imaging of light emission from the expression of luciferases in living cells and organisms

��

��

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Received 13 August 2001; accepted 16 October 2001

ABSTRACT: Luciferases are enzymes that emit light in the presence of oxygen and a substrate (luciferin) and which have been usedfor real-time, low-light imaging of gene expression in cell cultures, individual cells, whole organisms, and transgenic organisms. Suchluciferin–luciferase systems include, among others, the bacterial lux genes of terrestrial Photorhabdus luminescens and marine Vibrioharveyi bacteria, as well as eukaryotic luciferase luc and ruc genes from firefly species (Photinus) and the sea panzy (Renillareniformis), respectively. In various vectors and in fusion constructs with other gene products such as green fluorescence protein(GFP; from the jellyfish Aequorea), luciferases have served as reporters in a number of promoter search and targeted gene expressionexperiments over the last two decades. Luciferase imaging has also been used to trace bacterial and viral infection in vivo and tovisualize the proliferation of tumour cells in animal models. Copyright � 2002 John Wiley & Sons, Ltd.

KEYWORDS: luciferases; gene expression; low-light imaging; luciferase expression constructs

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From time immemorial, seamen and fishermen haveobserved ‘lights’ on the water. In the nineteenth century itwas realized that the most frequent cause of suchluminous oceanic phenomena are minute marine organ-isms emitting light—bioluminescence. About 35 yearsago, various luciferases began to be characterized (1, 2)which, in their many forms, in the presence of a substrate,a luciferin, emit light in the visible range underphysiological conditions. Some eukaryotic organisms,such as the firefly (Photinus), have their own luciferin–luciferase light-emitting systems. Many marine organ-isms, however, such as mid-depth fishes and invertebratessuch as molluscs, emit light because of symbioses withluciferase-producing bacteria occurring in highly specia-lized light organs. These luminescent bacteria includetaxa such as Photobacterium phosphoreum, P. leiognathi,Vibrio logei, V. harveyi and V. fischeri.

It is to be expected that a costly characteristic likebiological production of light would be retained only ifluminescent visualizing were advantageous. Biolumines-cence is used as a disguise for fleeing prey, for ventrallight emission to efface an organism’s shadow and render

it invisible from below (3, 4), for luring prey (ceratioidfish), for signalling for courtship and mating, and instress-induced light emission (bioluminescent plankton).One could argue that ever since such metazoanbioluminescent bacteria symbioses and other biolumi-nescent organisms appeared in the oceans with theirunique light emission systems, there has been in vivoluminescent ‘imaging’ or visualization.

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Marine bioluminescence may be considered one of themost widespread forms of communication on the planet.Organisms emit light that other organisms detect or‘visualize’ and to which they give some behavioralresponse (5). Behavior based on natural bioluminescenceimaging may be classified under three general headings (5):offence (luring, baiting); defence (startle, camouflage); andcommunication (courtship and mating). Some striking usesof natural bioluminescent ‘visualization’ include thefollowing: some squids with bacterial symbionts useshadow-effacing, or modulation of their ventrally-emittedlight, to match ambient sunlight or moonlight; crustaceans,similar to fireflies, may use a repetitive mating ‘Morsecode’ of blinks; some jellyfishes deposit an adhesive glowupon contact with predators, leaving the predator visibleand vulnerable; some squids flee, leaving a luminescentcloud of ‘ink’ in a predator’s face; some dragonfishes(Malacosteidae) emit blue-green light, but also emit a‘night-vision’ long-wavelength red light by which they can

Luminescence 2002;17:43–74DOI: 10.1002/bio.676

*Correspondence to: A. A. Szalay, Department of Biochemistry,School of Medicine and Department of Natural Sciences—BiologySection; Loma Linda University, Loma Linda, CA 92354, USA.Email: [email protected]

Contract/grant sponsor: Dept. Natural Sciences, Loma Linda Uni-versity, CA, USA.Contract/grant sponsor: Basic Science Research Grant from LomaLinda University, USA.

Copyright 2002 John Wiley & Sons, Ltd.

REVIEW

detect prey (using reverse fluorescence energy transfer)without their prey seeing them (6–12).

The purpose here is to review the representativescientific imaging applications to which these naturallyoccurring visible light bioluminescent systems, the genesencoding the proteins and their modifications have beenput. However, we first present an overview of theluciferin–luciferase light emission systems.

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Luciferase is a generic name because none of the majorluciferases share sequence homology with each other (5).Luciferases occur in bacteria, fungi, dinoflagellates,radiolarians and about 17 metazoan phyla and 700genera, mostly marine (5, 12, 13). These include Anne-lida (segmented worms), Chordata (some elasmobran-chiomorphs or sharks, many teleosts or bony fishes),Cnidaria (jellyfishes, anthozoans such as the sea pansy,Renilla), Chaetognaths (one species of arrow-worm),Crustacea (many, including ostracods and euphausiidshrimps or krill), Ctenophora (comb jellies), Echinoder-mata (sea stars, brittle stars), hemichordate worms,Insecta (fireflies, click beetles), Mollusca (squids,octopods, nudibranchs), Nemertean worms (one species),Pycnogonids (sea spiders), Urochordata (larvaceans,pyrosomes, and one tunicate), millipedes and centipedes(12). Phylogenetic analyses suggest that luciferin–luciferase systems have had more than 30 independentorigins (5, 14–16).

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Bioluminescence is a chemiluminescent reaction be-tween at least two molecules produced under physio-logical conditions within or in association with anorganism. The substrate molecule reacted upon, whichemits light in such a reaction, is called a luciferin.Luciferases are a wide range of enzymes that catalyse theoxidation of substrate luciferins to yield non-reactiveoxyluciferins and the release of photons of light (17–21).As luciferin substrates are used, they must be replenished,which usually occurs through diet. Some luciferinsrequire the presence of a co-factor to undergo oxidation,such as FMNH2

�, Ca2� or ATP (22). Complexes thatcontain a luciferase, a luciferin, and generally requiringO2 are also called photoproteins (12).

Although luciferin–luciferase bioluminescence isfound in hundreds of taxa across many phyla, there arefive basic luciferin–luciferase system (12):

� Bacterial luciferin is a reduced riboflavin phos-phate (FMNH2) that is oxidized by a luciferase in

association with a long-chain aldehyde and anoxygen molecule. It is found in luminescentbacteria, certain fish, pyrosomes, and in somesquids (e.g. Euprymna).

� Dinoflagellate luciferin resembles, and may bederived from, the porphyrin of chlorophyll. In thedinoflagellate Gonycaulax, this luciferin is con-formationally shielded from luciferase at the basicpH of 8 but becomes free and accessible tooxidation near the more acidic pH of 6. Amodification of this luciferin occurs in a herbivor-ous euphausiid shrimp, where it is apparentlyacquired by ingestion.

� Another luciferin, from the marine ostracod Var-gula, is called vargulin. It also seems to be acquiredby ingestion. It is also found in some fish species.

� Coelenterazine is the most widely known luciferin.It occurs in cnidarians, copepods, chaetognaths,ctenophores, decapod shrimps, mysid shrimps,radiolarians, and some fish taxa. Coelenterateluciferase activity is controlled by the concentra-tion of Ca2� and shares homology with thecalcium-binding protein calmodulin (5).

� Firefly luciferin (a benzothiazole) is found exclu-sively in fireflies (Photinus or Luciola). It has theunique property of requiring ATP as a co-factor toconvert it to an active luciferin (5). It was realizedearly that firefly luciferin–luciferase could be usedto determine the presence of ATP (23). This hasbecome a standard ATP assay. For one example,since nickel alloys have been shown to have anadverse effect on respiratory metabolism in eu-karyotic cell lines, the firefly luciferin–luciferasesystem has been used to document depressed levelsof ATP in cells exposed to the alloys (24).

The mechanisms of bioluminescence utilized byamphipods, bivalves, earthworms, fresh-water limpets,fungus gnats, larvaceans, nemertean worms, polychaeteworms and tunicates are currently unknown. Luciferin–luciferase bioluminescence systems are multiformphenomena and polyphyletic in origin.

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Science has entered into the field of bioluminescentvisualization in far more recent times. In the last fewdecades, many luciferase genes have been isolated,sequenced at least in part, and used to build DNAvectors. In Table 1 we summarize the DNA fragmentsand cDNAs that encode the different luciferases sig-nificant in scientific imaging.

The luciferases most commonly used in experimentalbioluminescent imaging applications include the bacterial

Copyright 2002 John Wiley & Sons, Ltd. Luminescence 2002;17:43–74

44 REVIEW L. F. Greer and A. A. Szalay

Tab

le1.

Asu

mm

ary

ofkn

own

luci

fera

sege

nes,

cDN

As,

and

prot

eins

.Am

ong

thes

ear

eth

epr

okar

yoti

clu

cife

rase

s(L

ux),

euka

ryot

iclu

cife

rase

s(L

uc,R

ucan

dth

eir

regu

lato

rypr

otei

ns)

both

ofw

hich

are

com

mon

lyus

edin

imag

ing

oflu

cife

rase

expr

essi

onin

livin

gce

lls,t

issu

es,a

ndor

gani

sms

Tax

aG

ene

–cD

NA

(siz

ein

bp)

Prot

ein

prod

uct

(siz

ein

num

ber

ofam

ino

acid

s)G

enB

ank

acce

ssio

nno

.(D

NA

and

amin

oac

id)

Ref

eren

ce

Vib

rio

harv

eyi

luxA

,106

7bp

�su

buni

t,35

5aa

M10

961

AA

A88

685

32

Vib

rio

harv

eyi

luxB

,947

bp�

subu

nit,

324

aaM

1096

1.1

AA

A88

686

223

Vib

rio

harv

eyi

luxE

,113

6bp

acyl

-pro

tein

synt

heta

se,

378

aaM

2881

5.1

AA

A27

531

223

Vib

rio

fisch

eri

luxA

,106

4bp

alka

nal

mon

o-ox

ygen

ase�-

chai

n,35

4aa

X06

758

CA

A29

931

224

Vib

rio

fisch

eri

luxB

,980

bpal

kana

lm

ono-

oxyg

enas

e�-

chai

n,32

6aa

X06

797

CA

A29

932

224

Vib

rio

fisch

eri

Lux

RIC

DA

BE

Gop

eron

�lu

xR,7

52bp

�lu

xI,5

81bp

�lu

xC,1

439

bp�

luxD

,923

bp�

luxA

,107

7bp

�lu

xB,9

93bp

�lu

xE,1

136

bp

�lu

xG,7

22bp

�re

gula

tory

prot

ein

Lux

R,2

50aa

�au

toin

duce

rsy

nthe

sis

prot

ein

Lux

I,19

3aa

�ac

yl-C

oAre

duct

ase

Lux

C,4

79aa

�ac

yltr

ansf

eras

eL

uxD

,307

aa�

alka

nal

mon

o-ox

ygen

ase�-

chai

nL

uxA

,354

aa�

alka

nal

mon

o-ox

ygen

ase�-

chai

nL

uxB

,326

aa�

long

chai

nfa

ttyac

idlu

cife

rin

com

pone

ntlig

ase

Lux

E,3

78aa

�pr

obab

lefla

vin

redu

ctas

eL

uxG

,236

aa

AF1

7010

4�

AA

D48

473

�A

AD

4847

4�

AF1

7010

4�

AA

D48

476

�A

AD

4847

7�

AA

D48

478

�A

AD

4847

9

�A

AD

4848

0

Kni

ght

T,P

apad

akis

N.

Vib

rio

fisch

eri

lux

oper

onSa

lI

dige

st(u

npub

lishe

d—di

rect

subm

issi

onto

Gen

Ban

k,19

99)

Pho

torh

abdu

slu

min

esce

ns=

Xen

orha

bdus

;si

nce

1999

redu

ced

tosy

nony

my

(225

)

Lux

CD

AB

Eop

eron

�lu

xC,1

442

bp�

luxD

,923

bp�

luxA

,108

8bp

�lu

xB,9

74bp

�lu

xE,3

47bp

�fa

ttyac

idre

duct

ase

Lux

C,4

80aa

�ac

yltr

ansf

eras

eL

uxD

,307

aa�

alka

nal

mon

o-ox

ygen

ase�-

chai

nL

uxA

,362

aa�

alka

nal

mon

o-ox

ygen

ase�-

chai

nL

uxB

,324

aa�

acyl

-pro

tein

synt

heta

seL

uxE

,116

aa

M62

917

�A

AA

6356

3�

AA

A63

564

�A

AA

6356

5�

AA

A63

566

�A

AA

6356

7

34


Imaging of light emission from luciferase expression REVIEW 45

Tab

le1.

Con

tinu

ed

Tax

aG

ene

–cD

NA

(siz

ein

bp)

Prot

ein

prod

uct

(siz

ein

num

ber

ofam

ino

acid

s)G

enB

ank

acce

ssio

nno

.(D

NA

and

amin

oac

id)

Ref

eren

ce

Pho

tinu

spy

rali

slu

c,23

87bp

Luc

ifer

ase,

550

aaM

1507

7A

AA

2979

522

6

Luc

iola

cruc

iata

luc,

1985

bpL

ucif

eras

e,54

8aa

M26

194

AA

A29

135

227

Var

gula

hilg

endo

rfii

(sea

firefl

y)�

vuc,

1834

bp�

vuc

mR

NA

,181

8bp

Var

gulin

,61

1aa

Var

gulin

,55

5aa

E02

749

M25

666

AA

A30

332

228 39

Aeq

uore

avi

ctor

iaae

q1,6

72(5

90)

bpae

qpre

c,56

8bp

aeq2

,531

bpae

q3,5

31bp

aq44

0,92

5bp

aqua

,587

bp

Aeq

uori

n1;

calc

ium

-bin

ding

prot

ein,

196

aaA

equo

rin

prec

urso

r,�1

89aa

Aeq

uori

n2,

177

aaA

equo

rin

3,17

7aa

Aeq

uori

n,ca

lciu

mbi

ndin

g-

196

aaL

umin

esce

ntpr

otei

nA

qual

ine,

�196

M16

103

AA

A27

719

M11

394

AA

A27

716

M16

104

AA

A27

717

M16

105

AA

A27

718

L29

571

AA

A27

720

E02

319

229

230

231

Opl

opho

rus

grac

ilor

ostr

islu

c,59

0bp

luc,

1079

bpO

plop

hori

n,ox

ygen

ase,

imid

azop

yraz

inon

elu

cife

rase

,19

6aa

Opl

opho

rin,

oxyg

enas

e,im

idaz

opyr

azin

one

luci

fera

se�3

59aa

AB

0302

46B

AB

1377

6A

B03

0245

BA

B13

775

52

Ren

illa

mue

ller

iru

c,12

08bp

Ruc

,311

aaA

Y01

5988

AA

G54

094

Szen

t-G

yorg

yiC

S,B

ryan

BJ.

cDN

Aen

codi

ngR

enil

lam

uell

eri

luci

fera

se(u

npub

lishe

dm

anus

crip

t).

