Ra#onal engineering of molecular excited states: towards novel indicators for high-­‐resolu#on imaging in cells RANIERI BIZZARRI
NEST, SCUOLA NORMALE SUPERIORE AND NANOSCIENCE INSTITUTE – CNR
PISA, ITALY
SIOF SCHOOL
OF
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
LOOKING
AT
CELL BIOCHEMISTRY
Fluorescence Microscopy
Ø  Poorly invasive
Ø  High sensitivity (down to single molecule)
Ø  High spatial resolution (≤250 nm...see
Superresolution talks...)
Ø  High temporal resolution (down to 1 ms)
Ø  Sensing
Ø  Fluorophore makes the difference
SIOF SCHOOL
OF
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
FLUORESCENCE
heat
excitation
0.1-1 fs
0.01-0.1 ns
0.1-10 ns
emission
Stokes’ shift:
sensitivity
heat
F = η ⋅ ϕ (λem )⋅ ε (λex )⋅ [ I 0 ⋅ C ]
Cuve%e F = η ⋅ ϕ (λem )⋅ ε (λex )⋅ [ I(x, y, z) ⊗ C(x, y, z)]
SIOF SCHOOL
OF
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
Microscope FLUORESCENCE READ-OUTS
Ø  Intensity
Ø  Wavelength (emission/excitation)
Ø  Lifetime
Ø  Depolarization (anisotropy)
14000
2000
Fluorescence emission
Fluorescence (counts)
12000
Monoexponential fit
Instrument response function
10000
1500
8000
1000
6000
4000
500
2000
0
0
0
1
2
3
4
5
6
7
8
9
10
Time (ns)
SIOF SCHOOL
OF
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
INTENSITY: CONCENTRATION
OR
INTRINSIC BRIGHTNESS
☞ Two configurations are
possible
☞ Concentrantions in
cells can not be
determined
SIOF SCHOOL
OF
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
RATIOMETRIC APPROACH: WAVELENGTH SHIFT
60x103
55
Extinction Coefficient (M-1·cm-1)
50
5.0
5.5
6.0
6.5
7.0
7.5
8.0
45
40
35
30
25
20
15
10
5
0
325
350
375
400
425
450
475
500
525
550
Wavelength (nm)
Fluorescence from state 1
S1/S2
Ratio image, concentrationindependent
SIOF SCHOOL
OF
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
Fluorescence from state 2
(anticorrelated)
FLUORESCENCE LIFETIME: RATIOMETRIC
14000
2000
Fluorescence emission
Fluorescence (counts)
12000
Monoexponential fit
Instrument response function
10000
1500
8000
1000
6000
& t )
F(t) = ∑α exp( − +
' τ *
4000
i
i
i
500
2000
0
0
1
2
3
€
0
4
5
Time (ns)
☞  Concentration-independent
☞  Intrinsecally ratiometric
SIOF SCHOOL
OF
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
6
7
8
9
10
FLUORESCENCE: A WONDERFUL MOLECULAR STOPWATCH
heat
excitation
0.1-1 fs
heat
0.01-0.1 ns
OF
Stokes’ shift:
sensitivity
Processes at excited state
Probing the
properties of the
nanoenvironment
Polarity
Viscosity
SIOF SCHOOL
0.1-10 ns
emission
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
Controlling the
photophysical state
of the molecule
Photochromism
FRET
Depletion
ENVIRONMENTAL
•  POLARITY
•  VISCOSITY
SIOF SCHOOL
OF
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
SENSITIVITY
WHAT
SIOF SCHOOL
OF
IS POLARITY?
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
+++++
++ SIOF SCHOOL
OF
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
SOLVATOCHROMIC FLUOROPHORES
Ø
Push-pull aromatic system
e-­‐ Electron-donor unit
SIOF SCHOOL
OF
Aromatic/polyenic spacer
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
Electron-Acceptor unit
ORIGIN
Ø
OF
SOLVATOCHROMISM: SOLVENT RELAXATION
If the dipole moment in the excited state changes compared to the ground state,
there will be rearrangement of polar molecules around the excited molecule
tr
Figure credit: J. Lakowicz, “Principles of Fluorescence Spectroscopy”
SIOF SCHOOL
OF
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
WHY
SIOF SCHOOL
OF
LOOK AT
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
POLARITY?
