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| Chemiluminescence
Labeling and Detection of
Bio-Rad Laboratories
Life Science Group
Chemiluminescence Systems
2000 Alfred Nobel Drive
Hercules, CA 94547
Enhanced Imaging of Chemilum
1-800-4BIORAD (1-800-424-6723)
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inescen
Tec
ce
hnical Support: lsg_techserv_us@bio-rad.com
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Signal Detection by Direct Digital Imaging vs X-ray
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e Quality Perception
Imaging chemiluminescent blots with charged-coupled device A common misperception when comparing images from CCD
(C
Enhanced Imaging of Chemiluminescence
CD) cameras is rapid, and the easiest, most accurate method. cameras to X-ray film is that the CCD image is not as good as the
Traditionally, the chemiluminescent signal from blots has been film image. A high quality image is one without saturated features,
d
E
et
n
ec
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ted
a
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c
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ra
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cenc
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e
nd the data throughout the entire sample range is linear. Film
le
Signal Detection by Direct
d to development of special films with enhanced sensitivity to the re
Image Quality P
aches saturation qu
erception
ickly, and does not indicate the degree
bl
Digital Imaging vs.
ue wavelengths of light
X-ray Film
emitted by chemiluminescent substrates. to
A
w
common
hich the e
misperception
xposure is beyo
when
nd satu
comparing
ration. Ther
ima
efor
ges
e, a b
from
road
CCD
IfS
Ima
qiguna
ging
natli tDaetiv
chemiluminescent
tec dtaiotan ibsy d Desirireecdt, Dfi
blots
limgi teaxl p
with
Iomsaegd i
charged-coupled
ntog c vhse mX-ilruamy inFeilsmce
device
nt raI
cameras
mngaeg eo fQ sui
to
gan
X-ray
laitlys Pape

pr
lm
ceea
is
prst
tha
isoinm
t the
ilar,
CCD
prev
ima
enti
ge
ng
is
di
not
rect
as
qu
good
antit
as
ativ
the
e
fi lm
sIim
(CCD)
gnaaglisn gi
cameras
s cimheamgeiludm
is
onin
ra
ea
pid,
s dcen

s
and
t ibtolomt
the
se tweri
easiest,
,t ha ncdh athr

ge
most
e di-gciota
accura
ul pimleda gd
te
ee va
method.
incae lyzed

cAo
ima
mcop
ge.
marmi

s
A
on
high
m(Figs
quality
puererc 2e,p u
ima
tipopne
ge
wr hp
is
ean
one
ecl)o. m
without
A pCaCriDng i m
sa
ima
tura
aggee,
ted
so nfr ot
fea
hme
tures,
oCtChD er
and
to(C
T
p
raditionally
CrDod) uccaem tehr
,
ea

s
the
fi nisa rl
chemiluminescent
adpaitda, . a Wndi tthh ere ecaesnite as
signal
dt,v manocse
from
t sa cinc ute
blots
racthen mol
has
oegthyo a
been
the data throughout the entire sample range is linear. Film reaches
detected by X-ray fi lm. The popularity of chemiluminescence
dn
has
. d hc
sa
aanm
tura
d,e wra
tion
isth t oit
quickly
sX -grraeya tfi
,
e

l
and
mr d iysn
does
tahmatic t
not
hraen Cg
indica
CeD, w imi
te
ll atyg
the
pei cisa
degree
lnlyo ht avse
to
g doao
which
tda as t
the
he
reT
led
rdaudci
to
tio
development
na ilnly ,t hthee c ochste
of
mof i
special
lcuomminpeust

ec
lms
resn at ns
with
dig nCaC
enhanced
l Dfr ocmam belorat
sensitivity
s ,h daisre bcet edn
to
igit
the
al dfi
exposure
islmtri bimutaegde
is
t.h Ar
beyond
ohuigh oquu
sa
ta tlh
tura
itey sim
tion.
amagp

el
Therefore,
e i sr aonngee w (Fi

ti
a
hgou
broad
uret s2a, tlu
range
orwateerd p
of
faena
signals
etul).r eAsn ,
adc
blue
eqtueicst
wa
ietiod
velengths
n b oy fX c-hraeym fi
of
illumm
light
. iTnhees
emitted
cpeonptu slaigr
by
nitay
chemiluminescent
lso fh cahse bmeicluomien epsrcae
substra
cnticea l.h A a
tes.
s