Ren

illa

reni

form

isru

c,11

96bp

Ruc

,oxy

gena

se,3

11aa

M63

501

AA

A29

804

59



luciferases (lux) from the marine genera Photobacteriumand Vibrio, firefly luciferase (Photinus), aequorin (lucifer-ase from the jellyfish Aequorea), vargulin (luciferase fromthe marine ostracod Vargula), oplophoran luciferase(deep-sea shrimp Oplophorus) and Renilla luciferase(anthozoan sea pansy, Renilla reniformis).

� Bacterial luciferase. Bacterial luciferase proteinswere purified and isolated from the light organs ofmid-depth fishes in the ocean (25, 26). It wasknown early that the catalytic site was on the �subunit (27). Belas et al. (1982) isolated andexpressed luciferase genes from Vibrio harveyi inE. coli (28). Olsson et al. (1988) characterized theactivity of the LuxA subunit of Vibrio harveyiluciferase by visualizing various luxA and luxBtruncations, as well as a luxAB fusion expressed inE. coli (29). Olsson et al. (1989) furthermore mademonomeric luxAB fusions and expressed them alsoin E. coli (30). The Vibrio harveyi luxA and luxBcDNAs were cloned and sequenced in the mid-1980s (31–33). The luxCDABE operon from theterrestrial bacterium Photorhabdus luminescenswas cloned and sequenced and its product, lucifer-ase, was characterized and published in 1991 (34).

� Firefly luciferase. The active sites and properties offirefly luciferase (Photinus) began to be character-ized about 35 years ago (35–37). Firefly luciferasewas purified and characterized in 1978 (19). ThecDNA encoding the luciferase (Luc) from thefirefly Photinus pyralis was cloned and expressedin E. coli by De Wet et al. (1985) (38).

� Vargulin. A cDNA for the luciferase gene from themarine ostracod Vargula hilgendorfii was cloned,sequenced and expressed in mammalian cells byThompson et al. (1989) (39). They also discoveredthat Vargula luciferase expression requires only itssubstrate and molecular oxygen (but no co-fac-tors), thus making it potentially more useful formammalian expression systems (40). The activityof Vargula luciferase is not dependent on apyrazine structure, as has been demonstrated bycross-reaction experiments with the Oplophorusluciferin (41).

� Aequorin. The aequorin protein was first extractedfrom the hydromedusa Aequorea, purified andcharacterized in part by Shimomura et al. (1962)(42). In 1975, Shimomura and Johnson describedwhat was known about the mechanisms of variouscoelenterate luciferins, including aequorin (22).Ward and Cormier (1975) reported the isolation ofvarious Renilla-type luciferins, including aequorin(43). A few years later, it was discovered thatRenilla luciferin analogues were catalysed byluciferase to excited energy states to transferenergy to a green fluorescence protein or GFP

(44). Ward and Cormier (1979) characterized theRenilla green fluorescence protein (RGFP) andshowed that a natural energy transfer was occur-ring from the isolated Renilla luciferase (Ruc)bioluminescence to RGFP (45). In 1985, the cDNAfor aequorin was cloned, sequenced and expressedin heterologous systems (46, 47). The aequoringene from the jellyfish Aequorea victoria wascloned in 1990 (48). It is now known that manycnidarians have GFPs that serve as energy-transferacceptors fluorescing in response to excited oxy-luciferin–luciferase complexes or to a Ca2�-acti-vated phosphoprotein. The cDNA encoding theGFP of Aequorea victoria has also been cloned andsequenced (49).

� Oplophorus luciferase. The general reaction mech-anisms and properties of the luciferin–luciferasesystem of the deep-sea shrimp Oplophorus graci-lorostris were reported by Shimomura et al. (1978)(50). An empirical formula and structure has beensuggested for Oplophorus luciferin using spectro-scopy and cross-reaction with the luciferase of theostracod Vargula hilgendorfii (40). By 1997,Oplophorus luciferase was known to have a moreintense light emission than either Renilla luciferaseor the recombinant aequorin. However the Oplo-phorus luciferase cDNA, not yet cloned, could notbe used as a reporter gene (51). Recently, Inouye etal. (2000) succeeded in cloning the Oplophorusluciferase cDNA (52).

� Renilla luciferase. In 1966, Hori and Cormierdescribed some of the properties and a hypotheticalpartial structure for the Renilla reniformis lucifer-ase protein (Ruc) (53). Kreis and Cormier (1967)showed that light could inhibit the activity of Ruc(54). The isolation of Ruc was first done andfurther properties elucidated by Karkhanis andCormier (1971) (55). DeLuca et al. (1971) demon-strated that the Renilla bioluminescent systeminvolves the oxidative production of CO2 (56). Itwas further shown that Ca2� triggered a luciferinbinding protein, thus inducing the Ruc system (57).Ruc was first purified and characterized byMatthews et al. (1977) (58). The cDNA of rucwas isolated and later expressed in E. coli byLorenz et al. (1991) (59). The ruc cDNA was alsoexpressed in a number of transgenic plant tissues(60). In 1996, Lorenz et al. expressed Ruc insimian COS-7 cells and in murine C5 cells (61).

In retrospect, it might be noted that since theirdiscovery, Luc (Photinus), aequorin (Aequorea) andGFP have been used in a multitude of successfulexperiments. In combination the three have even beenuseful in assaying or imaging the spatial–temporalconcentrations of Ca2� (62). Combinations of multiple



Tab

le2.

Asu

mm

ary

ofse

lect

edlu

cife

rase

cons

truc

tsan

dve

ctor

sus

eful

for

imag

ing.

(Con

stru

ct/v

ecto

rno

men

clat

ure

not

stan

dard

ized

inth

elit

erat

ure)

.

Con

stru

ct/v

ecto

rL

ucif

eras

ege

nes

orcD

NA

sPr

omot

ers/

enha

ncer

sO

rgan

ism

/cel

lsSu

bstr

ate

requ

irem

ent

Imag

ing

appl

icat

ion

and

refe

renc

e

pB10

1;pB

102;

pB10

5;pB

110;

pB12

3;pB

128;

pGM

C12

luxA

,lux

B(V

ibri

oha

rvey

i)Ph

age�

prom

oter

sP L

and

P RE

sche

rich

iaco

liD

ecan

alE

xpre

ssio

nof

luxA

,lux

Bin

E.

coli

(28)

—fir

sttr

ansg

enic

expr

essi

onof

lux

Tra

nspo

son

min

i-M

ulux

luxE

,B,A

,D,C

(Vib

rio

para

haem

o-ly

ticu

s)la

cpr

omot

erE

.co

liN

one

Vis

ualiz

atio

nof

Vib

rio

lux

gene

sin

E.

coli

(26)

pFIT

001;

pPA

LE

001

luxA

,lux

B(V

.ha

rvey

i)A

nti-

Tet

(P1)

,nif

D,n

ifH

prom

oter

sE

.co

li;

Bra

dyrh

izob

ium

japo

nicu

min

Gly

cine

max

Dec

anal

Vis

ualiz

atio

nof

N-fi

xatio

nin

soyb

ean

nodu

les

via

Bra

dyrh

izob

ium

japo

nicu

m(2

10)

Agr

obac

teri

umbi

nary

vect

orlu

xA&

B(V

.ha

rvey

i)T

Lpr

omot

erD

aucu

sca

rota

;N

icot

iana

taba

cum

Dec

anal

Vis

ualiz

atio

nof

tissu

e-sp

ecifi

cch

imae

ric

lux

expr

essi

on(1

69)

pDO

432;

pDO

435;

pDO

446;

pDO

445

luc

(Pho

tinu

s)C

aMV

35S

RN

Apr

omot

erN

icot

iana

taba

cum

Pho

tinu

slu

cife

rin

and

AT

Pin

solu

tion

(top

ical

lyde

liver

ed)

Vis

ualiz

atio

nof

luc

expr

essi

onin

toba

cco

plan

ts(1

68)

pPC

V70

1lux

A&

Blu

xA;

luxB

(V.

harv

eyi)

TR-D

NA

P1an

dT

R-D

NA

P2

mas

prom

oter

sN

icot

iana

taba

cum

;D

aucu

sca

rota

via

Agr

obac

teri

umtu

mef

acie

ns-

med

iate

dge

nede

liver

y

Dec

anal

(inj

ecte

d)D

ecan

al�

FMN

H2

Ass

embl

yan

dex

pres

sion

offu

nctio

nal

luxA

and

Bge

nes

inpl

ants

(170

)

pRSV

Llu

cbu

tim

aged

byim

mun

o-flu

resc

ence

Prom

oter

inR

ous

Sarc

oma

Vir

uslo

ngte

rmin

alre

peat

(RSV

LT

R)

Mon

key

kidn

eyce

lls(C

V-1

)N

AL

ucif

eras

epe

roxi

som

allo

caliz

atio

nvi

sual

-iz

edby

imm

unofl

uore

scen

ce(1

09)

pMR

P1;

pMR

D2

luxA

Bfu

sion

P1

prom

oter

Soyb

ean

(Gly

cine

max

)D

ecan

alSu

cces

sful

imag

ing

ofL

uxA

Bfu

sion

expr

essi

onin

soyb

ean

root

nodu

les

usin

gph

otog

raph

icfil

man

dlo

w-l

ight

inte

nsifi

edvi

deo

mic

rosc

opy

(211

)pS

CL

UC

→,p

SCL

UC��

,rV

V-l

uclu

c7.

5kD

avi

ral

prom

oter

,Vac

cini

atk

gene

frag

men

tsB

SC-4

0ce

lls(A

fric

angr

een

mon

key

kidn

eyce

lls)

Fire

flylu

cife

rin

Film

imag

ing

ofre

com

bina

ntva

ccin

iain

fect

ion

ofm

onke

yce

lls(2

12)

pLX

vect

orse

ries

luxA

;lu

xB;

luxA

B(l

uxF

)T

7pr

omot

erB

E21

(DE

3)ce

llsD

ecan

alIm

agin

gof

vari

ous

lux

trun

cati

ons

and

Lux

Ffu

sion

ince

lls(2

9)pR

S110

5lu

xA,l

uxB

(V.

harv

eyi)

End

oH,b

ldA

(leu

tRN

A),

Whi

G(u

ncha

ract

eriz

ed),

SapA

(all

Stre

ptom

yces

prom

oter

s)

Stre

ptom

yces

coel

icol

orD

ecan

alV

isua

lizin

glu

xA&

Bex

pres

sion

inSt

rept

omyc

es(9

1)

pLX

,pIC

LX

,pC

V70

2an

dp3

5Slu

xve

ctor

seri

eslu

xAB

;lu

xBA

fusi

ons

T7

gene

10an

dC

aMV

35S

prom

oter

E.

coli

;to

bacc

oca

lliD

ecan

alSh

owed

that

aL

uxA

Bfu

sion

ism

uch

mor

eac

tive

than

Lux

BA

(30)

pLX

vect

orse

ries

luxA

BT

7ge

ne10

prom

oter

E.

coli

Dec

anal

Bac

teri

allu

cife

rase

��fu

sion

func

tiona

las

am

onom

er(6

5)pM

W41

luc

CM

Vin

term

edia

te–e

arly

enha

ncer

/pr

omot

er;

tran

s-ac

tiva

ted

byH

IV-1

Tat

prot

ein

CO

S-7

cells

Luc

ifer

in(fi

refly

)an

dde

riva

tive

s:et

hoxy

viny

les

ter,

2-hy

drox

yeth

yles

ter,

3-hy

drox

y-n-

prop

yles

ter,

ethy

les

ter

Vis

ualiz

atio

nof

Luc

expr

essi

onin

indi

vidu

alm

amm

alia

nce

lls(1

12)

pSV

2-vl

luc

(Var

gula

luci

fera

se)

SV40

prom

oter

CH

Oce

llsL

ucif

erin

Vis

ualiz

atio

nof

luc

secr

etio

nin

CH

Oce

llsre

altim

eus

ing

anim

age-

inte

nsif

ying

tech

niqu

e(1

14)

pPC

VG

Lux

Aan

dB

luxA

and

BPr

omot

erle

sslu

xApC

aMV

35S

RN

A(l

uxB

),i.e

.pr

omot

erse

arch

vect

oras

say

Tob

acco

Dec

anal

Imag

ing

ofco

nstit

utiv

ean

dor

gan-

spec

ific

Lux

expr

essi

onin

tran

sgen

icto

bacc

o(1

72)

pAM

1224

;ps

bAI:

:luxA

Bco

nstr

uct

luxA

B(V

.ha

rvey

i)lu

xAB

prom

oter

less

,(to

bein

sert

eddo

wns

trea

mof

cyan

obac

teri

alpr

o-m

oter

);ps

bAI,

psbA

III

and

purF

prom

oter

s

Syne

choc

occu

ssp

.,ve

ctor

edby

conj

ugat

ion

with

E.

coli

Dec

anal

Coo

led-

CC

Dde

tect

ion

and

docu

men

tatio

nof

circ

adia

nrh

ythm

sin

cyan

obac

teri

a(9

8–10

0)

pPC

VG

luxA

and

Blu

xAan

dB

(V.

harv

eyi)

T-D

NA

prom

oter

sear

chve

ctor

;lu

xApr

omot

erle

ss,b

uten

hanc

edby

CaM

V35

Spr

omot

erin

fron

tof

luxB

Agr

obac

teri

umtu

mef

acie

nsT

-DN

A-

med

iate

dtr

ansf

erof

lux

into

tran

s-ge

nic

Nic

otia

nata

bacu

mcv

.SR

1(e

spec

ially

root

stem

,lea

fan

dflo

wer

tissu

es)

Dec

anal

Imag

ing

used

tolo

cate

deve

lopm

enta

llyre

gula

ted

prom

oter

sin

toba

cco

(96)

pWH

1520

–xyl

A–l

uxF

(Bac

illu

sG

ram

posi

tive)

expr

essi

onve

ctor

luxA

B(l

uxF

;V

.ha

rvey

i)X

ylos

e-in

duci

ble

prom

oter

–lux

Ffu

sion

Xyl

A–l

uxF

-tra

nsfo

rmed

B.

thur

ingi

en-

sis

inje

cted

into

Man

duca

sext

aar

vae

(6th

inst

arof

the

toba

cco

horn

wor

m)

Dec

anal

Use

ofph

oton

-cou

ntin

gto

visu

aliz

e(1

3):

�In

duci

ble

luxF

expr

essi

onfr

omB

.th

urin

-gi

ensi

san

dB

.m

egat

eriu

min

fect

ion

inle

-pi

dopt

eran

inse

ctla

rvae

and

envi

ronm

ent

(and

oflu

xFin

E.

coli

)

��



Tab

le2.