SOLVATOCHROMIC PROBES: APPLICATIONS
IN
CELLS
Ø  Sensor of endogeneous
biomolecules
Figure credit: P. Nalbant, Science 2004
SIOF SCHOOL
OF
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
SOLVATOCHROMIC PROBES: IN VITRO APPLICATIONS
Figure credit: G. S. Loving, Trends in Biotechnology 2011
SIOF SCHOOL
OF
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
MOLECULAR ROTORS
& VISCOSITY
SIOF SCHOOL
OF
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
MOLECULAR TWISTING
☞  Some molecules undergo structural twisting at excited state
SIOF SCHOOL
OF
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
PHOTOPHYSICS
OF
+ MOLECULAR ROTORS
-­‐ Locally excited (LE) state (planar)
kr= radiative rate constant
kd= non-radiative rate constant
krot= twisting rate constant
krot
-­‐ + * Twisted excited state (TS)
kr(LE)
kd(LE)
kd(TS)
Ground state (planar)
SIOF SCHOOL
OF
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
DEPENDENCE
ON
! VS $
η = η0 exp # &
" Vc %
" Vs %
knr = k exp $ −x '
# Vc &
0
nr
SIOF SCHOOL
OF
LOCAL VISCOSITY
$ V
Φ
k
= exp&& x
1− Φ k
% V
nr
0
0
nr
PHOTONICS
€ , MARCH 31ST-APRIL 3RD, 2014
f
'
)) = aη
(
x
logΦ ≅ x log η + a
log τ ≅ x log η + b
WHY
SIOF SCHOOL
OF
LOOK AT VISCOSITY?
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
VISCOSITY IN CELL ENVIRONMENTS
1 −ΔH
k∝ e
η
RT
Ø Molecular diffusion is a rate-­‐limi8ng step in many metabolic reac8ons and it is Influenced by factors including viscosity of intracellular media Ø Viscosity is probed by diffusion of molecules or by direct imaging SIOF SCHOOL
OF
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
EXPLOITING NATURE
POLARITY & VISCOSITY
COMBO-PROBE
SIOF SCHOOL
OF
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
INSPIRED
BY
NATURE: FLUORESCENT PROTEIN
All fluorescence information is
STORED in the gene/primary
sequence of the protein
SIOF SCHOOL
OF
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
GENETIC ENCODING
Ø
Ø
OF
FLUORESCENCE
Almost any protein can be stained
Usually no interference with
biological processes
SIOF SCHOOL
OF
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
From “Scholarpedia” GFP CHROMOPHORE: ENVIRONMENTAL SENSITIVE
Ø  GFP chromophore is fairly sensitive to its immediate environment
O
N
HO
N
GFP Figure credit: G. Patterson, J. Cell Sci. 2001
SIOF SCHOOL
OF
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
YFP GFP CHROMOPHORE: POOR
EMISSION
Ø  FP chromophore is non fluorescent in water or organic solution on
account of fast twisting at excited state
O
HO
N
N
*
Δ
OH
TRANS
hν
Δ
O
Δ
O
N
N
N
N
HO
CIS
G. Abbandonato et al. Eur. Biophys. J. 2011
V. Voliani et al. J. Phys. Chem. B 2008
SIOF SCHOOL
OF
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
Figure credit: S. Olsen, JACS 2010
BIOCONJUGABLE GE1 FLUOROPHORE
O
N
HO
N
O
X HO
N
*
N
O
X = N, O
X
Ar
(Push)Pull
Ø
Ø
Push(Pull)
Biofunctional group
Fairly high changes in dipole moment upon excitation (8÷10 D)
Biofunctional group allows for bioconjugation
G. Signore et al. Chem Comm 2013
SIOF SCHOOL
OF
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
SPECTRA
OF
GE1
IN
SOLVENTS
CCl4 DMSO O
O
H3CO
O
H3CO
Ø
Ø
Excitation at 405-458 nm, QY from 0.8 to 0.2
Peak red-shift upon increase of environmental polarity
SIOF SCHOOL
OF
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
OCH3
FS-SCALE
TRANSIENT SPECTRA
☞  Solvent rearrangement takes place within 50 ps
0.5
is os be s tic point
a t 5 7 0 nm
-­‐0.5
Δ T /T (% )
0.0
-­‐1.0
-­‐1.5
DMF: polar solvent in D MF ,
no s olve nt c orre c tion
-­‐2.0
400
500
600
700
W a v e le ng th (nm )
In collaboration with Dario Polli and Giulio Cerullo, Polytechnic of Milan
SIOF SCHOOL
OF
