Optical

molecular

imaging

for

systems

biology

from

molecule

to

organism

The

development

of

highly

efficient

analytical

methods

capable

of

probing

biological

systems

at

system

level

is

an

important

task

that

is

required

in

order

to

meet

the

requirements

of

the

emerging

field

of

systems

biology

Optical

molecular

imaging

-LRB-

OMI

-RRB-

is

a

very

powerful

tool

for

studying

the

temporal

and

spatial

dynamics

of

specific

biomolecules

and

their

interactions

in

real

time

in

vivo

In

this

article

recent

advances

in

OMI

are

reviewed

extensively

such

as

the

development

of

molecular

probes

that

make

imaging

brighter

more

stable

and

more

informative

-LRB-

e.g.

FPs

and

semiconductor

nanocrystals

also

referred

to

as

quantum

dots

-RRB-

the

development

of

imaging

approaches

that

provide

higher

resolution

and

greater

tissue

penetration

and

applications

for

measuring

biological

events

from

molecule

to

organism

level

including

gene

expression

protein

and

subcellular

compartment

localization

protein

activation

and

interaction

and

low-mass

molecule

dynamics

These

advances

are

of

great

significance

in

the

field

of

biological

science

and

could

also

be

applied

to

disease

diagnosis

and

pharmaceutical

screening

Further

developments

in

OMI

for

systems

biology

are

also

proposed

Introduction

Due

to

the

rapid

progress

in

the

field

of

molecular

biology

that

has

occurred

over

the

last

century

we

have

come

to

see

that

life

involves

thousands

of

genes

proteins

metabolites

etc.

However

life

is

not

invoked

by

simply

collecting

these

together

an

integrated

system

formed

from

those

components

is

required

It

is

vital

to

understand

biological

systems

such

as

molecular

networks

cells

tissues

and

even

the

organism

itself

at

the

system

level

This

shift

in

biological

paradigm

from

reductionism

to

integration

has

made

the

field

of

systems

biology

-LSB-

-RSB-

a

new

challenge

that

is

currently

a

hot

topic

Although

attempts

to

systematically

understand

biological

systems

can

be

dated

back

to

Wiener

's

time

-LSB-

-RSB-

research

into

systems

biology

represent

the

first

attempts

to

achieve

this

based

on

detailed

knowledge

of

molecules

It

promises

to

reveal

the

relationships

among

elements

of

systems

that

may

include

just

a

few

proteins

that

together

perform

a

defined

task

or

more

complex

molecular

machines

cells

and

groups

of

cells

with

the

goal

of

understanding

their

emergent

properties

-LSB-

-RSB-

It

is

widely

agreed

that

systems

biology

is

a

large

scientific

field

that

relies

greatly

on

collaborations

between

disciplines

such

as

the

life

sciences

information

science

system

engineering

and

analytical

technology

One

of

the

keys

to

successful

research

in

this

field

is

considered

to

be

work

being

done

in

analytical

chemistry

-LSB-

-RSB-

to

develop

innovative

analytical

methods

that

meet

the

needs

of

systems

biology

research

For

example

capillary

electrophoresis

has

been

recognized

as

being

the

gold

standard

in

genomics

for

DNA

sequencing

Similar

gold

standard

methods

are

available

in

transcriptomics

-LRB-

DNA

microarray

-RRB-

proteomics

-LRB-

multidimensional

separation

coupling

with

mass

spectrometry

-RRB-

and

metabolomics

-LRB-

microseparation

coupled

with

mass

spectrometry

and

nuclear

magnetic

resonance

-RRB-

However

more

of

these

methods

are

needed

As

well

as

revealing

genes

proteins

and

metabolites

from

those

omic

investigations

which

relate

to

the

basic

structures

of

systems

it

is

important

to

have

methods

capable

of

monitoring

their

localizations

connections

and

in

particular

their

dynamics

over

time

under

various

physiological

or

pathological

conditions

Optical

molecular

imaging

-LRB-

OMI

-RRB-

is

a

versatile

technique

that

can

be

used

to

investigate

the

dynamics

of

biological

events

in

molecules

cells

tissues

and

organisms

in

real

time

and

in

vivo

-LRB-

Fig.

-RRB-

-LSB-

-RSB-

Compared

with

other

imaging

approaches

like

magnetic

resonance

imaging

and

positron

emission

tomography

etc.

OMI

exhibits

the

great

advantages

of

high

temporal

-LRB-

picosecond

-RRB-

and

spatial

-LRB-

submicron

-RRB-

resolutions

high

sensitivity

-LRB-

single-molecule

level

-RRB-

and

minimal

invasion

and

shows

high

potential

for

systems

biology

This

review

highlights

recent

advances

in

OMI

focusing

especially

on

the

development

of

fluorescent

probes

such

as

fluorescent

proteins

and

semiconductor

nanocrystals

-LRB-

also

referred

to

as

quantum

dots

QDs

-RRB-

OMI

instrumentation

and

approaches

-LRB-

techniques

-RRB-

The

application

of

OMI

to

studies

of

localization

conformations

and

interactions

of

biomolecules

in

vivo

are

also

highlighted

In

particular

this

review

describes

the

application

of

OMI

for

tracing

the

dynamics

of

single

molecules

and

whole-body

living

organisms

which

strongly

suggest

that

OMI

is

an

informative

systems

biology

method

that

may

be

used

to

uncover

biological

events

at

different

system

levels

The

future

development

of

OMI

is

also

predicted

Fig.

of

OMI

in

life

science

research

OMI

can

investigate

the

dynamics

of

biological

events

in

real

time

from

molecules

-LRB-

left

-RRB-

cells

tissues

and

organisms

-LRB-

right

-RRB-

digitally

and

quantitatively

It

can

handle

a

wide

range

of

intensities

-LRB-

about

orders

of

magnitude

-RRB-

times

-LRB-

femtoseconds

to

years

-RRB-

and

spatial

dimensions

-LRB-

nanometers

to

centimeters

-RRB-

and

it

gives

high

spatial

and

temporal

resolution

of

the

targeted

cellular

structures

better

than

any

other

method

Advances

in

fluorescent

probes

Aequorea

fluorescent

protein

-LRB-

AFP

-RRB-

derived

mutants

Labeling

of

biomolecules

with

fluorescent

probes

or

other

dyes

has

facilitated

in

vitro

or

in

vivo

studies

of

biomolecular

structures

and

dynamics

as

well

as

their

interactions

which

is

critical

if

we

are

to

understand

the

biomolecular

mechanisms

of

cellular

function

However

traditional

methods

of

chemical

labeling

based

on

fluorescent

dyes

are

often

inadequate

for

biomolecular

labeling

repurification

and

reintroduction

into

cells

by

invasive

methods

like

microinjection

These

limitations

have

spawned

efforts

to

noninvasively

and

site-specifically

label

protein

in

living

cells

or

tissues

by

using

green

fluorescent

protein

-LRB-

GFP

-RRB-

and

its

variants

The

fluorescent

protein

presents

a

relatively

small

size

and

a

compact

single-domain

structure

which

allow

it

to

fuse

to

other

targeted

proteins

with

little

or

no

interference

in

native

protein

GFP

was

cloned

from

Aequorea

victoria

Excitation

of

the

wild-type

GFP

by

light

with

a

wavelength

of

nm

can

induce

the

emission

of

bright

green

fluorescence

peaking

at

nm

Through

continuous

efforts

to

perform

protein

mutation

of

GFP

scientists

have

developed

various

kinds

of

AFP

with

different

excitation

and

emission

wavelengths

enhanced

brightness

and

improved

pH

resistance

compared

to

the

original

wide-type

such

as

enhanced

GFP

-LRB-

EGFP

-RRB-

cyan

fluorescent

protein

-LRB-

CFP

-RRB-

and

yellow

fluorescent

protein

-LRB-

YFP

-RRB-

-LRB-

Fig.

-RRB-

-LSB-

-RSB-

The

original

YFP

exhibited

several

drawbacks

when

imaging

in

vivo

-LSB-

-RSB-

Later

generations

of

YFP

mutants

including

Citrine

-LSB-

-RSB-

and

Venus

-LSB-

-RSB-

exhibit

low

pH

-LRB-

-RRB-

resistance

halide

insensitivity

greatly

improved

photostability

brighter

fluorescence

and

faster

maturation

The

original

generation

of

CFP

mutants

also

displayed

several

spectroscopic

disadvantages

By

using

similar

methods

to

YFP

mutagenesis

a

CFP

mutant

named

Cerulean

-LSB-

-RSB-

was

obtained

with

an

improved

quantum

yield

a

higher

extinction

coefficient

and

a

longer

fluorescence

lifetime

Another

new

color

GFP

mutant

cyan-green

fluorescent

protein

-LRB-

CGFP

-RRB-

-LSB-

-RSB-

whose

excitation

and

emission

wavelengths

are

intermediate

between

those

of

CFP

and

EGFP

was

also

found

by

using

this

tactic

In

addition

PA-GFP

a

GFP

mutant

has

times

greater

fluorescence

than

the

original

and

remains

stable

for

days

-LSB-

-RSB-

These

fluorescent

proteins

were

termed

molecular

or

optical

highlighters

and

represent

perhaps

the

most

promising

tools

for

investigating

protein

lifetimes

transport

turnover

rates

and

so

on

Fig.

cVarious

FP

mutants

a

Introduction

of

the

mutation

of

in

GFP

results

in

significantly

red-shifted

maximum

excitation

and

emission

wavelengths

this

mutant

is

named

YFP

b

By

using

mutagenesis

the

original

tetrameric

DsRed

is

reconstructed

into

the

monomeric

DsRed

variant

c

Interchanging

the

amino

and

carboxyl

portions

of

GFP

and

rejoining

them

with

a

short

spacer

generates

cpGFP

Other

kinds

of

fluorescent

protein

Long-wavelength

fluorescence

can

provide

greater

tissue

penetration

and

better

spectral

separation

from

cellular

autofluorescence

Therefore

red

fluorescent

proteins

-LRB-

RFP

-RRB-

are

very

useful

in

applications

related

to

multicolor

protein-tracking

and

the

construction

of

sensors

The

first

RFP

with

a

red

emission

wavelength

was

cloned

from

Discosoma

genus

named

DsRed

-LSB-

-RSB-

Its

structure

is

similar

to

GFP

but

it

is

tetrameric

By

using

mutagenesis

different

derivations

exhibiting

several

advantages

have

been

generated

such

as

the

fluorescent

timer

of

DsRed

which

has

fluorescence

that

changes

from

green

to

red

over

time

-LSB-

-RSB-

the

monomeric

DsRed

named

-LSB-

-RSB-

-LRB-

Fig.