Con

tinu

ed

Con

stru

ct/v

ecto

rL

ucif

eras

ege

nes

orcD

NA

sPr

omot

ers/

enha

ncer

sO

rgan

ism

/cel

lsSu

bstr

ate

requ

irem

ent

Imag

ing

appl

icat

ion

and

refe

renc

e

Rec

ombi

nant

AcN

PV–l

uc(A

uto-

grap

hica

cali

forn

ica

nucl

ear

poly

-he

dros

isvi

rus

with

Pluc

)

luxF

(V.

harv

eyi)

;Pl

ucA

rabi

dops

isph

enyl

alan

ine

amm

onia

lyas

e(P

AL

)pr

omot

er–l

uxF

gene

fusi

on;

AcN

PVpo

lyhe

dron

late

prom

oter

(pN

I101

)–lu

c

Rec

ombi

nant

AcN

PV–l

uc-i

nfec

ted

Tri

chop

lusi

ani

(386

)ce

llsD

ecan

al;

luci

feri

n(fi

refly

)U

seof

phot

on-c

ount

ing

imag

ing

tovi

sual

ize

(93)

:�

AcN

PV–l

ucex

pres

sing

plaq

ues

inT

.ni

cells

pPC

VG

luxA

and

Blu

xAan

dlu

xB(V

.ha

rvey

i)lu

xApr

omot

erle

ss,b

uten

hanc

edby

CaM

V35

Spr

omot

erin

fron

tof

luxB

(i.e

.pro

mot

erse

arch

vect

or)

Agr

obac

teri

umtu

mef

acie

nsT

-DN

A-

med

iate

dtr

ansf

erof

lux

into

tran

s-ge

nic

Nic

otia

nata

bacu

mcv

.SR

1fo

rex

pres

sion

inre

spon

seto

vir

and

avir

infe

ctio

nby

Pse

udom

onas

syri

ngae

Dec

anal

Use

ofph

oton

-cou

ntin

gim

agin

gto

visu

aliz

e(9

3):

�Pr

omot

erse

arch

expr

essi

onof

Lux

Aan

dL

uxB

inN

.tab

acum

pPC

VG

luxA

and

Blu

xAan

dlu

xB(V

.ha

rvey

i)lu

xApr

omot

erle

ss,b

uten

hanc

edby

CaM

V35

Spr

omot

erin

fron

tof

luxB

(i.e

.pro

mot

erse

arch

vect

or)

Agr

obac

teri

umtu

mef

acie

nsT

-DN

A-

med

iate

dtr

ansf

erof

lux

into

tran

s-ge

nic

Ara

bido

psis

thal

iana

(RL

D)

for

expr

essi

onin

resp

onse

tovi

ran

dav

irin

fect

ion

byP

seud

omon

assy

ring

ae

Dec

anal

Use

ofph

oton

-cou

ntin

gim

agin

gto

visu

aliz

e(9

3):

�Pr

omot

erse

arch

expr

essi

onof

Lux

Aan

dB

inN

.tab

acum

pLT

u–lu

xFlu

xF(V

.ha

rvey

i)C

rate

rost

igm

apl

anta

gine

umdr

ough

tan

dA

BA

-reg

ulat

edpr

omot

er–l

uxF

Agr

obac

teri

umtu

mef

acie

nsT

-DN

A-

vect

ored

lux

intr

ansg

enic

Nic

otia

nata

bacu

mcv

.SR

1

Dec

anal

Use

ofph

oton

-cou

ntin

gim

agin

gto

visu

aliz

e(9

3):

�St

ress

and

AB

A-i

nduc

edL

uxF

expr

essi

onin

N.t

abac

um

pPC

V70

1–lu

xAB

luxA

B(V

.ha

rvey

i)A

grob

acte

rium

tum

efac

iens

auxi

n-re

gula

ted

man

nopi

nesy

ntha

sebi

-di

rect

iona

lpr

omot

ers

(mas

P1

and

P2)

Tra

nsge

nic

Nic

otia

nata

bacu

mcv

.SR

1D

ecan

alPh

oton

-cou

ntin

gvi

sual

izat

ion

ofau

xin-

indu

ced

activ

atio

nof

mas

prom

oter

sof

luxA

Bin

tran

sgen

icto

bacc

o(9

4)

pWH

1520

–xyl

A–l

uxF,

calle

dpW

H15

20SF

;pL

X70

3-fa

b9lu

xAB

(V.

harv

eyi)

B.m

egat

eriu

mxy

lose

isom

eras

ege

ne(x

ylA

)pr

omot

erlu

x-tr

ansf

orm

edB

acil

lus

thur

ingi

ensi

san

dB

.meg

ater

ium

Dec

anal

Mea

sure

dxy

lose

indu

ctio

nof

xylA

–lux

AB

intr

ansf

orm

edB

acil

lus

thur

ingi

ensi

san

dB

.m

egat

eriu

m(9

5)B

acte

rial

prok

aryo

ticpr

omot

erse

arch

vect

or:

35bp

luxF

–Ori

T–O

riV

–N

PT2–

tran

spos

ase

from

TN

5w

ithka

nr —35

bp

luxF

(V.

harv

eyi)

Ran

dom

prok

aryo

ticpr

omot

erre

gion

sE

.co

liD

ecan

alIm

age-

inte

nsifi

edlo

w-l

ight

sing

le-p

hoto

nvi

deo

imag

ing

(and

X-r

ayau

tora

diog

ra-

phy)

oftr

ansg

enic

orga

nism

sus

ing

bac-

teri

allu

cife

rase

s(9

7):

E.

coli

Vec

tor

cont

aini

ngG

al4

prom

oter

–lux

Flu

xF(V

.ha

rvey

i)G

al4

prom

oter

Sacc

haro

myc

esce

revi

siae

Dec

anal

Imag

e-in

tens

ified

low

-lig

htsi

ngle

-pho

ton

vide

oim

agin

g(a

ndX

-ray

auto

radi

ogra

-ph

y)of

tran

sgen

icor

gani

sms

usin

gba

c-te

rial

luci

fera

ses

(97)

:ye

ast

Tra

nsge

nic

rhiz

obia

(bac

teri

ods)

cont

aini

ngrh

izob

ium

nitr

ogen

ase

P1pr

omot

er–l

uxF

linka

ge

luxF

(V.

harv

eyi)

Con

stitu

tive

P1

prom

oter

Gly

cine

max

(soy

bean

plan

t)no

dule

sin

fect

edw

ithtr

ansg

enic

Bra

dyrh

izob

ium

Dec

anal

Imag

e-in

tens

ified

low

-lig

htsi

ngle

-pho

ton

vide

oim

agin

g(a

ndX

-ray

auto

radi

ogra

-ph

y)of

tran

sgen

icor

gani

sms

usin

gba

c-te

rial

luci

fera

ses

(97)

:tr

ansg

enic

rhiz

o-bi

umin

soyb

ean

nodu

les

Plan

tex

pres

sion

vect

or:

LB

–p–N

PTII

(neo

myc

inph

osph

otra

nsfe

rase

II)–

pA–p

–lux

F–pA

–RB

pWH

1520

SF–

xylR

–lux

AB

luxF

(V.

harv

eyi)

Aux

in-a

ctiv

ated

man

nopi

nebi

dire

c-tio

nal

1�,2

�(m

as)

prom

oter

s;no

pa-

line

synt

hase

prom

oter

Agr

obac

teri

um-d

eliv

ered

expr

essi

onve

ctor

into

expl

ants

ofN

icot

iana

taba

cum

Dec

anal

Imag

e-in

tens

ified

low

-lig

htsi

ngle

-pho

ton

vide

oim

agin

g(a

ndX

-ray

auto

radi

ogra

-ph

y)of

tran

sgen

icor

gani

sms

usin

gba

c-te

rial

luci

fera

ses

(97)

:tr

ansg

enic

toba

cco

pCV

701–

luxF

luxF

(V.

harv

eyi)

Aux

in-a

ctiv

ated

mas

prom

oter

Agr

obac

teri

um-d

eliv

ered

expr

essi

onve

ctor

into

Lyc

oper

sico

nes

cule

ntum

(tra

nsge

nic

tom

ato

tissu

es)

Dec

anal

Imag

e-in

tens

ified

low

-lig

htsi

ngle

-pho

ton

vide

oim

agin

g(a

ndX

-ray

auto

radi

ogra

-ph

y)of

tran

sgen

icor

gani

sms

usin

gba

c-te

rial

luci

fera

ses

(97)

:tr

ansg

enic

tom

atoe

sPl

ant

prom

oter

sear

chve

ctor

B:

Agr

o-ba

cter

ium

LB

–lux

A–N

OS p

A–a

P CaM

V35S–l

uxB

–NO

S pA

–PN

OS–

HPT

–g7 p

A–A

pr –RB

luxA

B(V

.ha

rvey

i)A

uxin

-act

ivat

edm

as1�

,2�p

rom

oter

sA

grob

acte

rium

-del

iver

edex

pres

sion

vect

orin

toSo

lanu

mtu

bero

sum

(pot

ato)

Dec

anal

Imag

e-in

tens

ified

low

-lig

htsi

ngle

-pho

ton

vide

oim

agin

g(a

ndX

-ray

auto

radi

ogra

-ph

y)of

tran

sgen

icor

gani

sms

usin

gba

c-te

rial

luci

fera

ses

(97)

:tr

ansg

enic

tom

atoe

s

��



Tab

le2.

Con

tinu

ed

Con

stru

ct/v

ecto

rL

ucif

eras

ege

nes

orcD

NA

sPr

omot

ers/

enha

ncer

sO

rgan

ism

/cel

lsSu

bstr

ate

requ

irem

ent

Imag

ing

appl

icat

ion

and

refe

renc

e

Plan

tpr

omot

erse

arch

vect

orA

:A

gro-

bact

eriu

mT

–DN

Ale

ftbo

rder

,LB–

luxF

–OS p

A–P

NO

S–H

PT–N

OS p

A–

Apr –R

B

luxF

(V.

harv

eyi)

Aux

in-a

ctiv

ated

mas

1�,2

�pro

mot

ers

Agr

obac

teri

um-d

eliv

ered

expr

essi

onve

ctor

into

Sola

num

tube

rosu

m(p

otat

o)

Dec

anal

Imag

e-in

tens

ified

low

-lig

htsi

ngle

-pho

ton

vide

oim

agin

g(a

ndX

-ray

auto

radi

ogra

-ph

y)of

tran

sgen

icor

gani

sms

usin

gba

c-te

rial

luci

fera

ses

(97)

:tr

ansg

enic

tom

atoe

sPl

ant

prom

oter

sear

chve

ctor

B:

Agr

o-ba

cter

ium

LB

–lux

A–N

OS p

A–a

P CaM

V35S–l

uxB

–NO

S pA

–PN

OS–

HPT

–g7 p

A–A

pr –RB

Mas

prom

oter

–lux

AB

(V.h

arve

yi)

fusi

onA

uxin

-act

ivat

edm

as1�

,2�p

rom

oter

;C

aMV

35S

prom

oter

Agr

obac

teri

um–d

eliv

ered

expr

essi

onve

ctor

into

Lyc

oper

sico

nes

cule

ntum

(tra

nsge

nic

tom

ato

frui

t)

Dec

anal

Imag

e-in

tens

ified

low

–lig

htsi

ngle

-pho

ton

vide

oim

agin

g(a

ndX

-ray

auto

radi

ogra

-ph

y)of

tran

sgen

icor

gani

sms

usin

gba

c-te

rial

luci

fera

ses

(97)

:tr

ansg

enic

tom

ato

frui

tPl

ant

prom

oter

sear

chve

ctor

A:

Agr

o-ba

cter

ium

T–D

NA

left

bord

er,L

B–

luxF

–OS p

A–P

NO

S–H

PT–N

OS p

A–

Apr –R

B

Mas

prom

oter

–lux

F(V

.har

veyi

)fu

sion

Aux

in-a

ctiv

ated

mas

1�,2

�pro

mot

erA

grob

acte

rium

-del

iver

edex

pres

sion

vect

orin

toN

icot

iana

taba

cum

Dec

anal

Imag

e-in

tens

ified

low

-lig

htsi

ngle

-pho

ton

vide

oim

agin

g(a

ndX

-ray

auto

radi

ogra

-ph

y)of

tran

sgen

icor

gani

sms

usin

gba

c-te

rial

luci

fera

ses

(97)

:tr

ansg

enic

toba

cco

stem

sPl

ant

prom

oter

sear

chve

ctor

A:

Agr

o-ba

cter

ium

T–D

NA

left

bord

er,L

B–

luxF

–OS p

A–P

NO

S–H

PT–N

OS p

A–

Apr –R

B

Mas

prom

oter

–lux

F(V

.har

veyi

)fu

sion

Aux

in-a

ctiv

ated

mas

1�,2

�pro

mot

erA

grob

acte

rium

-del

iver

edex

pres

sion

vect

orin

toD

atur

ast

ram

oniu

mD

ecan

alIm

age-

inte

nsifi

edlo

w-l

ight

sing

le-p

hoto

nvi

deo

imag

ing

(and

X-r

ayau

tora

diog

ra-

phy)

oftr

ansg

enic

orga

nism

sus

ing

bac-

teri

allu

cife

rase

s(9

7):

tran

sgen

icD

atur

aB

acte

rial

prok

aryo

ticpr

omot

erse

arch

vect

or:

35bp

luxF

–Ori

T–O

riV

–N

PT2–

tran

spos

ase

from

Tn5

with

kanr —

35bp

luxF

(V.

harv

eyi)

Ran

dom

prok

aryo

ticpr

omot

erre

gion

sP

seud

omon

asso

lana

caer

umvi

aco

n-ju

gatio

nw

ithan

E.