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
800
2 0 0
3 2 0
5 6 0
7 4 0
9 2 0
1 2 2 0
1 5 2 0
1 8 2 0
2 2 0 0
2 6 0 0
3 2 0 0
5 2 0 0
1 0 2 0 0
2 0 2 0 0
5 0 8 0 0
SPECTRA
OF
GE1
IN
SOLVENTS
CCl4 DMSO O
O
H3CO
O
H3CO
Ø  GP is concentration independent: fluorophores
suitable for intracellular ratiometric
measurements
SIOF SCHOOL
OF
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
GP =
OCH3
F480−525 − F540−580
F480−525 + F540−580
DEPENDENCE
FROM
DIELECTRIC CONSTANT
0.6
F
GP =
F
0.5
480 − 525
Generalized Polarization
0.4
480 − 525
−F
+F
540 − 580
= a + b log(ε )
540 − 580
0.3
0.2
0.1
€
0.0
-0.1
-0.2
-0.3
-0.4
-0.5
-0.6
0.4
0.6
0.8
1.0
log(ε)
SIOF SCHOOL
OF
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
1.2
1.4
1.6
1.8
VISCOSITY
DEPENDENCE OF
LIFETIME
increasing viscosity Ø  Lifetime of Ge1 is viscosity-dependent
SIOF SCHOOL
OF
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
FORSTER-HOFFMANN RELATIONSHIP
Ø  Slope of viscosiity-dependence is nearly independent of GP (dielectric
constant): universal calibration
SIOF SCHOOL
OF
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
WORKING MODEL
OF
GE1 PHOTOPHYSICS
* E
Viscosity dependent * Solvent
relaxation
50 ps
knr
* kr
k’nr
Very fast <ps
SIOF SCHOOL
OF
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
twisted
state
LET’S
SIOF SCHOOL
OF
GO IN VIVO...
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
INTRACELLULAR MAPS
OF
e
Ø  <e>(ER)=7.4!0.2 (15 cells)
Ø  <e>(NE)=7.5!0.5 (13 cells)
☞  First determination of NE/ER permittivity
SIOF SCHOOL
OF
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
TARGETING
SPECIFICALLY CELL MEMBRANE
Fluorogenic/rotor unit Membrane targe8ng unit G. Signore et al. Chem Comm 2013
SIOF SCHOOL
OF
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
POLARITY PROPERTIES
OF
CELL MEMBRANE
N=12
Ø
Ø
Ø
<e>(PM)=10.6!1.8 (25 cells)
Literature: e=9-11
Intermediate between Ld and Lo
☞  Dielectric value is relevant to
determine capacitance
☞  Correlation with proteins
SIOF SCHOOL
OF
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
DRUG DELIVERY
☞
☞
VIA
CELL PENENTRATING PEPTIDES
CPPs are positively-charged peptides that are known to cross cell membrane and may
thereby deliver biomolecules
Much effort in developing new delivery routes
Figure credit: G. Divita, CRBM, France; S. Fonseca, Adv. Drug Del. Rev. 2009
SIOF SCHOOL
OF
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
TRASACTIVATOR
☞
OF
HIV (TAT)
Tat peptide is by far the most studied CPP
Figure credit: E. Vives, J. Control. Release 2005
SIOF SCHOOL
OF
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
PROMOTING ENDOSOMAL ESCAPE
CPP (Tat) TAT Membrane destabilizing agent SIOF SCHOOL
OF
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
Payload (fluorophore) POLARITY PROPERTIES
Polarity-­‐sensi8ve dye OF
ENDOCYTIC VESICLE
Tat-­‐Alexa647 Colocaliza8on map SIOF SCHOOL
OF
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
MEMBRANE ORGANIZATION
Ø  Old fluid-mosaic model
SIOF SCHOOL
OF
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
MEMBRANE ORGANIZATION
Ø  Compartimentalization model
SIOF SCHOOL
OF
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
MODELING CELL MEMBRANE
☞  Incorporation of the polarity-sensitive probe in model membranes
O
O
O
O
O
N
H
O
O
O
P
O
O
O
OH
O
H3CO
S0
DPPC
SIOF SCHOOL
OF
Ld
POPC
POPC/POPE
POPC/POPS
POPC/Chol
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
Lo
DPPC7/Chol3
Counts
PHASE ORDER
10
4
10
3
10
2
GE1L CHCl3
DPPC, Lβ phase
DPPC/Chol 70/30, Lο phase
POPC, Lα phase
< τ > = 2.95 ns (1.84;3.39)
< τ > = 5.81 ns (2.88;6.09)
< τ > = 1.20 ns (0.7;2.27)
10
1
< τ > = 4.45 ns (2.57;5.15)
10
0
0
5
10
15
20
25
30