-RRB-

as

well

as

-LSB-

-RSB-

-LSB-

-RSB-

and

mCherry

-LSB-

-RSB-

which

have

chromophores

that

are

brighter

more

soluble

and

mature

faster

than

the

original

generation

Another

orange-emitting

fluorescent

protein

which

has

higher

pH-resistance

and

is

more

effective

for

imaging

was

discovered

in

Fungia

concinn

-LSB-

-RSB-

In

addition

Kaede

-LSB-

-RSB-

which

is

cloned

from

Trachyphyllia

geoffroyi

can

convert

from

green

to

red

fluorescence

and

brightens

under

UV

illumination

New

rearrangement

variants

of

fluorescent

protein

In

previous

studies

fluorescent

proteins

were

treated

as

an

indivisible

entity

usually

appended

to

the

amino

or

carboxyl

terminus

of

the

targeted

protein

However

several

rearrangements

of

GFPs

in

which

the

amino

and

carboxyl

portions

were

interchanged

and

rejoined

with

a

short

spacer

still

remained

fluorescent

-LRB-

Fig.

-RRB-

These

circular

permutations

exhibit

several

advantages

such

as

altered

pKa

values

orientations

of

the

chromophore

with

respect

to

a

fusion

partner

and

greater

absorbance

of

the

exciting

energy

-LSB-

-RSB-

Nongenetic

fluorescent

probes

However

there

are

some

disadvantages

of

using

FPs

to

perform

OMI

of

the

whole-body

Given

the

available

wavelengths

of

excitation

and

emission

and

the

power

of

the

FP

optical

imaging

with

a

depth

of

penetration

of

approximately

mm

limits

their

use

to

C.

elegans

Drosophila

or

surface

structures

in

small

animals

To

solve

these

problems

applying

QDs

-LRB-

quantum

dots

-RRB-

to

OMI

of

the

whole-body

could

allow

us

to

visualize

biological

events

in

deeper

tissues

with

better

image

quality

Compared

with

conventional

organic

dyes

and

fluorescent

proteins

semiconductor

nanocrystals

quantum

dots

exhibit

a

narrow

tunable

symmetric

emission

spectrum

and

better

photochemical

stability

Also

their

wavelengths

of

maximum

excitation

and

emission

shift

to

shorter

wavelengths

with

decreasing

size

-LSB-

-RSB-

QDs

present

several

great

advantages

for

whole-body

OMI

such

as

their

very

large

molar

extinction

coefficients

and

their

very

bright

emission

which

mean

that

the

emission

can

be

visualized

in

big

animals

-LSB-

-RSB-

the

ability

to

perform

simultaneous

multiple

color

imaging

of

targets

of

interest

-LSB-

-RSB-

and

long

excited

state

lifetimes

that

provide

a

way

to

separate

the

QD

fluorescence

from

background

fluorescence

-LSB-

-RSB-

Because

QDs

are

capped

with

a

monolayer

of

organic

ligands

and

are

hydrophobic

they

can

not

be

used

for

visualization

in

vivo

Goldman

et

al.

-LSB-

-RSB-

reported

that

QDs

capped

with

fusion

proteins

of

a

specific

antibody

protein

G

and

leucine

zipper

adaptor

protein

could

be

used

to

image

the

target

protein

in

vivo

-LRB-

Fig.

-RRB-

Since

then

bioconjugated

QDs

have

provided

new

approaches

for

the

ultrasensitive

and

multiple-color

imaging

of

targeted

molecules

in

vivo

In

addition

integrating

QDs

with

paramagnetic

substances

results

in

new

multimodality

imaging

probes

that

cuple

the

deep

imaging

capability

of

magnetic

resonance

imaging

with

ultrasensitive

OMI

-LSB-

-RSB-

these

would

be

very

powerful

probes

if

used

in

clinical

research

More

recently

a

new

QD

conjugate

was

found

to

emit

long

waves

in

the

absence

of

external

excitation

light

through

bioluminous

resonance

energy

transfer

-LSB-

-RSB-

Compared

with

existing

quantum

dots

self-luminous

quantum

dots

provide

great

sensitivity

when

imaging

small

animals

giving

high

signal-to-noise

ratios

Fig.

of

conjugating

QDs

to

target

proteins

The

pG-zb

acts

as

a

molecular

adaptor

connecting

the

QDs

with

the

target

protein

through

interactions

of

its

protein

G

portion

with

a

specific

antibody

as

well

as

interactions

of

its

positively

charged

tail

with

QDs

capped

with

a

negatively

charged

dihydrolipoic

acid

surface

OMI

techniques

General

imaging

microscopy

Laser

scanning

confocal

microscopy

-LRB-

LSCM

-RRB-

-LRB-

Fig.

-RRB-

and

wide-field

microscopy

-LRB-

WFM

-RRB-

are

the

tools

most

widely

used

for

in

vivo

OMI

The

major

difference

between

them

is

that

LSCM

only

collects

the

fluorescence

emission

of

in-focus

light

whereas

WFM

collects

all

signals

including

out-of-focus

light

-LSB-

-RSB-

LSCM

can

produce

superior

images

of

multiple-cell

samples

with

photobleaching

However

LSCM

can

not

perform

multiple-color

imaging

because

it

only

observes

in-focus

light

-LSB-

-RSB-

WFM

is

among

the

most

sensitive

of

all

such

methods

permits

minimal

exposure

of

the

sample

uniform

illumination

an

unlimited

choice

of

excitation

wavelengths

and

is

relatively

simple

-LSB-

-RSB-

One

of

the

disadvantages

of

WFM

is

that

disturbances

are

seen

in

observations

of

thicker

specimens

where

the

out-of-focus

signals

become

substantial

-LSB-

-RSB-

Fig.

fFluorescent

OMI

approaches

a

LSCM

only

collects

in-focus

emitted

light

b

The

principle

of

multiple

photon

excitation

is

based

on

the

use

of

pulsed

long

excitation

wavelengths

to

excite

fluorescence

c

FRET

occurs

between

a

donor

and

an

acceptor

that

are

in

molecular

proximity

if

the

emission

spectrum

of

the

donor

overlaps

the

excitation

spectrum

of

the

acceptor

d

FRAP

can

reveal

the

mobility

of

FP-labeling

proteins

These

images

illustrate

the

change

in

fluorescence

of

cells

expressed

with

YFP-hGR

before

and

after

photobleaching

Reproduced

from

-LSB-

-RSB-

with

permission

e

FLIM

can

measure

the

time-dependent

emission

intensity

The

histogram

represents

the

fluorescence

lifetime

distributions

for

the

donor

in

the

presence

of

interactions

-LRB-

red

-RRB-

or

not

-LRB-

yellow

-RRB-

Reproduced

from

-LSB-

-RSB-

with

permission

f

FCS

can

monitor

the

fluorescence

signals

emitted

from

the

ROI

The

cross-correlation

curve

-LRB-

black

-RRB-

indicates

a

higher

level

of

dimer

or

oligomer

formation

in

the

and

cells

Reproduced

from

-LSB-

-RSB-

with

permission

Furthermore

multiple-photon

laser

scanning

microscopy

-LRB-

MPLSM

-RRB-

has

also

been

used

in

OMI

-LSB-

-RSB-

The

principle

of

MPLSM

is

that

pulsed

long

excitation

wavelength

light

such

as

that

provided

by

an

infrared

-LRB-

IR

-RRB-

laser

beam

is

used

to

excite

a

molecule

at

the

focal

plane

by

multiple

photons

causing

fluorescence

-LRB-

Fig.