coli

bear

ing

Tn5

Dec

anal

Imag

e-in

tens

ified

low

-lig

htsi

ngle

-pho

ton

vide

oim

agin

g(a

ndX

-ray

auto

radi

ogra

-ph

y)of

tran

sgen

icor

gani

sms

usin

gba

c-te

rial

luci

fera

ses

(97)

:tr

ansg

enic

Pse

udo-

mon

as—

visu

aliz

ing

Plan

tpr

omot

erse

arch

vect

orB

:A

gro-

bact

eriu

mL

B–l

uxA

–NO

S pA

–aP C

aMV

35S–l

uxB

–NO

S pA

–PN

OS–

HPT

–g7 p

A–A

pr –RB

Het

erod

imer

iclu

xAB

(V.

harv

eyi)

Aux

in-a

ctiv

ated

mas

1�,2

�pro

mot

eror

phen

ylal

anin

eam

mon

ialy

ase

(PA

L)

prom

oter

Tra

nsge

nic

Sola

num

tube

rosu

m(p

otat

o)in

fect

edw

ithE

rwin

iahe

rbic

ola

(avi

rule

nt),

E.

caro

tovo

ra(v

irul

ent)

,Pse

udom

onas

syri

ngae

stra

into

mat

o(a

viru

lent

)

Dec

anal

Imag

e-in

tens

ified

low

-lig

htsi

ngle

-pho

ton

vide

oim

agin

g(a

ndX

-ray

auto

radi

ogra

-ph

y)of

tran

sgen

icor

gani

sms

usin

gba

c-te

rial

luci

fera

ses

(97)

:tr

ansg

enic

Pse

udo-

mon

as—

mon

itori

ngth

evi

rule

nce

ofpa

thog

enst

rain

spP

CV

701–

luxA

Blu

xAB

(V.

harv

eyi)

Aux

in-a

ctiv

ated

mas

1�,2

�pro

mot

ers

Tra

nsge

nic

Sola

num

tube

rosu

m(p

otat

o)in

fect

edby

Pse

udom

onas

syri

ngae

,Erw

inia

caro

tovo

raca

ro-

tovo

ra,E

rwin

iaca

roto

vora

atro

sep-

tica

,E

rwin

iahe

rbic

ola

Dec

anal

Imag

e-in

tens

ified

low

-lig

htsi

ngle

-pho

ton

vide

oim

agin

g(a

ndX

-ray

auto

radi

ogra

-ph

y)of

tran

sgen

icor

gani

sms

usin

gba

c-te

rial

luci

fera

ses

(97)

:tr

ansg

enic

pota

to

pWH

1520

SF–x

ylR

–xyl

A–l

uxA

Blu

xAB

(V.

harv

eyi)

–xyl

Apr

omot

erfu

sion

xylA

prom

oter

Bac

illu

sth

urin

gien

sis

and

Bac

illu

sm

egat

eriu

mD

ecan

alIm

age-

inte

nsifi

edlo

w-l

ight

sing

le-p

hoto

nvi

deo

imag

ing

(and

X-r

ayau

tora

diog

ra-

phy)

oftr

ansg

enic

orga

nism

sus

ing

bac-

teri

allu

cife

rase

s(9

7):

Bac

illu

sA

cNPV

–luc

luc

AcN

PVpo

lyhe

drin

late

prom

oter

Aut

ogra

pha

cali

forn

ica

nucl

ear

poly

-he

dros

isvi

rus-

vect

ored

Luc

inT

.ni

368

cells

and

Tri

chop

lusi

ani

3rd

inst

arla

rvae

Fire

flylu

cife

rin

Imag

e-in

tens

ified

low

-lig

htsi

ngle

-pho

ton

vide

oim

agin

g(a

ndX

-ray

auto

radi

ogra

-ph

y)of

tran

sgen

icor

gani

sms

usin

gba

c-te

rial

luci

fera

ses

and

firefl

ylu

cife

rase

(97)

:T

.ni

368

cells

and

T.n

i3r

din

star

larv

aelu

xAan

dB

(V.

harv

eyi)

,Fab

9,F

abcb

c9–

E.

coli

Dec

anal

Imag

ing

ofov

erex

pres

sion

ofG

roE

Lan

dG

roE

S-m

edia

ted

fold

ing

ofFa

b9–b

acte

rial

luci

fera

sefu

sion

sat

diff

eren

tte

mpe

ratu

res

(97)

:E

.co

lipC

EP4

–luc

luc

CM

Vpr

omot

erB

rach

ydan

iore

rio

(zeb

rafis

h)L

ucif

erin

(fire

fly;

0.1

mm

ol/L

)T

rans

geni

cze

brafi

shw

ithL

ucvi

sual

ized

bylo

w-l

ight

vide

o-im

age

anal

ysis

(195

)pP

VC

701–

ruc

ruc

(Ren

illa

reni

form

islu

cife

rase

)pC

aMV

35S

RN

AA

lfal

fa(M

edic

ago

sati

va),

tom

ato,

pota

to,t

obac

coC

oele

nter

azin

eIm

agin

gof

Ruc

intis

sues

oftr

ansg

enic

plan

ts(6

0)pM

W54

;pM

V53

;pM

V16

;pO

GS2

13lu

cH

IV-1

,HIV

-1LT

Ren

hanc

er–p

rom

o-er

elem

ents

and

CM

Vpr

omot

erH

eLa

cells

Luc

ifer

in(fi

refly

)V

isua

lizat

ion

ofH

IV-

and

hCM

V-p

rom

oted

expr

essi

onof

Luc

insi

ngle

mam

mal

ian

(HeL

a)ce

lls(1

39)

pCol

.luc;

pCM

V.lu

c;pC

MV

.Aqm

;pc

DN

AA

Ineo

.CL

100

luc;

aequ

orin

Col

lage

nase

and

CM

Vpr

omot

ers

CH

O.T

cells

Coe

lent

eraz

ine

and

beet

lelu

cife

rin

(for

Aeq

and

Pluc

resp

ectiv

ely)

Hig

h-in

tens

ityre

al-t

ime

phot

on-c

ount

ing

imag

ing

ofin

sulin

-ind

uced

MA

Pki

nase

sign

alin

gin

sing

lece

lls(1

37)

��



Tab

le2.

Con

tinu

ed

Con

stru

ct/v

ecto

rL

ucif

eras

ege

nes

orcD

NA

sPr

omot

ers/

enha

ncer

sO

rgan

ism

/cel

lsSu

bstr

ate

requ

irem

ent

Imag

ing

appl

icat

ion

and

refe

renc

e

pGL

101

luxF

(lux

AB

fusi

on)

Ara

bido

psis

PA

L1

(phe

nyla

lani

neam

mon

ia-l

yase

)pr

omot

erA

rabi

dops

isth

alia

naD

ecan

alSu

cces

sful

phot

on-c

ount

ing

imag

ing

oflo

caliz

edac

tivat

ion

ofPA

L1

(187

)pC

EP4

ruc–

gfp

fusi

on,a

ndin

divi

dual

lyC

MV

and

hum

an�-

actin

prom

oter

sL

M-T

K�

cells

;m

urin

eem

bryo

nic

stem

(ES)

cells

grow

th-s

uppo

rted

byST

Ofe

eder

cells

Coe

lent

eraz

ine

Vis

ualiz

atio

nof

Ruc

–GFP

expr

essi

onin

ES

cells

and

embr

yos

(69)

pCE

P4–R

uc;

pCE

P4–R

uc/G

FP;

pCE

P4–G

FP/R

uc;

pGE

M–5

z(�)

–Ruc

/G

FP;

pGE

M-5

z(�)

–GFP

/Ruc

ruc;

‘hum

aniz

ed’

gfp

(Aeq

uore

a)C

MV

,�-a

ctin

,(f

orpC

EP4

vect

or),

and

T7

(pG

EM

-5zf

�ve

ctor

)pr

omot

ers

LM

-TK�

(mur

ine

fibro

blas

tce

lllin

ew

ithth

ymid

ine

kina

sem

issi

ngbe

caus

eof

mut

atio

n)

Coe

lent

eraz

ine

Imag

ing

Ruc

–mod

ified

GFP

fusi

onin

mur

ine

cells

(70)

Mito

chon

dria

llyta

rget

edae

quor

inve

c-to

rsae

q(A

equo

rea)

–C

HO

.Tce

llsC

oele

nter

azin

eIm

aged

intr

amito

chon

dria

lC

a2�

ince

llsus

ing

reco

mbi

nant

aequ

orin

with

aC

CD

cam

era

(138

)R

ecom

bina

ntba

culo

viru

sco

nstr

ucts

Bm

NPV

luc

(Bom

byx

mor

inu

clea

rpo

lyhe

dros

isvi

rus)

and

AcN

PVlu

c[2

]

luc

[2]

Inre

vers

eor

ient

atio

nto

the

Aut

o-gr

apha

cali

forn

ica

nucl

ear

poly

-he

dros

isvi

rus

(AcN

PV)

poly

hedr

invi

ral

prom

oter

,pos

sibl

yun

der

OR

F629

prom

oter

Bom

byx

mor

iN

-4an

dSf

9ce

lls,

Tri

chop

lusi

ani

368

cells

Luc

ifer

in(fi

refly

)Im

agin

gof

bacu

lovi

rus-

vect

ored

Luc

inin

sect

cells

(218

)

pRL

uc6

and

pRL

uc6.

1;pM

CT

–Ruc

ruc

AdV

maj

orla

tepr

omot

er;

hCM

Vin

term

edia

te–e

arly

enha

ncer

/pr

omot

er

CO

S-7

cells

;C

5ce

llsC

oele

nter

azin

eIm

agin

gof

Ruc

expr

essi

onin

sim

ian

and

mur

ine

cells

(61)

Rec

ombi

nant

herp

es/

pseu

dora

bies

viru

sPr

VA

916

luc

Gly

copr

otei

ngG

earl

ypr

omot

erA

fric

angr

een

mon

key

kidn

ey(V

ER

O)

cells

Luc

ifer

inV

isua

lizin

gof

PrV

infe

ctio

nin

cultu

revi

aPl

ucus

ing

aph

oton

-co

untin

gca

mer

a(2

17)

CH

S::lu

cco

nstr

uct

luc

Frag

men

tof

chal

cone

synt

hase

prom

o-te

r;�

tran

slat

iona

len

hanc

erof

the

Tob

acco

Mos

aic

Vir

us(T

MV

)

Ath

alia

nase

edlin

gs(C

olum

bia

g/1)

and

inan

A.

thal

iana

C24

cell

line

Luc

ifer

inC

ompa

riso

nsbe

twee

nai

r-co

oled

CC

Dan

din

tens

ified

CC

Dca

mer

as(1

78)

LT

R–l

ucin

pGL

3lu

cSV

40pr

omot

eran

den

hanc

erin

LT

Rs

Neo

nata

lra

ts(R

attu

s);

mic

e(M

us);

hum

anT

cells

Luc

ifer

in(fi

refly

)In

duct

ion

ofSV

40pr

omot

er–l

ucif

eras

eex

pres

sion

inne

onat

alra

ts,i

nm

ice/

and

inhu

man

T-c

ells

(157

)pL

PKL

ucF

F,t

runc

ated

p�L

4–L

PKL

ucF

F

ruc

L-p

yruv

ate

kina

seno

rmal

ized

byco

n-st

itutiv

eC

MV

prom

oter

,req

uiri

ngup

stre

amst

imul

ator

yfa

ctor

2(U

SF2)

Hum

anis

let�-

cells

,der

ived

INS-

1ce

llsB

eetle

luci

feri

n,eo

elen

tera

zine

Sing

le-c

ell

CC

Dim

agin

gof

Ruc

-mar

ked

nece

ssar

yup

stre

amst

imul

ator

yfa

ctor

activ

ity(1

36)

pCK

218

luxA

B(V

ibri

ofis

cher

i)Pr

omot

erle

ssin

V.

fisch

eri;

unid

enti-

fied

stro

ngco

nstit

utiv

epr

omot

erin

Pse

udom

onas

puti

da

Vib

rio

fisch

eri

MJ-

1;lu

x-m

arke

dP

seud

omon

aspu

tida

Dec

anal

Com

pari

son

ofsi

ngle

-ba

cter

ium

low

-lig

htim

agin

gus

ing

acr

yoge

nica

lly-c

oole

dC

CD

cam

era

and

aph

oton

-cou

ntin

gca

mer

a(1

41)

pTK

Elu

c;pT

K6W

Elu

c;pS

VE

luc;

pMiw

luc;

pMiw

Elu

clu

c6W

enha

ncer

,TK

;SV

40;

RSV

(Rou

sSa

rcom

aV

irus

)L

TR

,�-a

ctin

prom

oter

s

Bov

ine

embr

yos

Luc

ifer

in(fi

refly

)D

etec

tion

oflig

htem

issi

onfr

omL

ucin

tran

sgen

icbo

vine

embr

yos

(200

)

–ru

c(R

enil

lare

nifo

rmis

)Pr

omot

erle

ssR

ucex

pres

sion

–pro

mot

erse

arch

vect

orT

rans

geni

cA

rabi

dops

is,T

abac

umor

othe

rpl

ant

calli

,ro

ots,

leaf

,st

em,

flow

ertis

sue;

pota

totu

bers

;se

di-

men

ted

tran

sfor

med

plan

tpr

oto-

plas

ts(l

ux,r

uc);

tran

sgen

ic

Dec

anal

;lu

cife

rin

2-be

nzyl

coel

ente

razi

neIm

agin

gof

plan

tpr

omot

erex

pres

sion

usin

gL

uxan

dR

ucre

port

ers

inpl

ant

cells

(182

)

pSP–

Luc

�ph

PRL

luc

hPR

L(h

uman

prol

acti

n)pr

omot

erR

atpi

tuita

rytu

mou

rG

H3

cells

Luc

ifer

in(fi

refly

)C

CD

phot

on-c

ount

ing

imag

ing

ofPR

Lpr

omot

erac

tivat

ion

ofL

ucin

indi

vidu

alce

lls(1

21)

RD

29A

–Luc

cons

truc

tlu

cR

D29

A;

CO

R47

;C

OR

15A

;K

IN1;

AD

H;

RA

B18

;R

D22

;R

D29

B;

LUC

;ac

tin—

all

cold

sens

itive

gene

prom

oter

s

Ara

bido

psis

thal

iana

Luc

ifer

in(fi

refly

)V

isua

lized

mut

ant

seed

lings

with

mut

ant

cold

-res

pons

ege

neH

OS-

1(1

81)

Synt

hetic

RB

CSB

gene

luc

(pro

mot

erle

ssin

cons

truc

t)R

BC

SBpr

omot

erju

xtap

osed

upst

ream

oflu

cby

cros

s-ov

erA

rabi

dops

isth

alia

naL

ucif

erin

(fire

fly)

Vis

ualiz

edcr

oss-

over

seed

lings

(184

)

p260

Ins.

