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Bio
ph
oto
nic
s??
Fu
sio
n t
ech
no
log
ies
of
ph
oto
nic
s an
d b
iote
chn
olo
gy a
nd
b
iom
ed
ical
en
gin
eeri
ng
en
ab
led
by i
nfo
rmati
on
tech
no
log
y
Bio
ph
oto
nic
s
-Im
agin
g:
X-r
ay,
OC
T,
pola
rim
etry
,D
OT
,T
IRF,
photo
nm
igra
tion,
endosc
opy,
confo
cal
mic
rosc
opy,
mult
iphoto
nm
icro
scopy,
mult
ispec
tral
imag
ing
-Bi
ose
nsi
ng:
IRsp
ectr
osc
opy,
fluore
scen
ce,
lum
ines
cence
,Ram
ansc
atte
ring,
surf
ace
pla
smon
reso
nan
ce,
pola
rim
etry
,ev
anes
cent
wav
e-bas
eddet
ecti
on
-La
ser-
tiss
ue
inte
ract
ion:
abla
tion,
coag
ula
tion,
cutt
ing,
wel
din
g,
PDT
,opti
caltr
appin
g
Ad
van
tag
es
of
the l
igh
twave
tech
no
log
y i
n b
iom
ed
ical
en
gin
eeri
ng
-N
on
-in
vasi
ve/m
inim
all
yin
vasi
ve
tech
niq
ues
(det
ecti
on
bas
edon
fiber
-opti
cs)
-R
eal-ti
me
feed
bac
kfo
rcl
inic
aldia
gnost
ics
and
visu
alguid
ance
for
surg
ery
-H
igh
sen
siti
vit
yan
dsp
eci
fici
ty:
single
mole
cule
sensi
ng,
mole
cula
rta
ggin
g
-W
ide
range
of
spati
al
scale
sth
atca
nbe
sam
ple
d:
«m
~cm
-A
bundan
ceof
intr
insi
c(h
emoglo
bin
)an
dex
trin
sic
(flu
ore
scen
tpro
tein
)ch
rom
op
ho
res
Bio
-im
ag
ing
Opti
cal
imag
ing
for
earl
ydet
ecti
on
and
monit
ori
ng
the
pro
gre
ssio
nof
adis
ease
Benef
its
of
opti
calim
agin
g:
•N
on-inva
sive
or
min
imal
lyin
vasi
ve•
No
har
mfu
lra
dio
acti
vity
(vs.
radio
isoto
pe
imag
ing)
•R
apid
real
-tim
em
easu
rem
ents
(vs.
MR
I)•
Hig
hsp
atia
lre
solu
tion
(vs.
ult
raso
und)
•H
igh
sensi
tivi
ty(v
s.x-r
ayim
agin
g)
•Po
rtab
ility
and
com
pac
tnes
s•Invitro,invivo
,an
dexvivo
spec
imen
s
Ref
eren
ces
htt
p:/
/mic
rosc
ope.
fsu.e
du/p
rim
er/
htt
p:/
/bam
a.ua.
edu/~
hsm
ithso
/cla
ss/b
sc_6
56/w
ebsi
tes/
light.
htm
l
Bio
-im
ag
ing
: Kö
hle
r Il
lum
inati
on
•A
bri
gh
t-fi
eld
illu
min
ati
on
syst
emth
atpro
vides
anev
enly
illum
inat
edfi
eld
of
view
.
•R
ecom
men
ded
by
all
man
ufa
cture
rsof
moder
nla
bora
tory
mic
rosc
opes
bec
ause
itca
npro
duce
spec
imen
illum
inat
ion
that
isunif
orm
lybri
ght
and
free
from
gla
re.
Bio
-im
ag
ing
: ep
i-fl
uo
resc
en
ce
Bio
-im
ag
ing
: Ph
ase
Co
ntr
ast
Mic
rosc
op
y
•Ph
ase
and a
mplit
ude
dif
fere
nce
s bet
wee
n d
iffr
acte
d
and u
ndif
frac
ted lig
ht
are
alte
red t
o p
roduce
inte
rfer
ence
or
contr
ast
enhan
cem
ent.