ima
a
n
ppears
adg eth cel ods
similar
aetra inth ar
,
o

p
preventing
upgehaoraunt cthee t o
direct
e nthtiere m
quantita
soarme pfalem
tive
rialinagr
comparison
eX -isra liyn fielamr. Fcia
(F
lm n
igure
be
CleC
If
dD
quantita
t oca dmeveeral
tive
o rpemq
da
uenir
ta
te so
is
fo sn
desired,
plye cthiaal tfi a

lm

w
lm
sr awp
exposed
ipthe de nbhloatn
to
bce
chemiluminescent
dp lsaecnesdit iovnit yt hte o the ar
2,
ceha

i
upper
cehveds sb
panel).
ayt utrraantiso

f
A
no r
CCD
qmuiincgk
ima
lyth, ea
ge,
nrde

pd
on
roees
the
se nntoa
other
t ioinnd ioc
hand,
fa tthee t h

C
with
eC dDe ig
its
mreae
grea
ge to
ter
imb
signals
laugei nwga vs
is
eulr
ima
efancge
ged
th, sa no
on
df lti
a
hgeh
densitometer
ti netmegitrtaetdio bny
,
(

ec
and
xhpeom
the
siulurem
digital
) itnimesec
ima
esnet
ge
. sTuhb
analyzed
esntr athtee s. et
d
xop
ynamic
wrehsisc hth te
range,
h ed aetxap

fo
will
rsoumr
typically
e m isid b reaynog
ha
ned
ve
asnad
da
tu hr
ta
aigtiho
distributed
enr. Tath egreafyo lr
throughout
ev, eals b trhoatd a
the
re
sIif
to
g qnua
produce
al nist iatantaivley
the
z dead

t.

a
nal
is
da
de
ta.
sir

e
With
d, film
recent
expo
advances
sed to che
in
mi
technolog
luminesce
y
n
and
t crlsample aonseg et oo fbrange lsaigckn.a(Flsigure appe2,ar slo swer imilpanel).ar, pre vAn enimatingge dirceloser ct quin anatppearance itative
sreduction ignals is imin athe gedcost on aof decomputers nsitometerand , andCCD the cameras,digital im direct age andigital alyzed c
to the more familiar X-ray fi
omparison (Figure 2, uppe
lm can be achieved by transforming the
r panel). A CCD image, on the other
Ftio
acquisition
lm p riso dau scen tsh
of
ieti vfi
chemiluminescent
en mal eddaiutam. Wforit hc arpetcu
signals
ernint ga dsviga
has
nnacles
become
s b iunt taenc hin
practical.
hoelorgeny t an

d
A
h
representa
and, with
tion
its g
of
rea
the
ter
CCD
dyna
ima
mic
ge
ra
to
ng
express
e, will ty
the
pica
da
lly
ta
h
from
ave d
mid
ata
range
drre
CCD
adwubcat
camera
icokn iisn itsh
requires
es icgomsot iodfa
only
cl oremsp
tha
uotne
t
sre
a
s ta
wra
on dlig
pped
ChCt, Dw ch
blot
aicmh e
be
rreassu
placed
, ltdsi rienc a t
on
dig
the
ital d
and higher a
istributed th
t gray levels tha
roughout the sa
t are c
mple
lose to black.
range (Figure 2, lower panFeilml). An
naa
ima
crqrou
ging
wis iltiinoen
surface,
a or fr ecshpe

om
and
nisluem
the
reingei
integra
osnce, ntht us
tion
sig lnima
(exposure)
listi nhga sit sb edcyon
time
mame icp
set.
rraacn

t
Then
gicea.l . A
the
image closer in appearance to the more familiar X-ray film can be
InC
signal is analyzed.
CcoDn ctramste, raa 1re6q buiitr

eCsC oDn clya tmhaetr aa swurcahp apse dth balto to nb et hpela Mceodle ocnu lathr e a
Summar
chieved by
y
transforming the representation of the CCD image to
Imimaaggeirn
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g VseurrsfacDeo,c a