Luc

FF;

pF71

1fo.

Luc

FF;

pCM

V–R

enlu

c;ru

cSR

Ean

dC

RE

ofth

ehu

man

insu

linpr

omot

er,H

erpe

ssi

mpl

exm

inim

alTK

,c-f

osan

dC

MV

prom

oter

s

MIN

6�-

cells

;C

HO

cells

;an

teri

orpi

tuita

ry-d

eriv

edA

tT20

Luc

ifer

in(fi

refly

);co

elen

tera

zine

Rea

l-tim

ein

tens

ified

CC

Dca

mer

aim

agin

gof

cons

titut

ive

gluc

ose

enha

ncem

ent

ofin

sulin

prom

oter

-act

ivat

edlu

cife

rase

activ

ity(1

23)

pcL

uc(n

on-t

arge

ted,

cyto

solic

luci

fer-

ase)

;pm

Luc

(pla

sma

mem

bran

eta

rget

edlu

cife

rase

)

luc

CM

Van

dSV

40in

term

edia

te–e

arly

prom

oter

sPr

imar

yra

tis

let�-

cells

;de

rive

dM

IN6

cells

Luc

ifer

in(fi

refly

)L

ucif

eras

eph

oton

-cou

ntin

gim

agin

gof

sub-

cellu

lar

com

part

men

tali

zatio

nof

AT

P(1

24)

��



Tab

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photoproteins as simultaneous reporters will probablybecome more common in the future.

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The papers cited above show that luciferases have beenvery useful as reporter genes in living cells and inbioluminescent immunoassays (63). In fact there wereabout 30 photoprotein fusions and conjugates reportedbetween 1988 and 2000 (63–65). The first such fusion wasthat of the Vibrio harveyi luxA and luxB into the luxAB(luxF) fusion, which was expressed as a monomer in E. coli,Bacillus, yeast and plant systems (30, 66). A luxAB fusionhas also been made from the luciferase of Photorhabdusluminescens (67). A gfp–luxAB fusion construct expressedin E. coli DH5� and Pseudomonas fluorescens SBW25 wasused to assay bacterial numbers and nutrient-based bacterialmetabolic activity in soil samples (68). Assaying the luxABexpression required luminometry (For more information,see section on Bacterial luciferase imaging in environ-mental health assays, below).

An ruc–gfp fusion construct was first engineered andexpressed in murine LM–TK� fibroblast cells, inembryonic stem (ES) cells and in early stage embryosby Wang et al. (1996) (69). A ruc-modified gfp fusionwas found to be functionally expressed in murine LM–TK� cells, whereas a reverse modified gfp–ruc fusionshowed no GFP expression, probably because ofmisfolding (70). In 1999, Wang et al. suggested thatchemiluminescent energy transfer Ruc to GFP could beused to image protein–protein interactions (71).

Using an ruc–gfp fusion construct, Wang et al. (2001)imaged the luminescence resonance energy transfer(LRET) phenomenon from Renilla luciferase (Ruc)emission to a humanized Aequorea GFP to documentprotein–protein interaction in eukaryotic cells (72).cDNAs of ruc and insulin-like growth factor bindingprotein 6 (Ruc–IGFBP-6) were expressed, along withfused cDNAs for gfp and insulin-like growth factor II(gfp–igf-II). The interaction of the recombinant IGF-IIand IGFBP-6 resulted in LRET (luminescence resonanceenergy transfer) from Ruc to GFP. In 2000, Liu et al.visualized and quantified protein secretion using an Ruc–GFP fusion in the COS-7 and Chinese hamster ovary(CHO) cell lines (73). In simian COS-7 cells, intensifiedCCD camera imaging has been used to detect LRET fromRuc to aequorin GFP in an Ruc–GFP fusion protein (72).

An important recent development is the use of P.luminescens luxCDABE gene fusions in a collection of8066 individual E. coli transformants to assay genome-wideexpression profiles in response to environmental stress (74).

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In 1989, Wick (75) reviewed the growing usage of single

photon-counting visualization being used to assay lumi-nescence in microtitre plates, to image metabolitedistribution in tumour tissues, to visualize single cellgene expression, and even to visualize the faint chemi-luminescence resulting from oxidative metabolism inphagocytes.

In a series of papers from 1992 to 2000, Stanleyreviewed the commercially available luminometers,radiometers, low-light imaging CCD cameras, immu-noassays, ATP rapid microbiology, hygiene monitoring,molecular probes, labels, nucleic acid hybridizations andreporter genes available for bio-imaging applications, aswell as units and standards of bioluminescence (76–86).

Hill and Stewart (1994) reviewed the applied devel-opments of bacterial luciferases as real-time, non-invasive reporters using low-light and photon-countingvideo cameras (87). They noted their sensitivity and real-time, non-invasive nature and their amenability toimaging by photon-counting and low-light video cam-eras. Contag et al. published two reviews on the variousmolecular imaging technologies for the detection andtracking of molecules and cells in vivo (88, 89). They alsodescribed briefly the work done in imaging tumours usingluciferase expression in vivo.

It is to the imaging experiments that we now turn (seeTable 2).

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We attempt to summarize the imaging of luciferaseexpression in individual cells and cell cultures, inindividual bacteria, yeasts, algae, insect cells, plant cells,or mammalian cells. We also focus on some of theapplications and types of questions that can be answeredby such imaging. These imaging experiments include theimaging of luciferases in transformed cells and cellsystems in real time.

Next, we consider imaging of luciferase expression inmulticellular organisms in vivo. This includes theexpression and visualization of luciferase in permanentlytransformed or transgenic organisms.

Finally, we present representative studies usingluciferase expression imaging to investigate host–patho-gen interactions in whole plant and animal models.

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Imaging of bacterial luciferase in bacteria and cell culturehas proved to be a fruitful venture. Some representative



papers are overviewed below. The monitoring ofbioluminescent bacterial pathogens and symbionts inhosts is discussed in the section on symbiosis and host–pathogen interactions.

In a landmark paper, Engebrecht and his co-workersshowed, in 1985, that the bacterial luciferase operonluxCDABE (i.e. luxICDABE under the control of luxRand luxI) from Vibrio fischeri could be vectored by atransposon and expressed in E. coli without the need toadd the lux substrate, decanal, because of the presence ofthe full operon (26). Ulitzur and Khun (1987) alsodiscussed the use of introduced luciferase genes inbacteria as an assay for the presence of particular bacteriaand their susceptibility to a given antibiotic (90). In 1988,Schauer et al. visualized Vibrio harveyi luxA and luxBexpression in Streptomyces coelicolor (91). Olsson et al.(1988) have shown that fusion gene products can beadded to the luxA of Vibrio harveyi as long as the N-terminal hydrophobic sequences of the �-subunit arepreserved intact, in order to retain enzymatic activity(29).

Olsson et al. (1989) constructed luxAB and luxBAfusions of the V. harveyi luciferase genes and expressedthem in E. coli (Fig. 1) and in calli of Nicotiana tabacum,indicating their possible application as reporter genes ineukaryotic cells (30). The authors showed that luxAB hashigher expression levels than luxBA. This was animportant first. Escher et al. (1989) described a fusionof the luxAB genes of V. harveyi and showed it to becapable of functioning as a monomer in E. coli (66).Using video-imaging and spectroscopy, they found thatluxAB has an emission spectrum comparable to the wild-type luxA and luxB, but is more sensitive to elevatedtemperature. In 1991, Langridge et al. provided anoverview of the bacterial luciferase gene expression

system and its applications, using low-light imaging inother bacteria and in eukaryotic cells, namely plant cells(92).

In a series of studies (93–97) it was shown that Vibrioharveyi luxA, luxB and a luxAB fusion could besuccessfully expressed under various bacterial, mamma-lian and viral promoters and visualized by photon-counting imaging in Gram-positive Bacillus thuringien-sis and B. megaterium (cf. Fig. 2), Arabidopsis thaliana,Nicotiana tabacum, Trichoplusia ni (cabbage looper)(386) cells and Manduca sextans (tobacco hornworm;Fig. 3a, b; see the section a Imaging of host–pathogeninteractions, below) (95). These lux constructs werevectored into transgenic plants by Agrobacteriumtumefaciens T-DNA. Through T-DNA integration, pro-moterless constructs bearing luxA and luxB wererandomly inserted into transgenic Nicotiana tabacum.Lux activity was visualized at different developmentalstages in different organs (Fig. 4) (96). Bacterialluciferase expression was also successfully imaged intomato leaves and fruit (Fig. 5a, b) (97). Firefly luciferase(Pluc) was also expressed in some of these experiments.

When promoterless V. harveyi luciferase (luxAB) wasintroduced downstream of the promoter for the cyano-bacterium Synechococcus psbAI gene (a photosystem IIprotein), its varying expression under constant bio-luminescent imaging revealed that prokaryotes alsohave circadian rhythms (98). Furthermore, when luxABwas inserted randomly by conjugation and subsequenthom-ologous recombination into the Synechococcusgenome and transformed clones were monitored by thethen newly developed cooled-CCD camera system, itwas found that luciferase expression in these cyano-bacteria exhibited not only circadian rhythmicity but a

Figure 1. Visualization of individual Escherichia colicolonies transformed by Vibrio harveyi lux genes (cf. 97).

Figure 2. Visualization of lux-transformed Pseudomonaspatches grown on agar medium using the Argus-100 low-lightimaging system. (cf. 97).



wide variety of amplitude and waveform cyclicity (99).Liu et al. also discovered that random insertion ofpromoterless luciferases by homologous recombinationis an extremely sensitive assay for differential geneexpression levels. Thus, bacterial luciferase imaging wasuseful in documenting a complex gene expressionphenomenon such as cyanobacterial circadian rhythms(100).

In 2000, Kunert et al. transfected Synechocystis withgfp and luxAB (64). Under different media conditions,luminescence imaging revealed that GFP expressioninitiated and dissipated at a slower rate, while LuxABexpression had a much more rapid response reactiontime. The advantage of GFP in Synechocystis is that it hasno substrate requirement. The advantage of LuxAB is itsrapid expression response time. So, in combination withanother reporter gene, such as GFP, LuxAB can be usedas a sensitive measure of gene expression in bacteria.

Waddell and Poppe (2000) developed a mini-transpo-son bearing Vibrio harveyi luciferase (luxAB) in order toproduce a luciferase-transfecting bacteriophage to detectE. coli strain 0157:H7 (101). The E. coli colonies ofinterest could be visualized by image quantifier about 1 hpost-transfection. The ability to use a vectored luxAB todetect the presence of bacteria has led to importantenvironmental and food safety applications, of which wereview a few.

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One of the earliest uses of luciferase imaging to detectmicrobial contamination involved eukaryotic firefly(Photinus) luciferase (Luc), not bacterial luciferase

Figure 3. Visualization of the progression of an infection ofBacillus thuringiensis, labelled with the xylA promoter–luxABfusion gene construct, in tobacco hornworm (Manduca sextans)larvae (A) through feeding or (B) after injection into thehaemolymph (95). Figure 4. Visualization of randomly inserted luxA and luxB

containing construct in transgenic N. tabacum corollas,indicating N. tabacum promoter-driven expression at differentdevelopmental stages (96).



(102). Since that time, both luc and bacterial luxABassays have been used in environmental sampling.

Prosser et al. (1996) reviewed how luminometry andCCD image-enhanced microscopy may be useful indetecting naturally luminescent bacteria in environmen-tal samples, to monitor their growth and metabolism onsoil particles, microbial survival and recovery, microbialpredation, plant pathogenicity, rhizosphere colonizationand the reporting of gene expression in environmental

samples (103). They suggested that this technology mayalso be used to distinguish genetically modified bacteriafrom indigenous bacteria in environmental samples.

Using a mycobacteriophage T5–luc construct, Sarkiset al. (1995) infected Mycobacterium smegmatis toproduce luciferase expression both in the bacteria andin the post-infection lysogen (104). Such luciferasereporter phages may be used to test for the presence ofdrug-resistant or drug-sensitive M. smegmatis strains, aswell as for the rapid identification of other effectiveantimycobacterial agents.

Loessner et al. showed (1996, 1997) that the Listeriamonocytogenes-specific bacteriophage A511, whentransformed with the Vibrio harveyi luxAB gene, can bea sensitive detector of viable Listeria cells in environ-mental and food samples within 24 h, rather than theusual 3 days required for traditional culturing (105, 106).Also, competitive PCR and imaging analysis can be usedto quantify the number of luciferase gene copies insediment samples to which Synechocystis 6803-luc cellswere added (107). The authors suggest that this techniqueshould have applications for quantifying geneticallymodified cyanobacteria in nature.

Although not directly imaging luciferase expression,Unge et al. used a mini-transposon construct bearing agfp–luxAB fusion to simultaneously assay bacterial cellnumbers and populational metabolic activities in specificpopulations of E. coli DH5� and Pseudomonas fluor-escens SBW25 in soil samples (68). Bacterial numberswere determined by flow cytometric monitoring of GFP-expressing cells. LuxAB expression, as determined byluminometry, was shown to be dependent on nutrientlevels and hence metabolic activity.

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A bioluminescent reporter strain of Escherichia coli(O157:H7) containing the full bacterial luciferase(luxABCDE) operon was inoculated in buffer and infecal slurry, which were both placed on surfaces of beefcarcases to determine the interaction between potentialbacterial pathogens and human food animal tissues (108).A sensitive photon-counting camera was used tovisualize the presence of bacteria in real time. The fulllux operon in O157:H7 renders substrate additionunnecessary.

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Imaging of luciferase expression in plant and animal celllines has found more diverse applications than hasimaging of luciferase in bacteria. These applicationsinclude the imaging of protein site-specific secretion,protein trafficking and protein targeting to the imaging of

Figure 5. Visualization of bacterial luciferase expression inthe leaves (A) and fruit (B) sections of transgenic tomatoLycopersicon esculentum) plants (97, 172).



transgenic promoter expression, real-time gene activa-tion, cell injury-induced expression and regulation, thedetermination of ATP and free Ca2� concentrations, andthe visualization of immune response.