•A
phas
e pla
te is
intr
oduce
d t
o
mak
e a
phas
e sh
ift
that
pro
duce
s des
truct
ive
inte
rfer
ence
.
•T
he
tech
niq
ue
suff
ers
from
hal
o a
rtif
acts
, is
res
tric
ted t
o
very
thin
spec
imen
pre
par
atio
ns,
and c
annot
take
ad
vanta
ge
of
the
full
conden
ser
and o
bje
ctiv
e ap
ertu
res.
Bio
-im
ag
ing
: Ph
ase
Co
ntr
ast
Mic
rosc
op
y
Dif
fere
nti
al
Inte
rfere
nce
Co
ntr
ast
(D
IC)
Mic
rosc
op
y
•The
opti
cal co
mponen
ts r
equir
ed f
or
DIC
m
icro
scopy
do n
ot
mas
k or
obst
ruct
the
obje
ctiv
e an
d c
onden
ser
aper
ture
s, t
hus
enab
ling t
he
inst
rum
ent
to b
e em
plo
yed a
t fu
ll N
A.
This
res
ult
s in
a d
ram
atic
im
pro
vem
ent
in r
esolu
tion, el
imin
atio
n o
f hal
o a
rtif
acts
, an
d t
he
abili
ty t
o p
roduce
ex
celle
nt
imag
es w
ith r
elat
ivel
y th
ick
spec
imen
s.•
When
a p
air
of
coher
ent
light
rays
pro
duce
d
by
the
bea
msp
litte
r en
counte
rs a
phas
e gra
die
nt,
due
to r
efra
ctiv
e in
dex
and/o
r th
ickn
ess
vari
atio
ns,
eac
h r
ay b
ecom
es
def
orm
ed a
nd e
xper
ience
s a
slig
htl
y dif
fere
nt
opti
cal pat
h d
iffe
rence
when
tra
vers
ing
thro
ugh t
he
spec
imen
. U
pon e
mer
gin
g f
rom
th
e sp
ecim
en,
the
rays
is
uneq
ual
in p
has
e.
The
dif
fere
nce
in o
pti
cal pat
h is
tran
slat
ed
then
into
a c
han
ge
in a
mplit
ude
in t
he
final
im
age
obse
rved
in t
he
eyep
iece
s.
htt
p:/
/ww
w.o
lym
pusm
icro
.com
/pri
mer
/tec
hniq
ues
/dic
/dic
intr
o.h
tml
Bio
-im
ag
ing
: Dark
Fie
ld M
icro
sco
py
•H
igher
inte
nsi
ty s
ourc
e is
nee
ded
than
bri
ght-
fiel
d s
ince
only
dif
frac
ted/r
efra
cted
lig
ht
is v
isib
le.
•Sa
mple
is
illum
inat
ed a
t an
angle
so t
hat
th
e ce
ntr
al lig
ht
is b
lock
ed a
nd o
nly
obliq
ue
rays
rea
ch t
he
spec
imen
.
•O
nly
the
rays
dif
frac
ted o
r re
frac
ted f
rom
th
e sp
ecim
en r
each
the
obje
ctiv
e.
Bio
-im
ag
ing
: Co
nfo
cal
Mic
rosc
op
y
•Bl
urr
ed o
ut-
of-
focu
s bac
kgro
und r
ejec
ted b
y a
con
foca
l ap
ert
ure
.
•O
pti
cal
3-D
re
con
stru
ctio
n/s
ect
ion
ing
can
be
achie
ved.
•H
igher
exci
tati
on p
ow
er n
eeded
, si
nce
the
confo
cal ap
ertu
re
reduce
s th
e fl
uore
scen
ce s
ignal
.
•H
igh e
xci
tati
on p
ow
er a
nd lar
ge
area
of
fluore
scen
ce e
xci
tati
on
incr
ease
s th
e poss
ibili
ty o
f p
ho
tob
leach
ing
.
•W
ith m
ost
flu
oro
phore
s ex
cite
d
by
one-
photo
n a
bso
rpti
on in t
he
UV
or
blu
e re
gio
n,
hig
h
atte
nuat
ion o
f lig
ht
in t
he
wav
elen
gth
ran
ge
limit
s th
e dep
th a
cces
s.