n dM tPh e5 i0n0te0g Sraytsiotenm (e, xhpaoss uar neo) ntismaetu sreat.e Tdh leinne athr e e
Digital
xpress
acquisition
the data from
of
m
chemiluminescent
id range and highe
signals
r at gray
has
leve
become
ls th
V
a
e
t
r

s
a
a
r
D
e
a
oc
res
F
isg
ilm
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is
sise
a
aonv
sensitive
aelry tzheed .sam
medium
e range
for
of s
ca
am
pturing
ples (F
signals
igure 1)
but
.
an inherent
practical technology that is easy to use. Compared to fi lm, CCD
drawback is its sigmoidal response to light, which results in a

clo
2
s
.7
e

to b
6
l
.
a
0
c

k.
21.2 71.3 202 706 1,840 6,903 16,907
Light units
1
F
0
i
0
lm
,00

0
is a seVnesrsiatDivoec MmPe5d00iu0m for capturing signals but an inherent
Fig. 2. Calibrated luminometer reference plate recorded on x-ray film and VersaDoc MP 5000.
drawback is
X-

R
it
a
s
y

F
s
il
i
m
gmoidal response to light, which results in a Film
n
1
a
0,
r
0
r
0
o
0
w linear response region, thus limiting its dynamic range.
In contrast, a 16 bit CCD camera such as that on the Molecular
Summary
Imager
®
VersaDoc

MP 5000 System, has a nonsaturated linear
Digital acquisition of chemiluminescent signals has become
VersaDoc
re
1
s
,0
p
0
o
0
nse over the same range of samples (Figure 1).
a practical technology that is easy to use. Compared to film,
n
i
t
s
2.7 6.0 21.2 71.3 202 706 1,840 6,903 16,907
CCD imaging has a greater d
Lig
y
h
n
t
a
un
m
its
ic range, which can be easily
L
i
g
h
t
u
100,000 displayed by adjusting the data expression.
100 VersaDoc MP 5000
FFigig. . 2.2. CaClibarlaibterda ltuemd inluomeinteor mreefetreer ncre fpelareten rcee coprdlaetde onre xc-oraryd feild m oan ndX V-errasy aDfoilcm MaPn 5d 000.
X-Ray Film
VersaDoc MP 5000.
10,000
10
S
ima
um
ging
mar
has
y
a greater dynamic range, which can be easily displayed
D
by adjusting the da
igital acquisition o
ta expression.
f chemiluminescent signals has become
1,000
0
a practical technology that is easy to use. Compared to film,
t
u
n
i
t
s
10 10 100 1,000 10,000 100,000
CCD imaging has a greater dynamic range, which can be easily
L
i
g
h
Adj. volume
displayed by adjusting the data expression.
100
Fig. 1. Linearity of signal.
narrow linear response
A calibrated luminometer reference plate with peak emission w
region, thus limiting its dynamic
avelength of
range.
440 nm was placed on Kodak
®
BioMax

XAR film for 30 seconds or imaged by a VersaDoc MP 5000
for 30 seconds. The film was subsequently scanned on a GS-800In contrast, a 16 bit CCD camera such as

calibrated densitometer. Volume that on the Molecular
analysis of the images was performed with Quantity One
Ima
1
ger
0
®
VersaDoc
TM
MP 5000 System
®
software.
has a nonsaturated linear
Biomax and Kodak are trademarks of Eastman Kodak Company.
response over the same range of samples (Figure 1). BioMax and Kodak are trademarks of Eastman Kodak Company.
0
10 10 100 1,000 10,000 100,000
Adj. volume
Fig. 1. Linearity of signal. A calibrated luminometer reference plate with peak emission wavelength of
440 nm was placed on Kodak
®
BioMax

XAR film for 30 seconds or imaged by a VersaDoc MP 5000
for 30 seconds. The film was subsequently scanned on a GS-800Protocol Guide ı 2008

calibrated densitometer. Volume View entire protocol online at www.biotechniques.com/protocol ı BioTechniques ı 15
analysis of the images was performed with Quantity One
®
software.
Biomax and Kodak are trademarks of Eastman Kodak Company.
BioRad labeling Protocol.indd 15 10/25/07 6:50:48 PM
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