-RRB-

The

advantages

of

MPLSM

are

high

tissue

penetration

ability

-LRB-

μm

-RRB-

low

photobleaching

or

photodamage

and

a

high

signal/noise

ratio

-LRB-

due

to

low

autofluorescence

-RRB-

-LSB-

-RSB-

Second-harmonic

imaging

microscopy

-LRB-

SHIM

-RRB-

SHIM

is

based

on

a

nonlinear

optical

effect

called

second

harmonic

generation

-LRB-

SHG

-RRB-

commonly

called

frequency

doubling

This

phenomenon

requires

that

intense

laser

light

passes

through

a

highly

polarizable

material

with

noncentrosymmetric

molecular

organization

-LSB-

-RSB-

Biological

macromolecules

often

assemble

into

large

ordered

noncentrosymmetric

structures

and

have

highly

polarizability

Therefore

high-resolution

SHIM

imaging

can

visualize

living

cells

or

tissues

This

method

provides

several

benefits

the

targeted

molecules

are

not

excited

photobleaching

does

not

occur

and

it

can

detect

information

related

to

pathology

In

addition

a

combination

of

SHIM

and

MPLSM

could

make

a

powerful

optical

imaging

technique

Optical

coherence

tomography

-LRB-

OCT

-RRB-

The

principle

of

OCT

imaging

is

analogous

to

that

of

ultrasound

B-mode

imaging

except

that

OCT

uses

light

rather

than

acoustic

waves

-LSB-

-RSB-

OCT

can

provide

cross-sectional

imaging

of

structures

below

the

tissue

surface

analogous

to

histopathology

By

using

a

state-of-the-art

laser

as

the

light

source

ultrahigh-resolution

imaging

with

axial

resolutions

as

fine

as

μm

can

be

achieved

-LSB-

-RSB-

However

the

penetration

depth

of

the

technique

in

most

tissues

is

limited

to

approximately

mm

-LSB-

-RSB-

Several

features

of

OCT

imaging

make

it

well-suited

to

imaging-based

diagnostics

and

surgical

guidance

such

as

its

high

resolution

its

ability

to

quantitatively

assess

and

its

ability

to

image

some

tissue

functions

Fluorescent

molecular

imaging

approaches

Fluorescence

resonance

energy

transfer

-LRB-

FRET

-RRB-

FRET

is

a

quantum

mechanical

phenomenon

that

occurs

between

a

fluorescence

donor

and

a

fluorescence

acceptor

with

a

favorable

dipole

dipole

orientation

that

are

in

molecular

proximity

to

each

other

provided

the

emission

spectrum

of

the

donor

overlaps

the

excitation

spectrum

of

the

acceptor

-LRB-

Fig.

-RRB-

-LSB-

-RSB-

Therefore

imaging

based

on

FRET

can

determine

the

proximity

-LRB-

within

the

nanometer

range

-RRB-

between

labeled

biomolecules

in

living

cells

-LSB-

-RSB-

FRET-based

microscopy

includes

intensity-based

detection

methods

and

fluorescence

decay

kinetics-based

detection

methods

-LSB-

-RSB-

Similar

to

general

fluorescence

imaging

microscopy

FRET

also

suffers

from

various

drawbacks

like

autofluorescence

detector

noise

optical

noise

and

photobleaching

In

addition

spectral

bleed-through

-LRB-

SBT

-RRB-

or

cross-talk

is

a

major

problem

in

FRET

-LSB-

-RSB-

FRET

is

currently

widely

used

in

studies

of

protein

colocalization

conformational

changes

protein

interactions

and

signal

transduction

-LSB-

-RSB-

Fluorescence

recovery

after

photobleaching

-LRB-

FRAP

-RRB-

FRAP

involves

observing

the

rate

of

recovery

of

fluorescence

resulting

from

the

movement

of

a

fluorescent

marker

into

an

area

which

contains

the

same

marker

that

has

been

rendered

nonfluorescent

via

an

intense

photobleaching

pulse

of

laser

light

-LRB-

Fig.

-RRB-

FRAP

has

proved

to

be

a

powerful

method

for

measuring

the

mobilities

of

target

molecules

in

various

membranes

cytoplasms

and

nuclei

-LSB-

-RSB-

For

example

the

mobility

of

the

hormone

receptor

in

the

nucleus

of

a

living

cell

-LSB-

-RSB-

the

mobility

in

subcellular

compartments

and

the

dynamics

of

vesicles

inside

the

synapses

of

cultured

hippocampal

neurons

-LSB-

-RSB-

have

been

studied

using

this

approach

Fluorescence

lifetime

imaging

microscopy

-LRB-

FLIM

-RRB-

FLIM

is

a

technique

by

which

the

mean

fluorescence

lifetime

of

a

chromophore

is

measured

at

each

spatially

resolvable

element

of

a

microscope

image

-LSB-

-RSB-

The

fluorescence

lifetime

is

an

inherent

property

of

a

chromophore

that

is

sensitive

to

environmental

and

physical

processes

Therefore

FLIM

can

detect

interesting

physical

processes

that

can

influence

the

excited

state

of

a

probe

in

vivo

-LSB-

-RSB-

In

addition

the

mean

lifetime

of

a

donor

is

shifted

to

shorter

lifetimes

in

the

presence

of

an

acceptor

in

FRET

-LRB-

Fig.

-RRB-

so

FLIM

can

detect

changes

in

FRET

-LSB-

-RSB-

although

this

may

not

be

valid

in

complex

cellular

systems

that

consist

of

several

different

lifetime

components

-LSB-

-RSB-

For

example

using

a

FRET

FLIM

method

the

regulation

of

the

activity

of

receptor

protein

tyrosine

kinase

-LRB-

RPTK

-RRB-

through

its

dimerization

was

demonstrated

in

living

cells

-LSB-

-RSB-

The

main

drawbacks

of

FLIM

are

that

it

has

relatively

low

sensitivity

and

requires

expensive

instrumentation

Fluorescence

correlation

spectroscopy

-LRB-

FCS

-RRB-

FCS

is

an

approach

that

has

emerged

only

recently

It

is

a

high-sensitivity

photon-counting

technique

that

permits

fluctuations

in

the

fluorescence

signal

due

to

changes

in

the

fluorescence

quantum

yield

to

be

measured

-LRB-

Fig.

-RRB-

-LSB-

-RSB-

Therefore

FCS

can

directly

measure

the

mobilities

of

biomolecules

and

it

can

monitor

the

average

association

and

dissociation

of

target

labeled

with

fluorescence

probe

which

vary

with

interaction

kinetics

complex

composition

and

complex

size

in

vivo

without

photobleaching

For

example

FCS

can

be

used

to

detect

the

clustering

of

somatostatin

receptors

-LSB-

-RSB-

and

the

binding

affinities

of

glucocorticoids

-LRB-

GC

-RRB-

in

different

subcellular

compartments

-LSB-

-RSB-

Applications

of

OMI

OMI

for

biomolecules

Originally

OMI

of

biomolecule

function

was

performed

on

dead

fixed

cells

or

tissue

sections

in

vitro

The

first

application

of

fluorescent

detection

in

situ

emerged

in

when

RNA

that

was

directly

labeled

with

fluorophore

was

used

as

a

probe

for

specific

DNA

sequences

-LSB-

-RSB-

However

this

method

is

not

sensitive

enough

Another

indirect

detection

method

allows

signal

to

be

significantly

increased

by

binding

secondary

reporter

to

the

hybridization

probes

that

bind

to

targeted

DNA

-LSB-

-RSB-

and

mRNA

-LSB-

-RSB-

Recently

Santangelo

et

al.

-LSB-

-RSB-

indicated

that

coinjection

of

two

beacons

based

on

FRET

could

be

used

to

map

the

localization

of

a

specific

hybridization

in

living

cells

Another

application

of

FISH

is

immunocytochemistry

-LRB-

IC

-RRB-

which

is

used

to

detect

target

protein

in

specimens

in

vitro

as

antigen

by

means

of

antibodies

labeled

with

fluorescence

probe

-LSB-

-RSB-

The

technique

of

FISH

is

still

being

developed

with

more

specific

fine-tuning

of

sensitivity

and

multiplicity

necessary

after

which

its

applications

should

gradually

move

from

being

in

vitro

to

in

vivo

-LSB-

-RSB-

In

addition

Fourier

transform

infrared

-LRB-

FT

IR

-RRB-

spectroscopy

allows

the

simultaneous

detection

of

nearly

all

organic

molecules

in

a

single

spectrum

and

hence

is

ideally

suited

for

the

investigation

of

complex

metabolic

pathways

However

this

advantage

is

only

useful

when

powerful

mathematical

methods

are

available

for

the

analysis

of

the

highly

complex

spectra

produced

Glycolytic

intermediates

In

yeast

extract

were

identified

with

FT

IR

spectroscopy

The

results

were

in

good

agreement

with

known

phase

relationships

in

oscillatory

glycolysis

-LSB-

-RSB-

In

another

experiment

glycogen

protein

lipid

and

nucleic

acid

concentrations

were

monitored

in

fish

liver

-LSB-

-RSB-

using

this

method

Single-molecule

OMI

is

a

very

young

field

that

holds

great

promise

It

is

not

a

technique

but

a

way

of

thinking

that

can

allow

us

to

detect

individual

molecular

interactions

protein

dynamics

and

signaling

transduction

pathways

in

living

cells

which

is

difficult

and

sometimes

impossible

to

achieve

using

conventional

techniques

-LSB-

-RSB-

For

example

Sonnleitner

et

al.

observed

that

the

voltage-gated

ion

channel

does

not

directly

open

or

close

under

the

conditions

of

single-molecular

interaction

using

this

approach

-LSB-

-RSB-

Karymov

et

al.

reported

a

method

that

could

be

used

to

directly

follow

junction

branch

migration

in

the

holiday

junction

at

the

single-molecule

level

and

they

detected

that

branch

migration

was

a

stepwise

random

process

where

the

overall

kinetics

were

dependent

on

the

concentration

-LSB-

-RSB-

Using

OMI

to

monitor

physiological

processes

in

living

cells

Monitoring

gene

expression

and

RNA

localization

The

fusion

of

gene

promoter

and

fluorescent

protein

cDNA

can

be

used

to

noninvasively

analyze

the

expression

pattern

of

a

target

gene

which

is

a

very

common

approach

used

to

study

gene

expression

in

vivo

and

is

widely

used

in

life

science

research

To

minimize

the

accumulation

of

the

background

fluorescence

that

is

generated

by

leaky

undegraded

level

-LRB-

i.e.

