF C RJQVQOWNVKRNKGT VWDG
2/6(QTCRTCEVKECNFGUETKRVKQPVJG2/6UCTGURGEKCNECOGTCUVJCVECPDGVWPGF
VQőUGGŒQPN[URGEKſEYCXGNGPIVJU
EQNQTUQHNKIJV/QUVEQPHQECNUJCXGOWNVKRNG
2/6UCPFECPTGEQTFVJGFKHHGTGPVEQNQTEJCPPGNUUKOWNVCPGQWUN[
The 7th RNCEG YKPPKPI GPVT[ QH ECTRGV ſDGTU YCU ECRVWTGF YKVJ C EQODKPCVKQP QH
EQPHQECN CPF RQNCTK\CVKQP VGEJPKSWGU
UGG VGEJPKSWG $GECWUG NCUGT NKIJV KU
inherently polarized, the light passing through the specimen is already oriented. The
light waves interact with the specimen and then pass through an analyzer which only
CNNQYUTGQTKGPVGFNKIJVVJTQWIJ6JKUſPCNKOCIGKUVJGPECRVWTGFYKVJCVTCPUOKVVGF
NKIJV2/6CVVJGUCOGVKOGCUVJGTGƀGEVGFƀWQTGUEGPEG
© James E. Hayden, RBC,FBCA
The Wistar Institute
11
-DKPKJ KJBK?=H
LDKPKJ?KJBK?=HIE?NKO?KLUEO=J=@R=J?A@BKNIKBOP=J@=N@?KJBK?=H
IE?NKO?KLU
SEPDPDA=>EHEPUPKQOALQHOA@
HKJCANS=RAHAJCPDOH=OANOPK
?NA=PAPDAAT?EP=PEKJHECDPQOA@PKOPEIQH=PAŃQKNAO?AJ?AEJPDAOQ>FA?P
)KJCANS=RAHAJCPDO?=JLAJAPN=PA@AALANEJPKPDAOLA?EIAJ=J@=NAHAOO
@=I=CEJCPKHEREJCPEOOQA
I=GEJCPDEOOUOPAIE@A=HBKNHKKGEJCEJPKPDE?G
OLA?EIAJO
HEREJCPEOOQA=J@?AHHO
1DA>=OE??KJ?ALPOKBŃQKNAO?AJ?AĠPA?DJEMQA
=J@?KJBK?=HĠPA?DJEMQA
=HH =LLHU
>QP PDA LQHOA@ H=OAN EO SD=P I=GAO PDA @EBBANAJ?A 1SK
QJEPOKBNA@HECDP
OL=?A@BAIPKOA?KJ@O=L=NP
LNKRE@APDAO=IA=IKQJP
KBAJANCU=OKJAQJEPKB>HQAHECDP1DEOIA=JOPDAAT?EP=PEKJHECDPEONA@
EJOPA=@KB>HQAEJKQNEHHQOPN=PEKJ
=HPDKQCDEP?NA=PAOPDAO=IA?KJPEJQKQO
CNAAJ ŃQKNAO?AJ?A @AIKJOPN=PA@ EJ PDA LNAREKQO ŃQKNAO?AJ?A =J@
?KJBK?=H@E=CN=IO1DANAOPKBPDAOUOPAIEOPDAO=IA=O=OP=J@=N@?KJBK?=H
IE?NKO?KLA=J@EI=CAO=NANA?KN@A@EJPDAO=IAS=U
1DAAT?ALPEKJ=H@AP=EHEJSEJJEJCEI=CAO=J@EO=HOK@QAEJL=NPPK
OLA?E=H LNK?AOOEJC OKBPS=NA SDE?D P=GAO IQHPELHA EI=CAO =J@ OA=IHAOOHU
?KI>EJAOPDAIOE@APKOE@AEJPK=OEJCHA
DECDNAOKHQPEKJEI=CA
© James E. Hayden, RBC,FBCA
The Wistar Institute
Credits
JAMES E. HAYDEN, RBP, FBCA
Manager, Microscopy Core Facility
jhayden@wistar.org
FREDERICK S. KEENEY
Assistant, Microscopy Core Facility
CHRISTINE DELAURENTIS
Graphic Artist
SOHEILA NIKPOUR
Web Developer
5PEBZT%JTDPWFSJFTo5PNPSSPXT$VSFT
3601 Spruce Street Philadelphia, PA 19104 www.wistar.org