Bio
-im
ag
ing
: Co
nfo
cal
Mic
rosc
op
y
Pu
rdu
e M
icro
sco
py S
eri
es
-T
he P
rin
cip
les
of
Co
nfo
cal
Mic
rosc
op
y-
Liv
e C
ell
Im
ag
ing
Ap
pli
cati
on
in
Co
nfo
cal
Mic
rosc
op
y
-A
pp
lica
tio
ns
of
Co
nfo
cal
Mic
rosc
op
y
Bio
-im
ag
ing
: Mu
ltip
ho
ton
Mic
rosc
op
y (
1)
•U
p-c
onve
rted
flu
ore
scen
ce (
thir
d-
ord
er n
onlin
ear
opti
cal pro
cess
) is
use
d t
o o
bta
in im
ages
.
•T
he
tran
siti
on p
roce
ss o
f tw
o-p
hoto
n
abso
rpti
on p
roce
ss is
pro
port
ional
to
the
squar
e of
the
inst
anta
neo
us
light
inte
nsi
ty,
nec
essi
tati
ng
extr
emel
y in
tense
lig
ht
sourc
e. T
o
keep
the
aver
age
pow
er v
ery
low
to
min
imiz
e th
erm
al d
amag
e of
the
bio
logic
al s
pec
imen
, ult
ra-s
hort
las
er
puls
es a
re u
sed.
Bio
-im
ag
ing
: Mu
ltip
ho
ton
Mic
rosc
op
y (
2)
•O
pti
cal
sect
ion
ing
may
be
poss
ible
wit
hout
usi
ng a
ny
confo
cal
aper
ture
.
•Ph
oto
ble
ach
ing
is g
reat
ly r
educe
d,
bec
ause
only
the
regio
n a
t th
e fo
cus
can b
e ex
cite
d.
•U
se o
f lo
ng
er
wavele
ng
thpen
etra
tes
dee
per
into
a t
issu
e.
•El
imin
atio
n o
f U
V im
pro
ves
the
tiss
ue
viab
ility
and les
s photo
dam
age.
•Lo
nger
wav
elen
gth
may
wors
en t
he
reso
luti
on.
•T
i:sa
pphir
e la
ser
~ 1
00
fs,
~ 8
00
nm
, pea
k pow
er ~
50
kW
To
tal
Inte
rnal
Refl
ect
ion
(T
IR)
Evan
esc
en
t W
ave: C
on
cep
t
Gla
ssA
ir
Gla
ss
Air
•O
bse
rved
into
talin
tern
alre
flec
tion
•A
wav
eth
atdec
ays
exponen
tial
lyw
ith
dis
tanceWikipedia.com
•A
wav
eth
atdec
ays
exponen
tial
lyw
ith
dis
tance
Evan
esc
en
t W
ave: C
on
cep
t
Gla
ssA
ir
Gla
ss
Air
•O
bse
rved
into
talin
tern
alre
flec
tion
Wikipedia.com
dye
100 n
m
To
tal
Inte
rnal
Refl
ect
ion
Flu
ore
scen
ce (
TIR
F)
J. A
. St
eyer
et
al.
Nat
ure
Rev
iew
s,
2001
a. P
rism
-typ
e (u
pri
ght)
b. O
bje
ctiv
e-le
ns-
type
(it
d)
To
tal
Inte
rnal
Refl
ect
ion
Flu
ore
scen
ce (
TIR
F)
To
tal
Inte
rnal
Refl
ect
ion
Flu
ore
scen
ce (
TIR
F)
To
tal
Inte
rnal
Refl
ect
ion
Flu
ore
scen
ce (
TIR
F)
To
tal
Inte
rnal
Refl
ect
ion
Flu
ore
scen
ce (
TIR
F)
Stoc
k et
al.