from

previously

expressed

fluorescent

protein

that

has

not

yet

degraded

-RRB-

several

chimeric

EGFPs

with

shorter

fluorescence

half-lives

were

constructed

by

fusing

protein

-LSB-

-RSB-

However

this

approach

leads

to

lower

sensitivity

and

can

not

reveal

low

levels

of

expression

To

overcome

these

drawbacks

a

new

approach

that

is

similar

to

the

amplification

of

an

enzymatic

reporter

was

applied

which

was

then

used

in

enhancer-trap

strategies

-LSB-

-RSB-

The

principle

behind

this

is

that

the

fluorescence

of

FP

revealing

targeted

gene

expression

in

different

tissues

is

magnified

through

a

transcript

factor

which

allows

weak

signals

to

be

seen

In

addition

DNA

and

RNA

sequences

and

other

molecules

could

be

visualized

indirectly

by

labeling

their

binding

proteins

with

FP

in

vivo

For

example

by

using

RNA

binding

protein

labeled

with

GFP

the

mobility

of

targeted

RNA

could

be

visualized

in

vivo

-LSB-

-RSB-

In

another

study

a

similar

method

was

used

to

observe

gene

expression

correlated

with

a

change

in

chromatin

structure

under

the

progress

of

transcription

in

real

time

-LSB-

-RSB-

Compared

with

FISH

in

vivo

the

disadvantage

of

this

method

is

the

introduction

of

a

protein

complex

of

considerable

size

Monitoring

protein

and

subcellular

organelle

dynamics

FP

can

also

be

used

to

monitor

the

behavior

of

the

targeted

protein

such

as

its

appearance

degradation

localization

translocation

and

interaction

in

vivo

For

example

by

using

a

fusion

protein

of

GR

and

FP

the

dynamics

of

the

exchange

of

GR

with

its

binding

sequence

array

can

be

monitored

-LSB-

-RSB-

In

addition

visualization

of

the

dynamics

of

the

GFP-GR

fusion

protein

in

vivo

in

more

physiological

conditions

was

achieved

by

using

GFP-GR

knock-in

mice

-LSB-

-RSB-

Another

interesting

application

involves

identifying

the

localization

of

an

unknown

protein

on

a

large

scale

a

method

termed

the

protein

trap

in

which

the

imaging

of

cells

containing

the

fusion

protein

of

FP

and

a

cDNA

library

was

used

to

screen

for

target

protein

at

the

localization

of

interest

-LSB-

-RSB-

Fusion

protein

that

targets

a

given

subcellular

organelle

is

often

used

to

study

the

dynamics

of

subcellular

organelles

For

example

Shaw

et

al.

-LSB-

-RSB-

revealed

the

dynamics

of

migration

across

the

cell

cortex

of

individual

cortical

microtubules

in

Arabidopsis

by

using

tubulin

fused

to

FP

In

a

similar

study

this

method

was

used

to

measure

the

rates

of

tubulin

polymer

growth

shortening

and

transition

-LSB-

-RSB-

Furthermore

new

subcellular

structures

can

also

be

discovered

by

visualizing

fusion

FP

such

as

the

novel

discrete

area

in

the

nucleus

in

which

phytochrome

species

were

induced

to

accumulated

by

light

-LSB-

-RSB-

Monitoring

general

protein

protein

interactions

Detecting

the

proximity

of

two

biomolecules

based

on

FRET

is

an

approach

widely

used

to

observe

protein

interactions

in

real

time

in

vivo

Most

commonly

CFP

and

YFP

are

respectively

fused

to

each

of

their

putative

interaction

proteins

which

are

coexpressed

in

one

living

cell

so

that

FRET

between

two

fused

FPs

of

interaction

can

be

detected

-LRB-

Fig.

-RRB-

A

variety

of

protein

interactions

in

different

cells

have

been

successfully

visualized

so

far

using

intermolecular

FRET

such

as

the

interaction

between

phytochrome

B

and

cryptochrome

-LSB-

-RSB-

the

rearrangement

of

G-protein

subunits

-LSB-

-RSB-

and

the

oligomeric

states

of

the

ligands

and

-LSB-

-RSB-

Protein

protein

interactions

can

also

be

imaged

via

protein

complementation

assays

The

putative

interaction

proteins

are

respectively

fused

to

two

complementary

fragments

of

one

fluorescent

protein

and

then

interaction

of

the

proteins

can

reinvoke

the

fluorescence

-LSB-

-RSB-

in

a

technique

which

is

analogous

to

yeast

two-hybrid

assays

-LRB-

Fig.

-RRB-

Using

this

method

Hu

et

al.

simultaneously

visualized

several

interactions

among

bZIP

and

Rel

family

transcription

factors

in

one

cell

-LSB-

-RSB-

Fig.

dGeneral

designs

of

FRET-based

fluorescent

probes

a

An

intermolecular

probe

consists

of

two

interacting

proteins

that

are

labeled

with

CFP

and

YFP

respectively

which

interact

and

result

in

FRET

b

An

intramolecular

probe

consists

of

CFP

and

YFP

fused

together

with

a

cleavable

linker

or

protein

which

can

be

cleaved

by

proteolysis

and

disrupt

FRET

c

An

intramolecular

probe

consists

of

sandwiching

two

domains

between

CFP

and

YFP

which

can

interact

after

phosphorylation

or

binding

to

calcium

resulting

in

a

change

in

FRET

d

An

intramolecular

probe

consists

of

CFP

YFP

and

a

protein/domain

which

permits

conformational

change

by

binding

to

another

biomolecule

leading

to

a

change

in

FRETFig

dSingle

FP-based

fluorescent

probes

a

The

probe

consists

of

the

fusion

of

two

interacting

proteins

to

two

complementary

fragments

of

one

FP

respectively

which

can

interact

and

reinvoke

the

fluorescence

b

Insertion

of

a

conformationlly

responsive

domain/protein

into

cpYFP

can

lead

to

a

change

of

fluorescence

when

its

conformation

is

changed

c

The

probe

consists

of

the

fusion

of

two

interacting

proteins/domains

to

the

amino

and

carboxyl

termini

of

cpGFP

which

can

interact

and

change

the

cpGFP

fluorescence

d

By

using

mutagenesis

AFP

can

be

engineered

to

be

directly

sensitive

to

a

small

molecule

such

as

Cl

H

Monitoring

protease

and

kinase

activity

The

first

reporter

of

proteolysis

consists

of

BFP

and

GFP

fusing

together

with

a

protease-sensitive

linker

which

exhibits

the

FRET

phenomenon

Proteolysis

can

disrupt

the

FRET

by

separating

the

donor

and

acceptor

fluorescent

proteins

-LSB-

-RSB-

In

recent

studies

fusion

proteins

of

CFP

YFP

and

a

specific

recognition

sequence

were

used

to

measure

the

different

caspase

proteolytic

activities

during

different

apoptosis

pathways

-LRB-

Fig.

-RRB-

-LSB-

-RSB-

In

another

study

YFP-Bid-CFP

fusion

protein

was

used

to

visualize

the

activation

of

Bid

protein

by

proteolytic

cleavage

and

translocation

of

the

cleaved

Bid

to

mitochondria

was

observed

directly

-LSB-

-RSB-

Reporters

for

the

activity

of

tyrosine

kinases

and

serine/threonine

kinases

have

been

made

by

sandwiching

a

substrate

peptide

for

the

kinase

of

interest

and

a

phosphoaminoacid-binding

domain

such

as

-LRB-

-RRB-

or

protein

between

two

FPs

-LRB-

Fig.

-RRB-

Phosphorylation

of

the

substrate

peptide

induces

the

formation

of

an

intramolecular

complex

with

the

phosphoaminoacid-binding

domain

which

leads

to

a

change

in

the

FRET

Meanwhile

these

indicators

also

report

the

opposite

phosphatase

activity

This

generic

concept

has

so

far

been

adapted

to

create

probes

for

the

phosphorylation

of

the

kinase

of

interest

in

different

signal

transduction

pathways

including

phosphorylation

of

protein

kinase

A

-LRB-

PKA

-RRB-

-LSB-

-RSB-

activation

of

serine/threonine

kinase

Akt

-LSB-

-RSB-

and

activation

of

Src

kinase

on

the

cell

membrane

-LSB-

-RSB-

Monitoring

changes

in

calcium

Genetically

encoded

fluorescent

indicators

for

calcium

without

cofactors

that

can

target

to

specific

intracellular

locations

were

first

constructed

by

Miyawaki

et

al.

-LSB-

-RSB-

This

indicator

was

termed

cameleon

The

cameleon

consists

of

the

tandem

fusion

of

CFP

calmodulin

the

calmodulin-binding

peptide

and

YFP

Binding

of

allows

calmodulin

to

wrap

around

the

domain

which

increases

the

FRET

between

flanking

FPs

-LRB-

Fig.