Keller et al. showed in 1987 that a firefly luciferin–luciferase cDNA construct could be expressed in monkeykidney cells and that the gene product was targeted to theperoxisomes via a putative peroxisomal targeting proteintranslocation sequence (109). Using cDNAs of luc withthe peroxisomal targeting sequences, Gould et al. helpedto demonstrate that such sequences for protein transportare conserved from yeasts to plants, insects and mammals(110, 111). The expression of firefly luciferase (Luc) hasbeen imaged in single COS-7 cells by White et al. (1990)(112).

A method for photographic film detection of fireflyluciferase expression regulated by the simian virus 40promoter in mammalian cells was developed for use witha polyester mesh replica plating technique to determineluciferase expression in mammalian cells (113).

Site-specific protein secretion from transformed Chi-nese hamster ovary (CHO) cells, containing luciferasefrom the marine crustacean ostracod Vargula hilgendorfii(Luc), was visualized in real time using an image-intensifying system (114).

Transient expression of luciferase fusion proteins hasbeen an important tool in cell imaging. Luciferase cDNAfused to the 5�-flanking region of the rabbit collagenasegene containing a wild-type promoter showed anincreased expression after mechanical injury to thesmooth muscle Rb-1 cell line (115). In hormone studies,a modified luciferase expression system was used for real-time measuring of gene expression in endocrine cells(116). Jausons-Loffreda et al. (1994) used single photon-counting technology to document steroid hormoneactivity in transformed cell lines expressing fireflyluciferase, activated by chimeric constructs of the bindingdomain of the Gal4 yeast protein fused to the hormone-binding domains of various steroid receptors (117). Usingpromoter–luciferase reporter constructs, dynamic geneexpression was visualized in real time and quantified innursing rat lactotrope cells by Castano et al. (118), whoproposed that similar constructs could be used to visualizegene expression in any normal cell type. The Renillareniformis luciferase cDNA was expressed transiently insimian COS-7 cells and stably in murine C5 fibroblastsand in extracts (61). A firefly luciferase–aequorin fusionprotein was used in HeLa cells to detect rapid changes inATP and free Ca2� levels, based on light emission inresponse to C9 complement attack (119). Quantificationof ATP concentrations has also been carried out in ratcardiac myocytes by Dorr et al. (1989) (120).

Rat pituitary tumour cells (GH3) were transformedwith a construct containing the firefly luciferase gene and5000 bp of the 5� flanking region of the human PRL(prolactin) gene, subjected to luciferin, and then imaged

by CCD photon-counting for time periods up to 72 h(121). Basal PRL promoter–luciferase activity wascompared to stimulated activity after the addition ofsuch stimuli of the PRL promoter as thyrotropin-releasing hormone (TRH), forskolin, calcium channelagonist Bay K8644, and basic fibroblast growth factor(bFGF). Individual cells could be imaged.

Visualization of longer-term transient luciferase ex-pression in mammalian cells has also been possible.Various modified polylysine constructs with couplingreagent sulpho-LC SPDP were transfected with luciferaseinto HuH7 human hepatoma cells and imaged 2–16 daysafter transfection (122).

Real time-intensified CCD camera imaging of fireflyluciferase (Luc) expression has been utilized to confirmthat glucose may induce insulin gene transcriptionthrough increases in intracellular Ca2� concentration inMIN6 �-cells when glucose, insulin or the Ca2� channelinhibitor verapamil were added (Kennedy et al. 1999a)(123). Furthermore, Kennedy et al. (1999b) usedrecombinant firefly luciferases and photon-countingimaging to visualize concentration changes in free ATPin subdomains of single living MIN6 and primary �-cells(124). When control was made for pH, free ATP levelscould be visualized in real time in the cytosol, at theplasma membrane and in the mitochondrial matrix byusing luciferases specifically targeted to these threesubdomains (cLuc, pmLuc, and mLuc), respectively.This was an excellent example of subcellular imagingof luciferase expression.

Chinese hamster ovary (CHO) cells with stable expres-sion of both a CRE–luciferase reporter construct and thehuman pituitary adenylyl cyclase-activating peptide (PA-CAP) receptor were exposed to receptor agonists for each.Visualization of luciferase light emission, as well asfluorescence, was used to visualize Ca2� mobilization andthe induction of adenylyl cyclase activity (125).

Pancreatic �-cell activation of phosphatidylinositol 3�-kinase has been visualized by photon-counting imagingwith an intensified CCD camera, using intranuclearinjection of recombinant promoter fused to firefly andRenilla luciferase cDNAs (126). Elevated glucose levelsinduced pre-proinsulin (PPI) and liver-type pyruvatekinase (L-PK) promoters fused to firefly and Renillaluciferase cDNAs, thus producing light emission whichwas used to visualize single cells (127).

By constructing a less stable (and fainter) luciferasewith a shorter functional half-life and implanting it intohuman breast cancer T-47D cells under the control ofoestrogen response elements, Leclerc et al. (2000) wereable to observe real-time gene expression in single livinghuman breast cancer T-47D cells (128).

Increases in Ca2� concentrations in HeLa cells andskeletal myotube cells caused by agonist addition havebeen shown to raise levels of ATP production (129).Ainscow and Rutter (2001) have shown the significance



of this by quantifying (via continuous photomultipliermonitoring and intensified CCD imaging of aequorin andfirefly luciferase) the expression introduced by adenoviralvectors in rat pancreatic islet (MIN6) cells and stimulatedby the addition of glucose (130). This further indicatedthat the expression of aequorin and Pluc is dependent onCa2� and ATP concentrations.

Synthetic esters of luciferin, a number of which weresubstrates for purified esterases, apparently hydrolysedinto luciferin in intact mammalian cells, where theycaused peaks in expression at levels six-fold higher thanwild-type luciferin (131). This suggests that such estersmay be used to assay for luciferase activity inmammalian cells where the concentration of luciferinwould be the rate-limiting factor.

Although imaging luciferase in individual plant celllines and cultures is more difficult, and therefore has notbeen done as often as in animal cell lines, several reportsare available. Polyadenylated luciferase mRNA electro-porated into tobacco protoplasts has been imaged byvideo at a wide range of levels of expression (132).

Another advance was the transformation of mouseembryonic stem (ES) cells with a cDNA constructexpressing the Ruc–GFP fusion protein, and its visual-ization (Fig. 6) (69).

Recently a gfp–luxABCDE reporter construct, underthe control of the XylA promoter, was used to monitor theexpression and temporal induction of the quorum-sensingaccessory gene regulator (agr) in S. aureus infectingbovine mammary epithelial MAC-T cells (133). The

reporter gene expression was occasioned by the virulencefactor-mediated escape of S. aureus from the hostendosome and its ensuing intracytoplasmic growth.

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A most exciting feature of luciferase expression imaging isthe ability to visualize gene expression in individual cellsin real time. Wood and DeLuca (1987) showed thatvectors can be checked for functional coding sequences byincluding a firefly luciferase gene probe, expressing it in E.coli, and detecting it by photographic film after theappropriate luciferin is added (134). Back in 1990, Hooperet al. reviewed low-light imaging with particular emphasison charge couple device (CCD) imaging of single cellsexpressing luciferase (135). Recombinant Vaccinia virusbearing firefly luciferase were added to cell culture.Virally transduced cells could be detected by imaging ofLuc expression at a level of one infected cell per million.Hooper et al. further suggested that imaging of luciferase-expressing viruses could be used to detect virus deletionmutants. Photon-counting CCD imaging of firefly lucifer-ase activity was used by Kennedy et al. to visualizeglucose L-pyruvate kinase (L-PK) promoter activity insingle living pancreatic islet �-cells at different glucoselevels (136). Detection of the L-PK promoter-driven fireflyluciferase activity was standardized using CMV promoter-controlled Renilla reniformis luciferase activity. Highintensity real-time photon-counting imaging was able to

Figure 6. Visualization of mouse embryonic stem (ES) cells transformed with a cDNA construct expressing the Ruc–GFP fusion protein (69). A and D are the light images; B and E are the images of green fluorescence under UV light. Cand F are overlays of A and B, D and E, respectively.



detect firefly luciferase and aequorin reporter genesactivated by insulin-induced MAP kinase signalling insingle CHO.T cells, even when under the control of weakpromoters, according to the findings of Rutter et al. (1995)(137). Rutter et al. (1996) also used CCD camera imagingto visualize intramitochondrial Ca2� concentrations inCHO.T cells, using recombinant, Ca2�-sensitive aequorin(138). Regulation of human cytomegalovirus (hCMV) andhuman immunodeficiency virus (hHIV) gene expression inindividual, intact HeLa cells has been imaged usingconstructs with a firefly luciferase reporter gene down-stream of viral promoters (139).

Single bacterial cell microscopic imaging also is anemerging field. Hill et al. (1994) were the first to reportthe imaging of bioluminescence in individual bacterialcells (140). Using photon-counting, they were not onlyable to visualize individual bacteria (such as Pseudomo-nas fluorescens) that had been transformed with luxAB-bearing plasmids and transposons, but were also able toimage naturally luminescent Photobacterium phosphor-eum. Furthermore, they were able to show that expressionvaried over time, due to cell cycle-related changes inmetabolic activity.

Two low-light imaging systems were compared toassess their efficiency in visualization of single cells ofVibrio fischeri (MJ-1) and of a strain of Pseudomonasputida both of which were expressing V. harveyi luxAB(141). The authors found that a slow scan liquid N2

cooled CCD (C-CCD) camera was preferable for higherresolution of single cell signal at longer exposure times,but that a photon-counting CCD (PC-CCD) camera wasto be preferred for living cells at shorter exposure times,even though the resolution was somewhat lower. Phieferand colleagues (1999) quantified relative photon fluxfrom individual cells of Vibrio fischeri and V. harveyiusing photon-counting microscopy (142). Vibrio fischeriluciferase was found to be more stable in expression,while V. harveyi luciferase was found to be much morevariable in its light emission.

Even the rapid flashing of individual bioluminescentorganelles (scintillons), within individual dinoflagellatesof the species Pyrocystis noctiluca, has now beensuccessfully imaged using video image intensifier lightmicroscopy (143). The flashing, which occurs withfraction of a second rapidity, was induced by concen-trated citric acid stimulation.

In another review (1994), Hooper et al. summarized ingeneral the new improvements in low-light imagingtechnology. They discussed the hardware and softwareavailable, noted the rapid non-invasive advantages oflow-light imaging in reporter (luc, lux) gene expression,in intracellular expression and in analysis of tissuesections, as well as immunoassays, gels and blots (144).Also in 1994, Nicolas reviewed the varied applications inthe biological sciences of low-light photon-countingimaging, from the large-scale (immunoassays, DNA

probes and in vivo imaging of expression and promoteractivity) to the small scale (in situ hybridization andcellular luciferase expression) (145).

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In some ways, the goal of luciferase imaging has alwaysbeen to monitor processes in living, multicellularorganisms non-invasively.

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As in cell and tissue cultures, a very wide variety ofapplications have been made of luciferase expressionimaging in multicellular animals—both living and post-mortem.

In 1989, Hohn-Berlage et al. showed that ATP in intactbrain cryosections can be imaged using firefly luciferin–luciferase, and that lactate concentrations can be likewiseimaged using V. fischeri luciferase and lactate dehydro-genase (146) (see also Paschen, 1985) (147). Luciferin–luciferase assays can also be used to image distributionsand abundances of respiratory metabolites such asglucose, ATP, and lactate in tissue sections (148).

Following liposome-mediated transformation of luci-ferse expression vectors, luciferase expression has beenmonitored in normal and atherosclerotic external iliacrabbit arteries (149). Replication-deficient adenovirus-bearing firefly luciferase and �-galactosidase genes weresuccessfully used to test the comparative efficiencies oftransgenic gene delivery to cultured Sprague–Dawley ratthoracic artery and aortic artery smooth muscle tissuecultures (150). Luminometer readings confirmed theluciferase assay differential.

In 1993, Mueller-Klieser and Walenta showed thespatial distribution and concentration of respiratorymetabolites (ATP, glucose and lactate) in rapidly frozentissue in absolute unit concentrations and at single-celllevel resolution, using photon-counting visualization ofluciferase expression coupled with any particular enzymeof interest (151). Single-photon count imaging ofluciferase assays was used to spatially quantify thedistribution of respiratory metabolites (ATP, glucose, andlactate) in cryosections of tumours and normal tissue(152). Luciferase light emission was proportional tometabolite concentration. These results were confirmedusing other metabolite quantification methods.

Rembold et al. (1997) used a replication-deficientadenoviral vector carrying an apo-aequorin cDNA with asarcoplasmic reticulum (SR) targeting sequence to infectintact rat tail arteries (153). In this way the authors wereable to measure fluctuation in the presence of free Ca2� inthe SR in the presence of coelenterazine, the apo-aequorin luciferin substrate.



Using a luciferase reporter, Thierry et al. (1997)showed that when a lipopolyamine, a neutral lipid and aplasmid DNA are associated in the formation of lamellarvesicles, they can be used to compare in vitro and in vivotransfection efficiencies in mouse tissues (154). Theselamellar vesicle–DNA complexes have a higher in vitrotransfection efficiency than that of previously reportedliposome transgenic delivery systems.

Making use of the fact that muscles, injected with apromoter–firefly luciferase cDNA fusion construct, retainLuc activity for up to 60 days, Davis et al. (1997) showedthat, when a luciferase construct was co-injected with ahepatitis B virus surface antigen (HBsAg)-expressingDNA, the luciferase expression time is shortened to about5 days of strong expression and a cut-off after 20 days(155). These findings suggest that luciferase can be usedto image indirectly the progress of immune-mediateddestruction of muscle myofibre tissue.

The subcutaneous injection of a lipid gadoliniumcontrast complex containing Luc was imaged by Wisneret al. (1997) using MRI (156). The Luc was also imaged.The successful insertion of Luc with the complex into thecell provided a possible method for the visualizing of atransfection event. MRI imaging provided an indepen-dent check on the luciferase imaging.

In 1997, Contag et al. successfully monitored in vivotransient expression of SV40 promoter/enhancer–Lucfusion constructs, using intensified CCD imaging on thelungs of neonatal rats (157). Cationic liposome deliveryof the vectors was used. The expression vector was alsoinduced in mice and in human T-cells.

Endogenous ATP was measured simultaneously withthe release of acetylcholine from the isolated superiorcervical ganglion of a rat using firefly luciferin–luciferase(158). Using imaging of luciferase light emission, Marchet al. (1999) showed the feasibility of catheter-basedpericardial local delivery of adenoviral vectors for genetherapy in dogs (159). One of the recombinant adenoviralexpression vectors encoded luciferase in the cytoplasm.This was visualized post mortem in pericardial tissues insacrificed animals.