J. M
icro
scop
y, 20
03
To
tal
Inte
rnal
Refl
ect
ion
Flu
ore
scen
ce (
TIR
F)
To
tal
Inte
rnal
Refl
ect
ion
Flu
ore
scen
ce (
TIR
F)
Gla
ssA
ir
Gla
ss
Air
Gold
Gold
Surf
ace
pla
smon r
esonan
ce
Gla
ssA
ir
Gla
ss
Air
•O
bse
rved
into
talin
tern
alre
flec
tion
•A
wav
eth
atdec
ays
exponen
tial
lyw
ith
dis
tanceWikipedia.com
To
tal
Inte
rnal
Refl
ect
ion
Flu
ore
scen
ce (
TIR
F)
To
tal
Inte
rnal
Refl
ect
ion
Flu
ore
scen
ce (
TIR
F)
Gla
ssA
ir
Gla
ss
Air
Gold
Gold
Surf
ace
pla
smon r
esonan
ce
100 n
m
To
tal
Inte
rnal
Refl
ect
ion
Flu
ore
scen
ce (
TIR
F)
Gla
ss
Air
Gold
Air
Gla
ssG
old
Gla
ss
Air
Gold
Air
Gla
ssG
old
100 n
m
Bio
-im
ag
ing
: To
tal
Inte
rnal
Refl
ect
ion
Flu
ore
scen
ce (
TIR
F)
Mic
rosc
op
y (
1)
•A
t TIR
, th
e fl
uore
scen
ce is
exci
ted
in a
ver
y th
in z
one
of
50
~ 1
00
nm
fr
om
a s
ubst
rate
by
an e
vanes
cent
wav
e.
•R
apid
ly d
ecay
ing n
ature
of
the
evan
esce
nt
fiel
d a
llow
s only
the
fluore
scen
t la
bel
s nea
r th
e su
bst
rate
to g
ener
ate
fluore
scen
ce
and t
o b
e im
aged
at
hig
h q
ual
ity
wit
h v
ery
low
bac
kgro
und
fluore
scen
ce, no o
ut-
of-
focu
s fl
uore
scen
ce, an
d n
arro
w d
epth
of
opti
cal se
ctio
n (
50
~ 1
00
nm
).
Bio
-im
ag
ing
: To
tal
Inte
rnal
Refl
ect
ion
Flu
ore
scen
ce (
TIR
F)
Mic
rosc
op
y (
2)
•Li
mit
ed lig
ht-
induce
d d
amag
e to
ce
ll vi
abili
ty
•M
uch
les
s ex
pen
sive
than
confo
cal
mic
rosc
opy
•Be
st s
uit
ed t
o im
age
and p
robe
a ce
llula
r en
viro
nm
ent
wit
hin
a
dis
tance
of
10
0 n
m f
rom
subst
rate
.
TIR
F M
icro
sco
py
Ap
pli
cati
on
s
TIR
F M
icro
sco
py
Ap
pli
cati
on
sBio
-im
ag
ing
: Flu
ore
scen
ce/F
örs
ter
Reso
nan
ce
En
erg
y T
ran
sfer
(FR
ET
) Im
ag
ing
(1
)
•FR
ET is
a dis
tance
-dep
enden
t in
tera
ctio
n b
etw
een t
he
elec
tronic
ex
cite
d s
tate
s of
two d
ye m
ole
cule
s in
w
hic
h e
xci
tati
on is
tran
sfer
red f
rom
a
donor
to a
n a
ccep
tor
wit
hout
emis
sion o
f a
photo
n.
•T
he
effi
cien
cy o
f FR
ET ~
d-6:
use
ful
ove
r dis
tance
s co
mpar
able
wit
h t
he
dim
ensi
ons
of
bio
logic
al
mac
rom
ole
cule
s.
•C
olo
caliz
atio
n o
f pro
tein
s an
d o
ther
m
ole
cule
s ca
n b
e im
aged
wit
h s
pat
ial
reso
luti
on b
eyond t
he
limit
s of
conve
nti
onal
opti
cal m
icro
scopy.
•htt
p:/
/ww
w.o
lym
pusf
luovi
ew.c
om
/applic
atio
ns/
fre
tintr
o.h
tml
Bio
-im
ag
ing
: Flu
ore
scen
ce/F
örs
ter
Reso
nan
ce
En
erg
y T
ran
sfer
(FR
ET
) Im
ag
ing
(2
)
Prim
ary
condit
ions
for
FRET
•D
onor
and a
ccep
tor
mole
cule
s m
ust
be
in c
lose
pro
xim
ity
(typ
ical
ly 1
0–1
00 Å
).