-RRB-

Replacing

YFP

with

cpYFP

-LSB-

-RSB-

Venus

-LSB-

-RSB-

or

EYFP

-LSB-

-RSB-

led

to

a

new

generation

of

cameleon

that

exhibited

better

spatial

and

temporal

resolution

as

well

as

better

environmental

resistance

In

order

to

measure

high

concentrations

of

the

CaM

interface

of

cameleon

was

re-engineered

to

reduce

its

affinity

to

which

improved

the

imaging

of

in

the

endoplasmic

reticulum

-LSB-

-RSB-

Other

indicators

for

calcium

that

are

not

based

on

FRET

have

also

been

reported

cpGFP

or

cpYFP

can

tolerate

the

insertion

of

another

entire

protein

and

conformational

change

of

the

fusion

cpFP

can

change

its

fluorescence

Therefore

the

insertion

of

calmodulin

into

cpYFP

-LRB-

called

camgaroos

-RRB-

-LRB-

Fig.

-RRB-

-LSB-

-RSB-

and

the

tandem

fusion

protein

of

cpGFP

and

calmodulin

-LRB-

termed

G-CaMP

-RRB-

-LRB-

Fig.

-RRB-

-LSB-

-RSB-

have

both

been

used

as

calcium

indicators

because

a

conformational

change

occurs

in

the

fusion

cpFPs

upon

calcium

binding

Monitoring

changes

in

cyclic

nucleotides

The

first

fluorescent

sensor

of

cAMP

consists

of

cAMP-dependent

protein

kinase

-LRB-

PKA

-RRB-

in

which

the

catalytic

subunit

and

the

regulatory

subunit

are

labeled

with

a

fluorescein

and

a

rhodamine

respectively

Binding

of

cAMP

to

the

labeled

regulatory

subunit

leads

to

the

dissociation

of

the

labeled

catalytic

subunit

which

disrupts

the

FRET

between

the

two

fluorescent

dyes

-LSB-

-RSB-

Later

this

system

was

developed

into

a

genetic

cAMP

indicator

with

BFP

and

GFP

replacing

the

fluorescent

dyes

-LSB-

-RSB-

The

most

recent

cAMP

indicator

was

constructed

by

sandwiching

a

full

length

between

CFP

and

YFP

-LRB-

Fig.

-RRB-

-LSB-

-RSB-

A

genetically

encoded

indicator

for

another

cyclic

nucleotide

cGMP

has

been

reported

by

Sato

et

al.

-LSB-

-RSB-

This

indicator

consists

of

a

tandem

fusion

of

BFP

nondimerizing

mutants

of

cGMP-dependent

protein

kinase

Iα

-LRB-

PKG

Iα

-RRB-

and

GFP

An

increase

in

FRET

between

the

two

FPs

can

be

detected

upon

the

cGMP-induced

conformational

change

of

PKG

Iα

Another

sensor

termed

-LSB-

-RSB-

was

constructed

to

have

high

selectivity

for

cGMP

over

cAMP

Here

the

truncation

of

PKG

Iα

consisted

of

residues

rather

than

reported

in

a

previous

study

-LSB-

-RSB-

and

it

exhibited

decreased

FRET

fluorescence

upon

the

binding

of

cGMP

Monitoring

changes

in

transmembrane

voltage

Measuring

electrical

activity

in

living

cells

with

high

spatial

and

temporal

resolution

is

a

fundamental

problem

in

studies

of

excited

cell

information

processing

To

address

this

problem

Siegel

and

Isacoff

-LSB-

-RSB-

first

constructed

a

novel

fluorescent

probe

that

could

be

used

to

measure

transmembrane

voltage

in

vivo

termed

the

fluorescent

shaker

or

FlaSh

in

which

the

modified

GFP

was

inserted

into

a

voltage-sensitive

K

channel

so

that

voltage-dependent

rearrangements

in

the

K

channel

fusion

protein

could

induce

changes

in

the

GFP

fluorescence

A

similar

voltage

sensor

was

generated

by

inserting

GFP

into

another

channel

the

rat

μI

skeletal

muscle

voltage-gated

channel

and

was

named

SPARC

-LSB-

-RSB-

In

other

studies

Sakai

et

al.

-LSB-

-RSB-

developed

a

new

voltage-sensitive

indicator

based

on

FRET

in

which

changes

in

membrane

voltage

can

lead

to

changes

in

FRET

Monitoring

changes

in

pH

in

living

cells

The

chromophore

of

a

FP

is

surrounded

by

a

hydrogen-bonding

network

within

the

β-barrel

which

means

that

the

FP

is

pH-sensitive

because

of

direct

interactions

between

its

hydrogen-bonding

network

and

external

protons

-LSB-

-RSB-

The

majority

of

GFP

YFP

and

their

circularly

permuted

constructions

described

in

previous

parts

of

this

review

exhibit

sensitivity

to

acid

-LRB-

Fig.

-RRB-

whereas

the

original

AFP

Renilla

reniformis

GFP

and

DsRed

are

insensitive

to

acid

Therefore

acid-sensitive

variants

can

serve

as

ideal

donors

in

pH

indicators

based

on

FRET

and

DsRed

is

the

most

desirable

acceptor

-LSB-

-RSB-

Another

pH

sensor

has

been

generated

by

using

a

new

group

of

pH-sensitive

GFP

variants

termed

deGFPs

which

exhibit

rapid

changes

in

emission

from

green

to

blue

as

the

pH

decreases

and

these

are

suitable

for

ratiometric

measurements

in

vivo

-LSB-

-RSB-

Monitoring

signal

transduction

from

cell

to

cell

Scientists

have

also

monitored

intercellular

changes

in

biomolecules

and

physiological

events

which

have

been

visualized

in

single

cells

in

real

time

in

a

cell

population

For

example

Bedner

et

al.

monitored

the

intercellular

permeability

to

cAMP

of

six

different

gap

junction

channels

by

using

sensors

that

are

highly

sensitive

to

cAMP

concentration

-LSB-

-RSB-

The

change

in

calcium

concentration

from

cell

to

cell

was

also

monitored

via

calcium

dyes

-LSB-

-RSB-

In

addition

to

these

biomicromolecules

the

propagation

of

intercellular

apoptotic

or

survival

events

in

monolayer

cells

mediated

by

the

gap

junction

channel

were

also

studied

through

OMI

-LSB-

-RSB-

Using

OMI

to

investigate

tissue

structure

and

function

It

has

recently

been

shown

that

SHIM

can

be

used

to

directly

detect

several

structural

protein

arrays

in

tissues

such

as

collagen

arrays

in

mouse

-LSB-

-RSB-

neurons

and

muscular

structures

of

the

pharynx

in

C.

elegans

-LSB-

-RSB-

without

the

need

for

fluorescent

labeling

SHIM

also

readily

retrieves

more

detailed

molecular

information

than

that

obtained

through

the

application

of

fluorescent

labeling

Therefore

SHIM

can

be

used

to

study

or

diagnose

several

diseases

that

are

related

to

the

assembly

polarity

of

microtubule

complexes

in

native

brain

tissue

-LSB-

-RSB-

In

addition

SHIM

has

proven

to

be

crucial

to

neuroscience

investigations

in

thick

tissue

preparations

For

example

the

fast

neuronal

membrane

potential

transient

in

mammalian

brain

slices

labeled

with

dye

was

investigated

by

this

method

-LSB-

-RSB-

By

labeling

with

another

dye

the

action

potential

was

recorded

with

high

temporal

and

spatial

resolution

on

soma

and

neurite

membranes

-LSB-

-RSB-

OCT

also

has

the

ability

to

perform

in

situ

real-time

imaging

of

tissue

pathology

and

it

can

be

used

to

guide

excisional

biopsy

in

order

to

reduce

false

negatives

caused

by

sampling

errors

In

particular

OCT

enables

the

internal

architectural

morphology

of

the

retina

to

be

visualized

noninvasively

and

it

can

be

used

to

diagnose

and

monitor

retinal

diseases

which

can

not

be

achieved

through

any

other

methods

-LSB-

-RSB-

The

development

of

high-speed

OCT

imaging

combined

with

small

fiber-optic

probes

has

enabled

in

vivo

endoscopic

imaging

such

as

the

visualization

of

the

oral

cavity

-LSB-

-RSB-

the

larynx

and

the

bladder

-LSB-

-RSB-

In

addition

OCT

can

perform

functional

imaging

of

tissues

such

as

brain

activity

-LSB-

-RSB-

inflammatory/neoplastic

morphologic

changes

-LSB-

-RSB-

Raman

spectroscopy

-LRB-

RS

-RRB-

is

another

powerful

diagnostic

tool

that

enables

tissue

identification

and

classification

as

demonstrated

by

measurements

of

the

brain

tissue

of

a

six-month-old

pig

by

fiber-optic

probes

-LSB-

-RSB-

and

analysis

of

the

molecular

composition

of

human

bronchial

tissue

structures

-LSB-

-RSB-

Also

a

combination

of

confocal

RS

and

LSCM

has

been

employed

to

obtain

detailed

information

about

the

subsurface

structures

in

the

skin

with

high

spatial

resolution

in

a

completely

noninvasive

manner

-LSB-

-RSB-

OMI

of

organisms

Employing

fluorescent

protein

When

performing

whole-body

OMI

of

organisms

the

utilization

of

genetically

encoded

fluorescence

probes

has

enabled

scientists

to

decipher

spatial

and

temporal

changes

in

biological

events

inside

complex

organisms

-LSB-

-RSB-

Over

the

past

few

decades

FPs

have

been

used

to

generate

various

fluorescent

probes

that

have

been

used

to

image

physiological

and

biochemical

events

in

the

monolayer

cells

Extracting

information

from

whole-body

imaging

is

more

relevant

to

real

physiological

conditions

but

up

to

now

only

a

few

fluorescent

indicators

have

been

used

in

complex

organisms

to

visualize

physiological

activity

in

vivo

To

study

the

feeding

behavior

of

C.

elegans

Kerr

et

al.