Time (ns)
SIOF SCHOOL
OF
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
35
40
45
50
55
PHASOR APPROACH TO
LIFETIME IMAGING
SIOF SCHOOL
OF
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
FLUORESCENCE LIFETIME IMAGING: CHALLENGES
Each pixel i,j
Fi, j (t) = ∑ Ak exp(−t τ k )
k
CHALLENGES OF FLIM
☞  At every pixel there are contributions by several species, and each one
could be multiexponential
☞  To make things worse, we can collect light only for a limited amount of
time which results in 500-1000 photon/pixel: this is barely enough to
distinguish a single exponential from a double exponential
☞  Analysis require global fitting procedures and high computational power
SIOF SCHOOL
OF
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
A NEW FLIM APPROACH: PHASOR PLOT
Each pixel i,j
g (ω ) =
∫I
s (ω ) =
∫I
i ,j
i ,j
€
i ,j
i ,j
i ,j
k
k
mon
si,j
k
g (ω ) =
1
1+ (ωτ )
s (ω ) =
ωτ
1+ (ωτ )
i ,j
ial
t
n
e
on
oexp
g (ω ) = ∑
i ,j
k
ntial
e
n
o
p
x
e
multi
s (ω ) = ∑
i ,j
SIOF SCHOOL
OF
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
gi,j
€
k
i ,j
(t)dt
(t)dt
2
si,2 j + (gi, j −1 2)2 = (1 2 )
€
i ,j
2
€
€
(1/2,0)
∫I
(t)sin(ωt)dt
i ,j
€
I (t) = ∑ A exp(−t τ )
∫I
(t)cos(ωt)dt
f
1+ (ωτ
2
k
f ωτ
1+ (ωτ
k
k
)
2
k
k
)
2
M. Digman & E. Gratton et al.
Biophys. J. 2008
€
PHASOR PLOT: TWO-STATE SYSTEMS
Each pixel i,j
g (ω ) =
∫I
s (ω ) =
∫I
i ,j
i ,j
€
i ,j
i ,j
(t)cos(ωt)dt
(t)sin(ωt)dt
∫I
∫I
i ,j
i ,j
(t)dt
(t)dt
€
I (t) = ∑ A exp(−t τ )
i ,j
k
k
k
Any mixture lays on the segment
joining A and B phasors and allows
for quantification
si,j
€
A (1/2,0)
SIOF SCHOOL
OF
B PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
gi,j
PHASOR PLOT: DETERMINING MEMBRANE ORDER
Ø Quan7fica7on of membrane order si,j
Lo
Ld gi,j
SIOF SCHOOL
OF
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
PHOTOCHROMISM
SIOF SCHOOL
OF
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
FLUORESCENCE: A WONDERFUL MOLECULAR STOPWATCH
heat
excitation
0.1-1 fs
heat
0.01-0.1 ns
OF
Stokes’ shift:
sensitivity
Processes at excited state
Probing the
properties of the
nanoenvironment
Polarity
Viscosity
SIOF SCHOOL
0.1-10 ns
emission
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
Controlling the
photophysical state
of the molecule
Photochromism
FRET
Depletion
PHOTOACTIVATABLE FLUORESCENT PROTEINS
Ø  ADDING AN INTERNAL
TEMPORAL DIMENSION
Ø  New tracking strategies
Ø  Innovative FRET patterns
Ø  Superresolution imaging
SIOF SCHOOL
OF
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
RELEVANCE
OF
RSFPS
Ø  They provide a further (temporal) dimension to fluorescence read-outs
Ø  RESOLFT concept
SIOF SCHOOL
OF
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
PHOTOACTIVATABLE FLUORESCENT PROTEINS
G. Patterson, Science, 2002
SIOF SCHOOL
OF
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
PHOTOACTIVATABLE FLUORESCENT PROTEINS
G. Patterson, Science, 2002
SIOF SCHOOL
OF
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
REVERSIBLE PHOTOSWITCHING
R.A.G. Cinelli, Appl Phys Lett, 2001
S. Habuchi, PNAS, 2005
SIOF SCHOOL OF PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
GFP CHROMOPHORE: POOR
EMISSION
Ø  FP chromophore is non fluorescent in water or organic solution on
account of fast twisting at excited state
O
HO
N
N
*
Δ
OH
TRANS
hν
Δ
O
Δ
O
N
N
N
N
HO
CIS
Figure credit: S. Olsen, JACS 2010
G. Abbandonato et al. Eur. Biophys. J. 2011
V. Voliani et al. J. Phys. Chem. B 2008
SIOF SCHOOL
OF
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
CIS-TRANS PHOTOISOMERIZATION
OF
CHROMOPHORE
HO
dN
= I ⋅σ ⋅ϕ
dt
O
N
j  = switching yield (1/
absorbed photons to
trigger isomerization)
N
jon
joff
trans Y 66 chrom ophore
h!