Similar imaging precision has been possible in livinganimals. In a landmark study by Edinger et al. (1999),HeLa cells stably expressing firefly luciferase wereintroduced via subcutaneous, intraperitoneal and intra-venous injection into SCID mice (160). Tumour cellkinetics and growth were monitored by whole-bodyphoton-counting visualization. Immediately postinjec-tion, the following tumour cell numbers could beobserved by low-light imaging: 1 � 103 cells in theperitoneal cavity; 1 � 104 cells at subcutaneous sites; and1 � 106 circulating cells.

Using a bispecific antibody, Reynolds et al. (2000)targeted an adenoviral vector encoding firefly luciferaseto the pulmonary endothelium of rats (161). Targeting ledto a 20-fold increase in target area luciferase expression

and a reduction in expression in non-targeted organs.Orson et al. (2000) imaged luciferase expressionlocalized in mice lungs by using an intravenouslyinjected, artificial, lung-targeting macroaggregated poly-ethyleneimine–albumin protein conjugates binding afirefly luciferase cDNA (162).

Sugihara et al. (2000) transfected the chloramphenicolacetyltransferase (CAT) gene and firefly luciferase (luc)expression cassettes by electroporation into the testiclesof living chickens (163). The authors were able to imageluciferase expression in and around the injection site. Aself-replicating sequence of the Epstein–Barr virus wasadded to stabilize luc expression in vivo.

Using a tetracycline-inducible promoter construct,Hasan et al. (2001) have shown that transgenic fireflyluciferase is suitable as a visualizing marker to monitorinduction of the expression of a second transgene inliving mice, in this case, Cre recombinase (164).

Recently, Lipshutz et al. (2001) tested prenatal, inutero delivery of recombinant adeno-associated virus(rAAV) vectors carrying firefly luciferase via intraper-itoneal injection in mice (165). Luciferase expressionwas visualized by whole-body imaging in all injectedanimals. At birth, the highest Luc expression was in theperitoneum and liver, with lower expression levels in theheart, brain and lung. Expression persisted for as long as18 months in the peritoneum. No antibodies against Lucor rAAV were detected and no liver cell damage wasreported. These data suggest that in utero DNA deliveryis a safe and effective method of prenatal gene therapy inanimal models.

Muramatsu et al. (2001) have demonstrated nutrition-ally-regulated transgene expression in mouse liver usinga liver-type phosphoenolpyruvate carboxykinase(PEPCK) gene promoter driving firefly luciferase ex-pression (166). Fasting induced a PEPCK-driven 13-foldincrease of luciferase expression in the liver, but nosimilar induction was found in muscle tissue for either thePEPCK promoter or a control SV40 promoter.

Yu et al. used the ‘Gene Switch’ progesteroneantagonist (RU486)-inducible system co-transformedwith a Renilla luciferase–gfp fusion construct (CMV–ruc–gfp) to visualize inducible gene expression in COScell culture, and also intramuscularly in male nu/nu mice(167). RU486-induced Ruc–GFP expression was visua-lized using low-light imaging.

In short, numerous studies have shown the efficacy ofluciferase imaging in modelling and observing complexgene activation events taking place in vivo in live animalsand in tissue sections.

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In many ways, the ultimate goal of luciferase imaging is



not only to monitor gene expression non-invasively andin real time in living, multicellular organisms, but also toreveal and document spatial, tissue and cell type-specificexpression in genetically altered (transgenic) organisms.Such applications of luciferase imaging have advancedrapidly especially in plants. By 1994, Langridge et al. hadaddressed the usefulness and convenience of using thebacterial luciferase (luxAB) system in a variety ofeukaryotic transgenic organisms and reporter geneapplications (97).

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Global imaging of luciferase-expressing transgenicplants has served further research in many geneexpression studies, from simple expression of luciferasesin transgenic plants, to regulation of developmental genesto the gene expression of wound response, and to thedetection of expression in response to thermo-osmoticstress. Photographic film was used by Ow et al. (1986) tovisualize transient and stable firefly luciferase (Pluc)expression in Nicotiana tabacum (168). In 1986, Konczand Schell imaged tissue-specific chimaeric LuxA andLuxB expression in Daucus carota and Nicotianatabacum (169). By 1987, Koncz et al. (170) haddemonstrated the successful assembly and expression ofLuxA and LuxB from Vibrio harveyi in transgenic carrot(Daucus carota) and tobacco (Nicotiana tabacum) plantsvia Agrobacterium-mediated T-DNA or direct DNAtransformation. Furthermore, Koncz et al. (1990) dis-cussed the advantages and disadvantages of expressingbacterial (Lux) and firefly (Luc) luciferases as reportergenes in transgenic plants (171).

A promoterless luxA gene was fused to the 5� end of aT-DNA, adjacent to a cauliflower mosaic virus 35Spromoter-driven selectable marker, and inserted intotobacco leaf explants to generate transgenic tobaccoplants. Some of these transformed plants were found, bylow-light imaging, to express luciferase in only oneorgan, e.g. a floral corolla. This system thus served as apromoter search assay to find organ-specific promoters(172, cf. 96) (Fig. 7a, b). Sequencing of linked genomicDNA from these plants allowed for the isolation ofdevelopmental genes and their regulatory elements.

By 1994, Mayerhofer et al. had visualized stableRenilla luciferase expression in transgenic tobaccoleaves, tomato fruit, and potato tubers (173). In afollow-up study, Mayerhofer et al. (1995) used Agro-bacterium-mediated transfection to create transgenicalfalfa (Medicago sativa), tomato (Lycopersicon escu-lentum), tobacco (Nicotiana tabacum) and potato (Sola-num tuberosum) plants (Fig. 8) with high levels ofRenilla luciferase (Ruc) (60). The authors found that Rucexpression levels are substantially higher than that ofeither firefly (luc) or bacterial (Lux) luciferase.

Firefly luciferase (luc) was fused with an Arabidopsiscircadian regulator, cab2 (chlorophyll binding protein 2)promoter, and the activity of Cab2 was inferred byvisualizing luc expression both spatially and temporallyin seedlings by low-light video-imaging (1992) (174). Aluc fusion construct carried by transgenic Arabidopsisplants was later (1995) used to identify plants withmutant long- and short-period circadian cycle genotypes,namely plants with mutations in toc1, a gene involved inthe timing of Cab regulation (175). Another group (1998)used luciferase Cab promoter–luciferase fusion vectorsand video-imaging to visualize transgenic expression in

Figure 7. Visualization of the expression levels of abidirectional mas P1,P2 promoter–luxA and luxB gene constructwithin specific tissues of transformed N. tabacum, such as (A)in flowers (non-transformed flower image on the left) and (B) inleaves. Placement of transgene accomplished via Agrobac-terium-mediated T-DNA or direct DNA transformation (172).



tobacco seedlings, to determine how the photoreceptorphytochrome circadian oscillator regulates expression ofcab genes, which drive early seedling development (176).Recently, Schutz and Furuya (2001) monitored Cabsignalling in the cotyledons of Nicotiana tabacum usingcab-luciferase reporter genes (177).

Luciferase expression in transgenic plants has beenused to evaluate imaging equipment and technology.Mutants of transgenic seedlings of Arabidopsis thaliana,containing native promoter-inserted luciferase con-structs, were monitored by low-light imaging (178).When compared, they found that a cooled CCD camerawas more efficient than an intensified CCD camera indetecting mutants in the screen.

Firefly luciferase (luc) under the control of the stress-responsive RD29A promoter was introduced into Arabi-dopsis plants by Ishitani et al. (179, 180). Induction of theendogenous RD29A gene was visualized using highthroughput in vivo luminescence imaging. Under differ-ent conditions, expression of the various stress/osmoticresponse pathways was monitored. These pathwaysincluded those that are both phytohormone abscisic acid(ABA)-dependent and ABA-independent. Further workby Ishitani et al. (1998) established that inducibleluciferase expression in Arabidopsis could be used tofind a temperature cold-response gene (HOS-1) mutant(181).

The general utility of imaging luciferase reporters intransgenic plants has been amply demonstrated, not onlyfor eukaryotic Luc but also for bacterial Lux. Langridgeand Szalay (1998) used low-light intensified photon-counting imaging to visualize bacterial luciferase (Vibrioharveyi) and eukaryotic luciferase (Renilla reniformis)

light emissions as markers for transformation andreporters of gene expression in transgenic Arabidopsisthaliana and Nicotiana tabacum. Both lux and ruc servedas promoter search reporter genes (182). Langridge et al.have also imaged bacterial luciferase expression in thetomato, Lycopersicon esculentum (see Fig. 5a, b), and inthe potato, Solanum tuberosum (97).

The success of luciferase expression in plants has beenextended to unicellular algae. Anthozoan Renilla reni-formis luciferase (Ruc) has been successfully expressedin the chloroplast of the alga Chlamydomonas reinhardtii(183). A cryogenic CCD camera was used to image thelight-emitting transgenic algal colonies.

Firefly luciferase (Luc) imaging has been used inevolutionary studies in Arabidopsis. Jelesko et al. (1999)documented unequal meiotic crossing over in Arabidop-sis thaliana plants with a synthetic rbcsb gene clustercomposed of �rbcs1b::luc–rbcs2b–rbcs3b, instead of thewild-type rbcs1b–rbcs2b–rbcs3b (184). Over 1 millionF2 generation seedlings screened by low-light photon-counting imaging yielded three light-emitting seedlingswith a chimeric �rbcs1b::luc–rbcs3b gene cluster,expressing luciferase and a predicted rbcs26 duplication.These results were confirmed by molecular methods.Luciferase imaging was thus used to assay directly thefrequency of evolutionary gene conversion in A. thaliana(�3 � 10�6).

Xiong et al. (1999a, b) utilized an efficient method ofhigh throughput genetic screening of hormone andenvironmental stress signal transduction mutants ofArabidopsis thaliana, using the firefly luciferase (luc)gene regulated by a cold, osmotic stress and ABA-responsive promoter (185, 186). A thermoelectrically-cooled CCD camera was used to image the plants underaddition of ABA and change of temperature conditions.The system allowed screening and recognition of high-and low-expression mutants.

Luciferase imaging, in addition to being used tovisualize plant promoter–gene response to thermo-osmotic pressures, has also been used to visualize plantgene response to infection. In 1996, Giacomin and Szalayutilized Pseudomonas infection of Arabidopsis thalianato induce expression of the phenylalanine ammonia-lyase(PAL1) promoter fused to the luxF gene (187). In anotherstudy, transgenic Arabidopsis carrying a gst1::luctransgene were used to image the spatial and temporalconcentrations of reactive oxygen intermediates (ROIs)in response to an assault by infectious Pseudomonassyringae pv. tomato (188) (for more details, see thesection on Imaging of host–pathogen interactions,below).

Transgenic seedlings were engineered by Urwin et al.(2000) to carry a bicistronic gene with both GFP andfirefly luciferase ORFs linked by the encephalomyocar-ditis (ECM) IRES element and regulated by the CaMV35S promoter (189). Both GFP and Luc were expressed

Figure 8. Visualization of Renilla luciferase gene expressionin transformed potato tubers (Solanum tuberosum) (cf. 60, 173).



and detected by in vivo imaging, indicating that the ECMIRES element facilitates the translation of the secondORF in vivo. Northern blot analysis also confirmed thepresence of both GFP and luciferase products.

Transgenic tobacco plants (Nicotiana tobacum L.)expressing firefly luciferase (luc) driven by the Arabi-dopsis phenylalanine ammonia lyase 1 (PAL1) promoterhave been imaged in vivo for up to 24 h after the additionof exogenous luciferin (190). Wounding enhanced the lucexpression in these plants, suggesting that there arebarriers in plants to ordinary luciferin uptake intransgenic luciferase assays.

Using a fusion of the GA5 promoter (growthphytohormone gibberellin) and firefly luciferase (ga5–luc) cDNA, Meier et al. (2001) used imaging to show thatGA5 promoter regulation of GA occurs at the level oftranscription (191). Imaging allowed the investigators toidentify recessive mutants with high Pluc expression.

Recently, van Leeuwen and colleagues (2001), usingthe CaMV 35S, modified CaMV 35S and the Arabidopsisthaliana lipid transfer protein gene promoters inconjunction with the firefly luciferase (luc) gene, wereable to show that variant levels of transgene promoteractivation result not only from the integration site(position effect) but also from spatial and temporalpromoter regulation (192). These patterns are inheritedby the next generation. Expression levels were monitoredin leaves of individual transgenic plants during a 50 dayperiod, both by imaging and by assaying local mRNAlevels. Further work has shown that matrix-associatedregions (MAR) elements in proximity to the transgenecause a varying effect on the temporal regulation of thetransgene expression between individual plant transfor-mants (193).

In summary, luciferase imaging of transgenic plantshas thus been used in the study of whole plant geneexpression and regulation, as well as in the screening ofmutants.

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Whole-body imaging of luciferase expression in trans-genic animals has not proceeded as rapidly as in plants,largely because of the greater difficulty of generatingtransgenic animals. However, during the past decade,significant work has been carried out, particularly inimaging of gene expression during development intransgenic embryos.

In 1990, Tamiya et al. first imaged the distribution offirefly luciferase expression in transgenic zebrafish(Brachydanio rerio) (194). Zebrafish have been afavourite transgenic model because zebrafish eggs areeasily accessible to DNA injection. Mayerhofer et al.visualized the spatial distribution of firefly luciferaseexpression in transgenic zebrafish using low-light video-image analysis (195).

Using photon-counting imaging, Matsumoto et al.(1994) found that mouse embryos from transgenicparents carrying the paternally inherited chicken �-actinpromoter–luc construct emitted detectable light at thetwo-cell stage (196). Luciferase mRNA was found evenat the one-cell stage. Thus, luciferase imaging helped todemonstrate the very early onset of embryonic geneexpression.

Expression of secreted Vargula luciferase (Vuc) wasimaged in live pre-implantation mouse embryos fromhomozygous transgenic mice containing luc and vuccDNAs, using image intensifiers connected to a CCDcamera (197). In this way, developmental gene expres-sion modulation could be observed and assessed inindividual embryos by two different luciferases.