-LSB-

-RSB-

introduced

a

calcium

indicator

based

on

cameleon

to

C.

elegans

which

allowed

them

to

measure

the

signals

evoked

in

pharyngeal

muscles

and

individual

neurons

under

the

stimulation

of

feeding

in

vivo

-LRB-

Fig.

b

-RRB-

Similar

visualization

of

cameleon

localized

in

other

tissues

was

used

to

study

the

functions

of

serotonin

and

G

proteins

-LSB-

-RSB-

the

role

of

G

proteins

in

signal

transduction

-LSB-

-RSB-

and

the

ASH

neuron

response

-LSB-

-RSB-

in

C.

elegans

The

spatial

and

temporal

representation

of

odorant-evoked

signals

in

the

Drosophila

brain

was

explored

using

cameleon

-LSB-

-RSB-

and

G-CaMP

-LRB-

Fig.

f

-RRB-

-LSB-

-RSB-

Calcium

imaging

was

also

used

in

other

animals

such

as

zebrafish

-LRB-

Fig.

i

-RRB-

-LSB-

-RSB-

and

mouse

-LSB-

-RSB-

In

addition

an

indicator

with

pH

sensitivity

was

used

to

visualize

spatial

patterns

of

defined

neuronal

activity

in

the

mouse

-LSB-

-RSB-

More

recently

the

proteolytic

activity

of

calpain

was

visualized

by

introducing

fluorescent

indicator

into

living

mouse

muscle

which

gave

the

first

imaging

of

FRET

in

vivo

-LSB-

-RSB-

Fig.

kWhole-body

OMI

using

FPs

and

QDs

Imaging

of

signals

in

pharyngeal

muscles

under

the

conditions

of

noncontraction

-LRB-

a

-RRB-

and

contraction

-LRB-

b

-RRB-

-LRB-

red

color

indicates

higher

calcium

-RRB-

in

transgenic

C.

elegans

expressing

cameleon

Reproduced

from

-LSB-

-RSB-

with

permission

c

f

signals

evoked

by

different

odors

in

Drosophila

brain

expressing

G-CaMP

Reproduced

from

-LSB-

-RSB-

with

permission

Imaging

of

a

three-day-old

transgenic

fish

-LRB-

g

-RRB-

carrying

the

cameleon

and

its

RB

neurons

-LRB-

h

-RRB-

by

confocal

optical

section

and

the

change

in

the

fluorescence

ratio

-LRB-

i

-RRB-

-LRB-

representing

the

calcium

concentration

-RRB-

in

an

RB

neuron

under

electrical

stimulation

of

the

skin

Reproduced

from

-LSB-

-RSB-

with

permission

j

Simultaneous

multicolor

imaging

in

a

mouse

injected

with

QD-encoded

microbeads

k

QD

imaging

of

a

prostate

tumor

in

the

mouse

Reproduced

from

-LSB-

-RSB-

with

permission

Employing

QDs

Due

to

several

advantages

of

QDs

over

FP

QDs

have

been

used

to

image

large

animals

in

vivo

which

holds

great

promise

for

clinical

applications

especially

in

the

imaging

of

tumors

Akerman

et

al.

-LSB-

-RSB-

report

the

application

of

QDs

coated

with

specific

targeting

peptides

to

the

imaging

of

different

tissues

in

tumor

in

vitro

but

this

QD

probe

can

not

be

detected

in

living

animals

Gao

et

al.

-LSB-

-RSB-

report

on

a

new

class

of

multifunctional

QDs

that

were

encapsulated

with

an

ABC

triblock

copolymer

and

then

linked

with

a

tumor-targeting

antibody

Using

subcutaneous

injection

or

systemic

injection

of

these

QDs

into

the

mouse

simultaneous

multicolor

fluorescence

imaging

of

the

prostate

tumor

with

efficient

background

removal

and

precise

delineation

of

weak

spectral

signatures

based

on

wavelength

resolution

was

achieved

-LRB-

Fig.

k

-RRB-

More

recent

research

showed

that

the

utilization

of

QDs

in

combination

with

GFP

labeling

can

differentiate

tumor

vessels

from

both

perivascular

cells

and

the

matrix

and

QDs

linked

to

bone

marrow-derived

precursor

cells

can

visualize

tumor

vasculature

-LSB-

-RSB-

These

OMI

techniques

as

well

as

their

applications

at

different

biological

levels

are

summarized

in

Table

Table

in

systems

biologyTechniquesPhysical

processesResolutionSensitivityPenetrationObservation

methodsProbesIndicator

localizationReferencesWFM

LSCM

MPLSMRNA

transcription

and

expressionHighHighHighDFI

FRETFPsIn

vitro

cells

tissues

organism

-LSB-

-RSB-

Protein

localization

and

dynamicsDFIFPsIn

vitro

cells

tissues

organism

-LSB-

-RSB-

Subcellular

organelle

dynamicsDFI

FRAPFPsCells

-LSB-

-RSB-

Protein

interactionFRET

FLIM

FCSFPs

cpFPsCells

mouse

-LSB-

-RSB-

Protease

and

kinase

activityFRETFPs

-LSB-

-RSB-

CalciumDFI

FRETFPs

cpFPsCells

C.

elegans

fish

mouse

Drosophila

-LSB-

-RSB-

cAMP

cGMPFRETFPsCells

-LSB-

-RSB-

Electrical

activityDFI

FRETFPsCells

-LSB-

-RSB-

pHDFI

FRETFPsCells

mouse

-LSB-

-RSB-

TumorDFIQDsMouse

-LSB-

-RSB-

SHIMBiomacromoleculesLowHighLowNI

Mouse

C.

elegans

brain

tissue

-LSB-

-RSB-

Membrane

potentialNIDyesCells

brain

tissue

-LSB-

-RSB-

OCTMorphology

of

tissueHighLowHighEI

Human

-LSB-

-RSB-

Activity

and

disease

of

organEI

Human

-LSB-

-RSB-

RSComposition

of

structureLowHighLowNI

Brain

tissues

bronchial

tissue

-LSB-

-RSB-

FT-IRKinetics

of

reaction

intermediatesLowHighLowNI

Yeast

extract

fish

liver

-LSB-

-RSB-

Abbreviations

DFI

Direct

fluorescent

imaging

NI

nonlinear

imaging

EI

light-echo

imaging

Future

development

As

described

above

OMI

is

a

feasible

analytical

technique

for

monitoring

the

dynamics

of

biological

events

in

vivo

and

in

real

time

anywhere

from

single-molecule

to

whole-body

level

which

makes

it

well-suited

to

systems

biology

measurements

Its

noninvasive

ultrasensitive

high-resolution

and

real

time

nature

has

resulted

in

the

widespread

use

of

OMI

However

its

disadvantages

are

also

very

apparent

such

as

its

high

cost

its

low

throughput

and

its

limited

detectable

depth

which

is

a

particular

hindrance

in

whole-body

analysis

It

should

be

pointed

out

that

OMI

is

still

far

from

fulfilling

all

of

the

requirements

of

systems

biology

-LRB-

as

concluded

in

our

previous

paper

-LSB-

-RSB-

-RRB-

in

terms

of

sensitivity

selectivity

or

specificity

linear

range

of

quantitation

throughput

robustness

flexibility

and

cost

that

are

definitely

required

in

genomics

transcriptomics

proteomics

and

metabolomic

profiling

and

dynamics

although

it

it

clear

that

OMI

is

a

promising

method

for

these

types

of

bioanalysis

In

the

near

future

several

routes

to

improving

OMI

for

systems

biology

need

to

be

followed

Firstly

it

is

crucial

to

develop

better

molecular

probes

for

OMI

in

other

words

probes

that

have

improved

biocompatibilities

and

high

quantum

yields

are

easy-to-tag

display

multiple

colors

and

have

better

penetration

capabilities

The

importance

of

the

molecular

probe

to

OMI

can

not

be

overstressed

Current

endeavors

in

QD

development

are

heading

in

a

good

direction

but

this

field

is

still

in

its

infancy

-LSB-

-RSB-

However

this

does

n't

mean

that

the

development

of

small

organic

dyestuffs

and

biogenetic

FP

is

less

important

on

the

contrary

applications

of

these

two

kinds

of

molecular

probes

will

still

constitute

most

OMI

studies

-LSB-

-RSB-

Secondly

the

integration

of

several

modalities

or

approaches

should

be

emphasized

since

it

makes

OMI

much

more

informative

Thirdly

efforts

to

improve

OMI

instrumentation

are

also

important

since

they

will

result

in

better

time

and

space

resolution

and

better

sensitivity

For

example

the

technique

has

improved

the

spatial

resolution

of

OMI

down

to

the

mid-nanometer

level

which

is

over

the

diffraction

limit

The

rapid

progress

currently

being

achieved

in

nanotechnology

and

nano/microelectromechanical

systems

looks

set

to

lead

to

the

manufacture

of

low-cost

and

high-efficiency

OMI

instruments

-LSB-

-RSB-

Finally

the

application

of

OMI

to

the

life

sciences

is

important

it

is

the

reason

behind

the

development

of

OMI

In

particular

the

application

of

OMI

to

systematic

measurements

of

biological

systems

will

be

of

great

interest

which

is

also

the

aim

of

systems

biology

B I I O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O

optical molecular imaging
fluorescent protein
quantum dots
systems biology
review