h!', "
Ø  Fundamental condition:
(joff, jon) >> jbl
O
Ø  Number of cycles ≈
jon(off) / jbl
N
N
jbl
HO
Ø  FPs: jbl ~ 10-6÷10-5
Ø  Y66 Chro: joff, jon ~
10-1 ÷ 1
V. Voliani, J Phys Chem, 2008
cis Y 66 chrom ophore
R. Nifosì, J Phys Chem, 2003
SIOF SCHOOL
OF
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
RATIONAL APPROACH
TO
PHOTOSWITCHING
1.  Residues around chromophore must play a
role in hampering the intrinsical
photochromism of GFP chromophore
2.  Looking for residues that take part in the
photophysics of the chromophore
Glutamic acid 222
SIOF SCHOOL
OF
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
E222:
A
SPECIAL RESIDUE
IN
GFP PHOTOPHYSICS
4.65 Å
2.66 Å
K. Brejc, PNAS, 1997
R. Bizzarri et al. Biochemistry, 2007
G. Jung et al. Micr Res Tech, 2006
SIOF SCHOOL
OF
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
E222Q: INDUCING PHOTOSWITCHING
IN
GFPS
R. Bizzarri, JACS, 2010
SIOF SCHOOL
OF
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
SCHEME
OF
PHOTOISOMERIZATION
Preconversion, Preconversion, p
H 7
.7 Preconversion, ppH H 77.7 .7 Postconversion, Postconversion, p
pH H 7
7.7 .7 pH pH JJump, ump, p
pH H 9
9.6 .6 Relaxed, pH 9.6 R. Bizzarri, J. Am. Chem. Soc, 2010
S. Abbruzzetti, Photochem Photobiol Sci 2011
SIOF SCHOOL
OF
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
SCHEME
OF
PHOTOISOMERIZATION
N
pK t= 9.38 (M ut2Q )
pK t= 9.87 (E Y Q 1)
O
N
+ H
0.1-1 m s
N
O
N
OH
O
Bt
At
!, very slow
(hours)
0.1-1 m s
N
! 25"40 s
h#'
pK= 6 (M ut2Q )
O H pK= 6.87 (E Y Q 1)
O
N
h#
O
N
O
N
A
B
ü joff ≈ 10-4÷10-3 jon≈ 10-3÷10-2
R. Bizzarri, J. Am. Chem. Soc, 2010
S. Abbruzzetti, Photochem Photobiol Sci 2011
SIOF SCHOOL
OF
Ø  Yellow: T203Y/E222Q (EYQ1), S65T/
T203Y/E222Q (EYQ2)
Ø  Green: S65T/E222Q (EQ1), E222Q (Q1)
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
E222Q: TARGETED PHOTOSWITCHING
TRPV1-E222Q
In collaboration with Prof. Alberto Diaspro and Dr. Francesca
Cella – IIT Nanophysics
☞  fatigue: 3-4% (max
100 cycles)
B. Storti et al., J. Biol. Chem, 2012
SIOF SCHOOL
OF
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
☞  Functional imaging
TRPV1-mTUBULE INTERACTIONS: OLID-FRET
☞  OLID: Optical Lock-In Detection
[G. Marriott et al., PNAS, 2008]
☞  Excellent for FRET where D or A
is photochromic
ρ ( x,y ) = ∑
t
{I(x,y,t ) − µI }{R(t ) − µR }
σ Iσ R
☞  R(t) is the “reference” waveform
EYQ1
TagRFP (Evrogen)
B. Storti et al., J. Biol. Chem, 2012
SIOF SCHOOL
OF
PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
TRPV1-mTUBULE INTERACTIONS: OLID-FRET
Donor channel (520-530) FRET channel (640-720 nm) OLID Image (FRET) ρ ( x,y ) = ∑
t
{I(x,y,t ) − µI }{R(t ) − µR }
σ Iσ R
G. Abbandonato et al., 2012
SIOF SCHOOL
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PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
Gerardo Abbandonato Alberto Diaspro Fabio Beltram Francesco Cardarelli Barbara Stor. Giovanni Signore Enrico GraCon Vinod Subramanian SIOF SCHOOL
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Cris7ano Viappiani PHOTONICS, MARCH 31ST-APRIL 3RD, 2014
Paolo Bianchini