Transgenic mice (adults and neonates) with HIV LTR-luc (firefly luciferase) constructs were found to expressluc luminescence after intraperitoneal, topical absorption,or topical electroporative delivery of D-luciferin substratein DMSO (198). Both near the surface and deeper,visualization of luc expression was possible using inten-sified CCD (ICCD) and cooled CCD (cCCD) imaging.

Transgene integration efficiency was determined bybioluminescent visualization screening in microinjectedbovine embryos (199). A murine HSP70.1 promoter waslinked to a firefly luciferase (luc) cDNA and micro-injected into zygote pronuclei produced in vitro. Plucexpression was then visualized in the resulting embryos.

Various promoter/enhancer–Pluc constructs weremicroinjected into pre-implantation bovine embryosand their expression assayed by luminometer andphoton-imaging at 2 and 6 days postinjection (200).These experiments tended to document the persistence ofearly somatic cell promoter activation during embryonicdevelopment.

Utilizing transgenic mice that carry an integratedmurine heme-oxygenase 1 (HO-1)-luc cistron, Zhang etal. (1999) were able to visualize levels of tissueoxygenation in real time using intensified CCD cameraimaging (201). The changing O2 concentrations weretriggered by intraperitoneal injections of CdCl2. Bothhypoxic and hyperoxic tissue conditions altered HO-1promoted luciferase expression.

Taking advantage of the recently discovered fact thatthe Cytomegalovirus immediate–early gene 1 (CMV IE-1) enhancer–promoter is selectively expressed in onlycertain brain cells, Sigworth et al. imaged Pluc expres-sion in brain sections from two lines of transgenic C3H/B6 mice, using a liquid nitrogen-cooled CCD camera(202). One line of mice contained the human CMV::lucfirefly luciferase construct and the other contained thehuman c-fos::luc firefly luciferase construct. TheCMV::luc mice brain sections maintained at 30C or36C showed discrete expression patterns, especially inthe dorsal suprachiasmatic nucleus circadian pacemakerof the hypothalmus.



Making use of an E1-deletion adenovirus expressingPluc under the control of a cytomegalovirus promoter,Wu et al. (2001), utilizing cooled CCD imaging, wereable to visualize the location, magnitude and persistenceof Pluc expression in Swiss Webster mice (203).

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Using photoproteins such as luciferase and GFP to imagethe progression of tumour growth in vivo is an importantand rapidly advancing field (204–206). We summarizeselected papers published recently. Contag et al. (2000)have discussed the use of GFP and firefly luciferase asreporter in real time, in vivo imaging of tumours. Theyalso discussed the use of these reporter genes in studyingcellular and molecular aspects of neoplastic disease,growth and regression under therapy (207). The authorsshow that photon imaging is sensitive enough now todetect 1000 luciferase-labelled tumour cells spreadthroughout a mouse peritoneal cavity. The authors furthernote that, while GFP is adequate for high-resolutionanalyses after tumour localization in vivo, luciferase-labelling is superior in tracing the progress of neoplasticgrowth from a few cells to extensive metastases. Infuture, luciferase reporter monitoring of cancer genetherapy is a suggested application.

In a recent tumour imaging study, Rehemtulla et al.(2000) used 9L rat gliosarcoma cells stably transfectedwith firefly luciferase (9LLuc) to produce orthotopic braintumors (208). Other luciferase-transformed tumour lineshave been used in intraperitoneal, subcutaneous andintravascular models to visualize the kinetics of tumourgrowth and response to therapy. Cooled CCD camera andmagnetic resonance imaging (MRI) showed an excellent0.91 correspondence between imaged photon emissionand MRI-measured tumour volume. In evaluatingchemotherapeutic treatment modalities, CCD and MRIconfirmed each other to a p = 0.951 confidence level. It issignificant and promising that luciferase in vivo imagingcompared so favourably with MRI as a tool for assessingthe spatial extent of in vivo tumours.

In a recent important luciferase imaging study,Honigman et al. tested a number of parameters of invivo imaging in mice and rats (209). After injectingvarious plasmid vectors, recombinant viruses and trans-fected tumour cell-lines (see Table 2), the authors imagedbladder, bone, dermis, liver, muscle, peritoneum, pros-tate, salivary glands, teeth and testis in mice and rats.They used visualization of Luc expression to check organspecificity, efficiency of substrate delivery, long-termmonitoring of tumour growth, promoter specificity, andefficiency of injection methods using image-intensified(ICCD) and cooled (CCCD) charge-couple devicecameras. Location, magnitude and duration of Luc

expression were simply and reproducibly determined byCCCD photon-counting methods. Luminometry servedto monitor Luc activity within organ and cell extracts.

In spite of the remarkable progress made, much moreremains to be done with luciferase visualization oftumours in vivo. Tumour imaging in general is animportant interface between basic research and theclinical applications. Luciferase imaging promises asignificant role in this burgeoning field, perhaps even-tually in the visualization of tumours in humans.

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Many of the marine bioluminescent organisms are foundto exist in symbiotic metazoan–bacterial interactions andso are involved in natural bioluminescent in vivo‘visualization’ (3). However, most of the applied scientificuses of luciferase imaging directed at such interactionshave tended to study host–pathogen relationships.

One of the earliest and most exciting areas of luciferasein vivo imaging has been the real-time visualization ofbacterial infection in living organisms, both plants andanimals. Back in 1986, Legocki and co-workers usedtransformed Bradyrhizobium japonicum carrying luxAB(V. harveyi) under the control of the B. japonicumnitrogenase nifD promoter to monitor the presence of N-fixing bacteria in soybean root nodules, and so indirectlyto visualize N-fixation (Glycine max var. Wilkin) (210).Cell extracts of root nodules grown on plants without N2

were assayed for LuxAB activity by luminometer,indicating nif-driven LuxAB synthesis. LuxAB-trans-formed E. coli bacteria were used as a negative control.LuxAB fusion expression in transformed B. japonicum-infected soybean root nodules was successfully visual-ized using photographic film (Fig. 9a, b) and low-lightintensified video microscopy (211). Using N. tabacumtransformed with an auxin-stimulated bidirectional maspromoter–luxAB gene fusion construct, it was possible tovisualize the spread of Pseudomonas syringiae infectionin N. tabacum leaves (Fig. 10) and of Agrobacterium-induced crown gall in N. tabacum stem sections (Fig. 11)(97).

In 1993 Wang et al. imaged the expression of luxAB-bearing Bacillus thuringiensis within sixth instar larvaeof Manduca sextans (tobacco hornworm) 10 min post-injection and also after larval ingestion of the bacteria(Figure 3a, b) (95). The B. thuringiensis bacteria weretransformed with a plasmid containing luxAB under thecontrol of the XylA promoter (B. megaterium). Arecombinant baculovirus (polyhedrin promoter–luc) wasdeveloped for use as a rapid luminescent plaque assay tooptimize concentrations of recombinant baculoviralinfection in insect cell cultures and larvae (93).



Figure 9. (A) Visualization of transformed Bradyrhizobium-infected soy-bean plant cells (Glycine max) in cross-sections of root nodules through LuxABlight emission (211). (B) Visualization of light emission in root nodules ofsoybean plants Glycine max grown in the absence of N2 in the gross medium.The bacterial inoculant was Bradyrhizobium japonicum, stably transformedwith the nifD promoter–luxAB fusion gene construct (211, cf. 210).



Figure 10. Visualization of the spread of pathogens, Pseudomonas syringiae, in N. tabacum carrying the bidirectionalmas promoter-luxAB gene fusion construct (97, 170).



Expression of Pluc within invading recombinantvaccinia virus has been imaged in African green monkeyBCS-40 kidney cells (212). Time exposure with sensitivefilm was used to capture the images of the light-emittingviral plaques in cell culture. Although not imaged, therecombinant luc-bearing vaccinia virus could be assayedin target organs in BALB/c mice.

Luciferase expression in transgenic Arabidopsis thali-ana transformed with a phenylalanine ammonia-lyasepromoter (PAL1)–luxF fusion was used to imagelocalized activation of PAL1 by infection of Pseudomo-nas syringae pathovar tomato, using photon counting(187). The PAL gene encodes phenylalanine ammonialyase, which catalyses the first step in a plant develop-ment pathway responding to environmental stresses, suchas infectious invasion.

In 1995, Contag et al. showed that bacterial infectionscould be traced non-invasively in vivo in mice usingStaphylococcus typhimurium transformed with a vectorcarrying constitutively expressed bacterial luciferase(lux) (213). Recently, Francis et al. (2000) showed thatStaphylococcus aureus transformed with the rearrangedPhotorhabdus luminescens lux operon (luxABCDE) can

be used to visualize the presence of infection and theeffectiveness of antibiotic treatment by direct whole-body imaging of mice after intramuscular injection of therecombinant bacteria (214). A similar method forStreptococcus pneumoniae has since been developed byFrancis et al. (2001), using a Gram-negative transposonbearing the luxABCDE and the kanamycin resistancegene (kanr) in one promoterless operon allowingtransformed, luminescent, kanamycin-resistant bacteriato be non-invasively visualized in vivo in murine models(215). Rocchetta et al. (2000) have used clinical E. coliEC14 transformed with the P. luminescens lux operon tovisualize bacterial infection in rat thigh muscles, using anintensified CCD camera system (ICCD) (216). Theimaging system was sensitive enough to achieve goodstatistical correlation between luminescence and viablebacterial cell numbers, both with and without thepresence of antimicrobial agents. Rocchetta et al. proposethe use of this technology for in vivo, therapeutic testingof antimicrobial agents.

Viral infection has also been visualized by luciferaseimaging. By using recombinant herpes/pseudorabiesvirus bearing luc, it is possible with ultra-high-sensitivity

Figure 11. Visualization of the auxin-activation of the bidirectional mas promoter-luxAB fusion construct in the stemsections of transgenic N. tabacum, which carry a crown gall tumour caused by the wild type Agrobacterium tumefaciens,strain C58 (93).



photon-counting enhanced video imaging to visualize inreal-time the progress of viral infection and destruction ofmammalian cells in cell culture, even down to single cellresolution, according to Mettenleiter and Grawe (1996)(217) who used recombinant herpes/pseudorabies virus inAfrican green monkey kidney (VERO) cells. Theysuggest that in vivo monitoring of the spread of viralinfection in a living animal is now feasible. This has infact been done in insects. Langridge et al. (1996)successfully imaged not only recombinant baculovirusexpression of firefly luciferase in insect cell culture(Trichoplusia ni) 386 cells and Bombyx mori N-4 and SF9 cells, but also tracked in vivo the progress of a Pluc-transformed baculovirus infection in cabbage looperlarvae (Trichoplusia ni), using low light photon-countingvideo imaging (218).

Digital imaging microscopy has also been used (in2001) to determine the difference in transductionefficiencies on human A549 cells between two recombi-nant adenoviral vectors, AdCMVLuc and Ad5LucRGD(219).

Another recent development has been the direct in vivoimaging of bioluminescent CD4� T cells in a murinemodel (220). In a murine analogue of multiple sclerosis,experimentally-induced autoimmune encephalomyelitis,T lymphocytes transduced to express luciferase by pGCretroviral vectors were visually tracked by low-lightimaging cameras into the central nervous system. Long-

term transgene expression in the central nervous systemwas confirmed by histology.

Recent work has shown that injected bacterial infec-tions of Vibrio, Salmonella and E. coli transformed withan expression construct containing the P. luminescensluxCDABE operon can be visualized over many days inC57 mice (Fig. 12) and Sprague–Dawley rats (221). Low-light imaging was used to visualize bacterial infections inmuscles, in specific organs such as the kidney and liver invivo, and in excised hearts. Luminescent bacteria couldalso be observed through the mouse skull in vivo andthrough excised rat tibia.

Recombinant vaccinia virus (rVV-RG, LIVP strain),bearing a Renilla luciferase–gfp fusion gene under thecontrol of the vaccinia strong synthetic early–latepromoter (rVV–PE/L–ruc–gfp), have been used to imagerVV infection in CV-1 African green monkey kidneycells and in athymic nu/nu mice (222).

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Natural in vivo luciferase ‘visualization’ has taken place inmarine organisms since time immemorial in the oceans ofthe world. Scientific instrumental imaging of luciferaseexpression in living cells, tissues and organisms has madesignificant advances over the last few decades. Thisprogress was only possible because of fundamental

Figure 12. Visualization of intramuscularly-injected bacteria carrying the complete luxCDABE operon from P.luminescens in a C57 mouse (221).



research. The mechanisms of the luciferin–luciferasebioluminescence systems are being elucidated and theirphylogenetic relationships are being worked out. Variousluciferase genes and cDNAs have been isolated andcloned. Advancement in visualization of luciferaseexpression in individual cells, in somatic plant and animaltissues and in transgenic plants and animals has been madepossible because of: (a) recombinant DNA construction ofvarious promoter–luciferase gene constructs and fusiongene products; (b) more efficient and precise delivery oftransgenic DNA and exogenous substrate; and (c)development of highly sensitive and versatile imagingtechnologies. Luciferase has been a useful reporter genefor imaging singly and in concert with other photoproteins,notably green fluorescence protein (GFP). Luciferaseimaging has also begun to play a crucial role in imagingof tumours and metastases. Other possible applicationsinclude the imaging of LRET between luciferases andGFPs in documentation of intracellular protein–proteininteractions. It seems likely that the most significant stepforward, although still in its infancy, is the ability ofluciferase imaging technology to resolve real-time geneexpression in individual cells. Perhaps the imaging of real-time gene expression within individual cells within livingorganisms may be a wave of the future.

� ��

One chimaeric dream of modern science has been toobserve the world without disturbing it. In a small way, invivo, non-invasive, real-time imaging of luciferaseexpression and light emission in living cells andorganisms is at least in the spirit of that dream.

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We would like to thank former and present members ofthe Szalay laboratory, some of whose unpublishedimaging data we have included here; Dr Phil Hill forproviding us with a pre-publication manuscript on thework in his laboratory with intracellular infections ofStaphylococcus aureus; and various members of theSzalay laboratory for their help during the preparation ofthe manuscript, as well as Dr Bruce Wilcox, Dr WilliamLangridge, Dr William Hayes and others who madehelpful suggestions. LG was the recipient of a graduateresearch assistantship from the Department of NaturalSciences, Loma Linda University. The imaging researchin A. A. Szalay’s laboratory was supported by differentgrants cited in earlier publications. The present researchand unpublished images were supported with the help offunding by BSRG research grants provided by LomaLinda University to A. Szalay. We apologize to theauthors whose papers we were not able to include due toour own limitations.

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imaging of light emission from the expression of luciferases in living cells and organisms

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