NF-κB : a new player in angiostatic therapy Angiogenesis is considered a promising target in the treatment of cancer . Most of the angiogenesis inhibitors in late-stage clinical testing or approved for the treatment of cancer act indirectly on endothelial cells . They either neutralize angiogenic growth factors from the circulation or block the signaling pathways activated by these growth factors . Another group of angiogenesis inhibitors are the direct angiostatic compounds . These agents have a direct effect on the endothelium , affecting cellular regulatory pathways , independently of the tumor cells . The reason that this category of agents is lagging behind regarding their translation to the clinic may be the lack of sufficient knowledge on the mechanism of action of these compounds . The transcription factor NF-κB has been recently connected with multiple aspects of angiogenesis . In addition , several recent studies report that angiogenesis inhibition is associated to NF-κB activation . This is of special interest since in tumor cells NF-κB activation has been associated to inhibition of apoptosis and currently novel treatment strategies are being developed based on inhibition of NF-κB . The paradigm that systemic NF-κB inhibition can serve as an anti-cancer strategy , therefore , might need to be re-evaluated . Based on recent data , it might be speculated that NF-κB activation , when performed specifically in endothelial cells , could be an efficient strategy for the treatment of cancer . Introduction The NF-κB/Rel proteins are a family of transcription factors that include five proteins , p50 , p52 , p65 or RelA , RelB , and c-Rel , that exist as homo - and hetero-dimers . The most common NF-κB heterodimer is composed of p50 and p65 . In resting cells , NF-κB is mainly sequestered in the cytoplasm by its association with proteins belonging to the IκB inhibitor family . Stimuli such as the proinflammatory cytokines tumor necrosis factor -LRB- TNF -RRB- - α , and interleukin-1 -LRB- IL-1 -RRB- , or bacterial products such as lipopolysaccharide -LRB- LPS -RRB- can activate NF-κB . In the canonical pathway , these stimuli activate IκB kinases -LRB- IKKs -RRB- , which in turn phosphorylate the main NF-κB inhibitor , IκBα . This phosphorylation step leads to the ubiquitination and subsequent degradation by the proteasome of IκBα . The NF-κB complex translocates to the nucleus where it binds to κB enhancers present in the regulatory regions of various genes and where it activates transcription -LSB- 1 -RSB- . The NF-κB target genes are involved in a wide range of biological functions including proliferation , survival , and inflammation -LRB- Fig. 1 -RRB- . Fig. 1Schematic NF-κB pathway . In the canonical activation pathway , NF-κB -LRB- often the dimer composed of p50/p65 -RRB- is sequestered by its main inhibitor IκB-α . Upon stimulation , cell surface receptors activate IKK complex , which then phosphorylate IκB-α . These phosphorylations lead to its degradation by the proteasome and the entry of NF-κB in the nucleus , which turns on target genes NF-κB activation has been connected with multiple aspects of oncogenesis , including the control of tumor cell proliferation , migration , cell cycle progression , and inhibition of apoptosis -LSB- 2 -- 4 -RSB- . Indeed , NF-κB is constitutively activated in several types of cancer cells and it is generally regarded as an anti-apoptotic and pro-oncogenic signal . The most studied and well-established functions of NF-κB in promoting oncogenesis are its ability to -LRB- i -RRB- induce growth promoting genes such as cyclin D1 and c-myc and -LRB- ii -RRB- induce anti-apoptotic genes such as c-IAP-1 , c-IAP-2 , or XIAP -LSB- 3 -RSB- . Therefore , activation of NF-κB in cancer cells by chemotherapy or radiation therapy is often associated with the acquisition of resistance to apoptosis . This has emerged as a significant impediment to effective cancer treatment . In combination with chemotherapy , inhibitors of the NF-κB pathway -LRB- e.g. , proteasome inhibitors -RRB- were recently used with success as treatment against cancer -LSB- 5 -RSB- . Next to this direct effect , it has also been reported that NF-κB activity can be tumorigenic by activation of pro-angiogenesis genes , such as VEGF , IL-8 , and MMP-9 -LSB- 6 -RSB- . In contrast to the negative effects of NF-κB activation , recent reports suggest that in certain situations NF-κB can promote apoptosis and may be viewed as a tumor suppressor gene . For example , blockade of NF-κB predisposes murine skin to squamous cell carcinoma -LSB- 7 -RSB- . This observation could be explained by the fact that in normal human epidermal cells , NF-κB activation induces cell cycle arrest -LSB- 8 -RSB- . In addition , Ryan et al. describe the role of NF-κB in p53-mediated programmed cell death . The tumor suppressor p53 inhibits cell growth through activation of apoptosis and cell cycle arrest . Using a p53-inducible Saos-2 cell line , it was demonstrated that induction of p53 causes activation of NF-κB . Furthermore , inhibition of NF-κB abrogated p53-induced apoptosis demonstrating that inhibition of NF-κB in tumors that retain wild-type p53 may reduce a therapeutic response -LSB- 9 -RSB- . Loss of p65 can also cause resistance to different agents that induce apoptosis through p53 -LSB- 10 -RSB- . Independently , it was demonstrated that activation of NF-κB is essential for the cytotoxic effect of doxorubicin and its analogs -LSB- 11 -RSB- . Many hypotheses have been put forward to explain this dual activity . The overall conclusion that is emerging is that the final outcome of NF-κB activation depends on cell type , the stimulus , and the context of activation -LSB- 12 -- 14 -RSB- . The dual activity of NF-κB complicates the systemic use of broad spectrum NF-κB inhibitors for the treatment of cancer and it has been suggested to design better therapeutics that specifically unleash the pro-apoptotic activity of NF-κB -LSB- 15 , 16 -RSB- . NF-κB in ongoing angiogenesis NF-κB signaling has been found to regulate endothelial cell integrity and vascular homeostasis in vivo . Treatment of zebrafish embryos with NF-κB inhibitors provokes vascular leakage and alters vessel morphology -LSB- 17 -RSB- . The role of NF-κB signaling in tumor angiogenesis has also been recently investigated . Inoculated tumors grow faster in transgenic mice expressing mutated IκBα , under control of the Tie-2 promoter , resulting in endothelial repression of NF-κB -LSB- 18 -RSB- . Histological analysis revealed a striking increase in tumor vascularization in these mice . This study highlighted , for the first time , the in vivo role of NF-κB in tumor angiogenesis , indicating an inhibitory role for NF-κB in tumor angiogenesis . Based on this study , NF-κB activation in endothelial cells appears to be a way to block angiogenesis . However , it is unknown how specific activation of NF-κB in endothelial cells can be realized and through which mechanisms NF-κB activation leads to inhibition of angiogenesis . Angiogenesis occurs in a coordinated series of steps , which can be divided into a destabilization , a proliferation , and a maturation phase . Whereas inhibition of angiogenesis can prevent diseases with excessive vessel growth such as cancer , diabetes retinopathy , and arthritis , stimulation of angiogenesis would be beneficial in the treatment of diseases such as coronary artery disease and critical limb ischemia in diabetes -LSB- 19 -RSB- . One of earliest events in angiogenesis is the degradation of the vascular basement membrane and the remodeling of the extracellular matrix -LRB- ECM -RRB- . The role of NF-κB in the regulation of these systems is well documented . In line with a pro-oncogenic activity , NF-κB promotes expression of several matrix metalloproteinases -LRB- MMPs -RRB- , including MMP-2 , -3 , and -9 -LSB- 20 -- 22 -RSB- . However , NF-κB could also inhibit endothelial cell migration via the up-regulation of tissue inhibitors of metalloproteinase-1 -LRB- TIMP-1 -RRB- as described in astrocytes -LSB- 23 -RSB- . Next to the MMPs , plasmin is a broad-spectrum protease that also hydrolyzes many extracellular proteins , the most notable of which is fibrin . Plasmin is produced from an inactive precursor called plasminogen . uPA -LRB- urokinase plaminogen activator -RRB- and tPA -LRB- tissue-type plasminogen activator -RRB- are two proteases with high affinity for plasminogen . The activation of plasminogen into plasmin could be negatively regulated by the physiological inhibitors , namely plasminogen activator inhibitor -LRB- PAI -RRB- -1 and -2 -LSB- 24 -RSB- . In endothelial cells , it has been described for both reactive oxygen species as well as for TNF-α induced expression of PAI-1 via NF-κB -LSB- 25 -RSB- . In addition , activation of NF-κB by TNF-α can also lead to the inhibition of the tPA expression -LSB- 26 -RSB- . These data suggest that NF-κB activation could impair angiogenesis via a decrease in ECM degradation capacity . Integrins are the principle adhesion receptors used by endothelial cells to interact with the extracellular environment and are necessary for cell migration , proliferation , and survival -LSB- 27 -RSB- . It has recently been demonstrated that the interaction of αVβ3-integrin with the ECM activates NF-κB by activation of the IKK complex and degradation of IκB-α . This activation triggers a pro-survival signal for example in rat aortic endothelial cells -LSB- 28 -RSB- . Many soluble molecules control the balance between cell proliferation and cell death . While angiogenic factors such as VEGF and bFGF are mitogenic and act as survival factors , angiostatic agents induce cell cycle arrest and promote endothelial cell death -LSB- 29 -RSB- . Activation of NF-κB in endothelial cell leads to the expression of angiogenic and angiostatic factors . VEGF expression is up-regulated by hypoxia-induced mitogenic factor through activation of the NF-κB pathway -LSB- 30 -RSB- . On the contrary , vascular endothelial growth inhibitor -LRB- VEGI , reported to inhibit endothelial cell proliferation -RRB- has also been found