•T
he
abso
rpti
on s
pec
trum
of
the
acce
pto
r m
ust
ove
rlap
the
fluore
scen
ce e
mis
sion s
pec
trum
of
the
donor.
•D
onor
and a
ccep
tor
tran
siti
on d
ipole
ori
enta
tions
must
be
appro
xim
atel
y par
alle
l.
Bio
-im
ag
ing
: Flu
ore
scen
ce/F
örs
ter
Reso
nan
ce
En
erg
y T
ran
sfer
(FR
ET
) Im
ag
ing
(3
)
•A
stan
dar
dtu
ngst
en-h
alogen
lam
phouse
exam
ines
and
reco
rds
the
cells
usi
ng
stan
dar
dbri
ghtf
ield
,phas
eco
ntr
ast,
or
dif
fere
nti
alin
terf
eren
ceco
ntr
ast
(DIC
)ill
um
inat
ion.
•The
argon-k
rypto
nla
ser
bea
mis
firs
tfi
lter
edth
rough
anac
oust
o-o
pti
ctu
nab
lew
avel
ength
dev
ice
tose
lect
spec
ific
exci
tati
on
wav
elen
gth
sbef
ore
pas
sing
toth
eco
nfo
calsc
anhea
d.
Bio
-im
ag
ing
: Flu
ore
scen
ce L
ifeti
me I
mag
ing
M
icro
sco
py (
FLIM
)
•FL
IM p
rovi
des
a s
pat
ial lif
etim
e m
ap o
f fl
uoro
phore
s w
ithin
a c
ell or
tiss
ue.
•Fl
uore
scen
ce lif
etim
e is
hig
hly
sen
siti
ve
to t
he
loca
l en
viro
nm
ent.
•T
empora
l re
solu
tion p
rovi
des
an
opport
unit
y to
stu
dy
dyn
amic
org
aniz
atio
n o
f a
livin
g s
yste
m.
•Fl
uore
scen
ce lif
etim
e is
indep
enden
t of
fluore
scen
ce inte
nsi
ty,
conce
ntr
atio
n,
and
photo
ble
achin
g.
Bio
-im
ag
ing
: Flu
ore
scen
ce L
ifeti
me I
mag
ing
M
icro
sco
py (
FLIM
)
•Fl
uoro
phore
s w
ith s
imila
r sp
ectr
a m
ay
hav
e dif
fere
nt
lifet
imes
in d
iffe
rent
envi
ronm
ents
; lif
etim
e is
a m
ore
sen
siti
ve
pro
be
of
the
envi
ronm
ent.
•In
ord
er t
o r
emove
the
auto
-flu
ore
scen
ce•
Ult
ra-s
hort
las
er a
ppro
aches
(TD
)•
Freq
uen
cy d
om
ain a
ppro
aches
Bio
-im
ag
ing
: Flu
ore
scen
ce L
ifeti
me I
mag
ing
M
icro
sco
py (
FLIM
)
From
GE
Amer
sham
Bio
-im
ag
ing
: Flu
ore
scen
ce L
ifeti
me I
mag
ing
M
icro
sco
py (
FLIM
)
FLIM
-FR
ET
FLIM
-FR
ET
Bio
-im
ag
ing
: Flu
ore
scen
ce C
orr
ela
tio
n
Sp
ect
rosc
op
y (
FC
S)
•In
a s
yste
m w
her
e par
ticl
es o
nly
under
go t
ransl
atio
nal
dif
fusi
on,
fluct
uat
ions
cause
d b
y dif
fusi
on o
f m
ole
cule
s dep
end o
n t
he
size
. R
apid
ly d
iffu
sing s
mal
l m
ole
cule
s pro
duce
rap
id inte
nsi
ty
fluct
uat
ions.
In c
ontr
ast,
lar
ge
mole
cule
s an
d b
iopoly
mer
s (p
rote
ins
and p
rote
in b
ound lig
ands)
exhib
it s
low
ly f
luct
uat
ing p
atte
rns
of
burs
ts o
f fl
uore
scen
ce.
Bio
-im
ag
ing
: Flu
ore
scen
ce C
orr
ela
tio
n
Sp
ect
rosc
op
y (
FC
S)
Imag
ing
FC
S