to be induced by NF-κB -LSB- 31 -RSB- . In addition , the promoters of thrombospondin-1 and -2 , which are among the first naturally occurring angiostatic agents discovered , contain NF-κB binding sites -LSB- 32 -RSB- . The role of NF-κB in the cell cycle progression has been also investigated -LSB- 33 -RSB- . NF-κB induces expression of activators of the cell cycle such as cyclin D or - E -LSB- 34 -RSB- as well as expression of inhibitors such as p21/cip1 -LSB- 35 -RSB- demonstrating that the overall effect of NF-κB on cell proliferation is difficult to predict . To our knowledge , there are no reports on a direct relationship between NF-κB activation and proliferation in endothelial cells . Programmed cell death or apoptosis occurs mainly by two connected pathways . The extrinsic pathway involves activation of caspase-8 by cell surface death receptors , while the intrinsic pathway , involves cytochrome-c release from mitochondria and subsequent caspase-9 activation . As previously described for tumor cells , diverse activities have been observed in endothelial cells . Silibinin , a cancer chemopreventive agent , was found to induce apoptosis in endothelial cell line by inhibiting NF-κB -LSB- 36 -RSB- and a report describes that pro-survival effect of VEGF is mediated by NF-κB activation -LSB- 37 -RSB- . However , a large body of evidence exists that NF-κB activation plays a pro-apoptotic role in endothelial cells . A high concentration of glucose activates the production of reactive oxygen species and induces caspase-3 activation in endothelial cells -LSB- 38 -RSB- . Recently , it has been demonstrated that this is mediated via NF-κB and subsequent c-Jun N-terminal protein kinase activation -LSB- 39 -RSB- . In human cardiac microvascular endothelial cells , IL-18 induces activation of both the intrinsic and extrinsic apoptotic pathways via NF-κB activation -LSB- 40 -RSB- . Angiopoietin-1 inhibits endothelial cell apoptosis induced by growth factor deprivation . This effect is mediated via the activation of an endogenous inhibitor of NF-κB , namely A20 binding inhibitor of NF-κB -LRB- ABIN-2 -RRB- -LSB- 41 -RSB- . In addition , in endothelial cells , it was observed that A20 inhibition and subsequent activation of NF-κB , effectively leads to reduced tube formation in a matrigel assay -LSB- 42 -RSB- . A role for NF-κB in angiostatic therapy ? The observations described above indicate that NF-κB is involved in the regulation of migration and/or proliferation/survival of endothelial cells and suggest a strong link between NF-κB in angiostatic agent signaling . The following section will highlight the role of NF-κB in the angiostatic agent signaling . Indeed , several angiostatic compounds , already described to block tumor growth , have been reported to act on endothelial cells via NF-κB activation -LSB- 29 -RSB- . Platelet factor-4 -LRB- PF4 -RRB- is an α-chemokine naturally secreted by platelets and is known to inhibit angiogenesis -LSB- 43 -RSB- . PF4 promotes the expression of E-Selectin in HUVEC . Data provide direct evidence that the NF-κB -- binding site is required for PF4-mediated activation of the E-selectin promoter . In addition , EMSA experiments demonstrate that PF4 treatment of HUVECs results in binding of NF-κB to the E-Selectin promoter already after 1 hour of stimulation -LSB- 44 -RSB- . The angiostatic properties of angiostatin , a cleavage product of plasminogen , have also been linked with NF-κB . Chen et al. have analyzed the global action of angiostatin in endothelial cells . By microarray screening , they have found an altered expression of 189 genes after treatment with angiostatin . These genes are mainly involved not only in growth , apoptosis , migration but also in inflammation -LSB- 45 -RSB- . Even though no direct evidence points a role of NF-κB , angiostatin promotes mRNA expression of RelB as well as many NF-κB target genes , namely E-selectin , intracellular adhesion molecule-1 -LRB- ICAM-1 -RRB- , Cyclin D1 , p21/cip1 , and FasL -LRB- for the complete list of altered gene expression , see reference -LSB- 45 -RSB- -RRB- . Based on these data , there is a strong suggestion that NF-κB is also activated by angiostatin . The 16 kDa N-terminal fragment of prolactin -LRB- 16 K PRL -RRB- is a potent angiostatic agent in various in vivo models and has been shown to inhibit endothelial cell migration and proliferation -LSB- 46 -- 48 -RSB- . We have demonstrated that NF-κB activation is required for 16 K hPRL-induced caspase-8 and -9 activation and subsequent apoptosis -LSB- 49 -RSB- . In addition , it is interesting to note that NF-κB activation appears to be a very proximal event . The angiostatic agent Neovastat , which is currently in phase III clinical studies , inhibits angiogenesis through an increase in tPA activity and it has been shown that this induction is NF-κB dependent -LSB- 50 -RSB- . Finally , administration of statins has been shown to decrease tumor growth and angiogenesis -LSB- 51 -RSB- . Statins up-regulate the expression of endothelial and inducible nitric oxide synthase through NF-κB activation -LSB- 52 -RSB- . Based on all these reports , it is suggested that the activity of angiostatic compounds is dependent on activation of NF-κB . Therefore , activation of NF-κB specifically in endothelial cells might be an attractive therapy . TNF-α is one of the most potent NF-κB activators . The clinical use of TNF-α as an anti-cancer drug is limited to local treatment -LRB- e.g. , isolated limb perfusion -RRB- because of its systemic toxicity -LSB- 53 -RSB- . To circumvent this problem , targeted delivery of TNF-alpha to tumor vessels was achieved by coupling this cytokine with cyclic CNGRC peptide , an aminopeptidase N -LRB- CD13 -RRB- ligand that targets the tumor neovasculature . Administration of this compound leads to a reduced toxicity , a marked endothelial cell apoptosis , destruction of blood vessels , and improvement of the anti-tumor activity of doxorubicin -LSB- 54 -RSB- . These studies indicate that NF-κB activation , specifically in endothelial cells , can be an efficient strategy for the treatment of cancer . In conclusion , while in tumor cells NF-κB is mostly described as an oncogenic factor , up-regulation of NF-κB in endothelial cells is associated with angiostatic activity . It might therefore be warranted to revisit anti-cancer therapies based on inhibition of NF-κB activity for effects on angiogenesis . An indirect anti-tumor activity of NF-κB through circumvention of endothelial cell anergy Next to a direct anti-tumor activity of NF-κB through inhibition of tumor angiogenesis , the activation of NF-κB could also be connected with an indirect anti-tumor activity through reversal of endothelial unresponsiveness to inflammatory signals , a process called endothelial cell anergy . The latter is defined as the inability of tumor endothelial cells to express adhesion molecules such as ICAM-1 / -2 , vascular endothelial cell adhesion molecule-1 -LRB- VCAM-1 -RRB- or E-selectin , in response to inflammatory cytokines such as TNF-α , interferon-γ and interleukin-1 . These adhesion molecules mediate leukocyte rolling along , adhering to , and diapedesis through the vessel wall , and thus have an important role in the selection of an inflammatory infiltrate -LSB- 55 -RSB- . The observation of a reduced number of infiltrated leukocytes in the tumor -LSB- 56 -RSB- has been correlated with the fact that tumor endothelial cells display a reduced expression of adhesion molecules -LRB- ICAM-1 and ICAM-2 -RRB- as compared with normal endothelial cells -LSB- 57 , 58 -RSB- . In vitro and in vivo studies on endothelial cell anergy have demonstrated that this reduced expression is caused by exposure to angiogenic growth factors such as VEGF and bFGF -LSB- 59 -RSB- . It has been recently described that bFGF down-regulates ICAM-1 expression via NF-κB inhibition -LSB- 60 -RSB- . Furthermore , we have demonstrated that suppressed leukocyte-vessel wall interactions in tumor vessels can be normalized by angiostatic compounds , such as endostatin , angiostatin , anginex , and 16 K hPRL , as well as by treatment with chemotherapeutic agents -LSB- 61 , 62 -RSB- . This normalization has been correlated with up-regulation of ICAM-1 , VCAM-1 , and E-selectin in endothelial cells -LSB- 44 , 63 -RSB- . Therefore , activation of NF-κB by angiostatic therapy does not directly affect angiogenesis but also has an indirect effect via expression of adhesion molecules and subsequent reversal of endothelial cell anergy . Therefore , such activation of NF-κB resulting in stimulation of anti-tumor immunity but also in inhibition of angiogenesis clearly results in an anti-tumor outcome . Where to go from here ? Inhibition of angiogenesis is a promising therapeutic approach to fight cancer . From several recent findings it is likely that activation of NF-κB is a common mechanism of angiostatic agents , resulting in both inhibition of angiogenesis and stimulation of anti-tumor immunity . While these data raised a cautionary note about the pharmaceutical agents that block NF-κB , they also suggest that a targeted activation of NF-κB , specifically in endothelial cells , could represent a new and promising strategy in cancer treatment -LRB- Fig. 2 -RRB- . Further studies remain necessary to fully understand the molecular mechanisms induced by angiostatic agents and the role of NF-κB in endothelial cells . Fig. 2Model of the dual role of NF-κB in tumorigenesis . In tumor cells , activation of NF-κB leads to tumor growth by both direct and indirect mechanisms . The direct mechanisms involve expression of anti-apoptotic and pro-proliferative molecules while the indirect mechanism involves promotion of angiogenesis . In endothelial cells , activation of NF-κB can block tumor progression by both , angiostatic activity , via the production of pro-apoptotic molecules and by improvement of immune response via expression of adhesion molecules

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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O 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