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Articles
nAture methods | ADVANCEONLINEPUBLICATION |
membrane proteins are a large, diverse group of proteins,
serving a multitude of cellular functions. they are diffcult
to study because of their requirement of a lipid membrane
for function. here we show that two-photon polarization
microscopy can take advantage of the cell membrane
requirement to yield insights into membrane protein structure
and function, in living cells and organisms. the technique
allows sensitive imaging of G-protein activation, changes in
intracellular calcium concentration and other processes, and is
not limited to membrane proteins. conveniently, many suitable
probes for two-photon polarization microscopy already exist.
Many molecular processes taking place in living cells can be visu-
alized
1,2
by using genetically encoded fluorescent probes (usually
relying on various fluorescent proteins) and techniques of fluores-
cence microscopy, such as fluorescence resonance energy trans-
fer (FRET), fluorescence lifetime imaging, fluorescence recovery
after photobleaching or fluorescence anisotropy imaging
3,4
. While
developing a genetically encoded probe of cell membrane voltage,
we decided to investigate whether anisotropic optical properties
of fluorescent proteins could be used to observe molecular proc-
esses involving membrane proteins.
Optical properties of most molecules are anisotropic. For
single-photon electronic absorption, the absorption properties
of a molecule are characterized by a vector, called the transition
dipole moment (TDM). The rate of light absorption by a molecule
is proportional to the squared cosine of the angle between the
electric field vector (polarization) of the excitation beam and
the TDM vector of the molecule
5
. The TDM direction therefore
represents, in the reference frame of the molecule, the direction
of excitation light polarization with maximum absorption rate.
To our knowledge, the direction of only one fluorescent proteins
TDM has been determined
6
. Two-photon absorption is described
by an absorptivity tensor, and effects of molecular orientation
are generally complex
7
. For some molecules (rod-like)
8
, how-
ever, two-photon absorption rate is proportional to the cosine
to the fourth power of the angle between the excitation light
two-photon polarization microscopy reveals protein
structure and function
Josef Lazar
14
, Alexey Bondar
1,2
, Stepan Timr
5
& Stuart J Firestein
4
polarization and a vector (which we here term two-photon
pseudo-TDM) describing the molecular orientation. Little is
known about fluorescent-protein absorptivity tensors, but GFP
has been shown
6,9
, in vitro, to exhibit anisotropic two-photon
absorption. Light emission is characterized by another TDM
(often similar in orientation to the excitation TDM), with fluores-
cence preferentially emitted in directions perpendicular to the
emission TDM and polarized in a TDM-containing plane.
Anisotropic optical properties of molecules can be observed
in orientationally biased molecular assemblies. The cell mem-
brane can provide sufficient orientational bias to dye molecules
10
,
but fluorescent protein optical properties, linker flexibility and
limited photon counts might prevent observing anisotropic
properties in fluorescent proteinlabeled membrane proteins.
Molecular rotation between excitation and emission, differences
between excitation and emission TDMs, and depolarization by
an objective lens can hamper observations of fluorescence polari-
zation. They should, however, have little effect on observing
dependence of fluorescence intensity on direction of excitation
light polarization or linear dichroism (LD).
Here we show that LD can indeed be observed in many
fluorescent proteintagged membrane proteins by two-photon
polarization microscopy (2PPM). The 2PPM technique pro-
vides information on molecular orientation and can be used for
sensitive monitoring and quantification of protein-protein
interactions and conformational changes. We illustrate its uses
by monitoring G-protein activation and changes in intracellular
calcium concentration.
results
mathematical modeling
To investigate the possibility of using anisotropic proper-
ties of fluorescent proteins to observe cellular processes, we
developed a mathematical model based on geometrical optics
(Supplementary Fig. 1). We modeled absorption
11
of a spherical
cell (approximating an oocyte, a yeast cell, a protoplast or, crudely,
a mammalian cell) and a cylindrical cell (a generic elongated cell,
1
Laboratory of Cell Biology, Institute of Nanobiology and Structural Biology, Global Change Research Centre, Academy of Sciences of the Czech Republic, Nove Hrady,
Czech Republic.
2
Department of Systems Biology, Institute of Physical Biology, University of South Bohemia, Nove Hrady, Czech Republic.
3
Department of Biochemistry
and Molecular Biology, Faculty of Sciences, University of South Bohemia, Ceske Budejovice, Czech Republic.
4
Department of Biological Sciences, Columbia University,
New York, New York, USA.
5
Department of Physics, Faculty of Nuclear Sciences and Physical Engineering, Czech Technical University in Prague, Prague, Czech Republic.
Correspondence should be addressed to J.L. (lazar@usbe.cas.cz).
Received13ApRil2010;Accepted17MAy2011;publishedonline3July2011;doi:10.1038/nMeth.1643

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| ADVANCEONLINEPUBLICATION | nAture methods
Articles
a neuronal projection or a small section of an irregularly shaped
cell). We kept the TDMs of fluorophore moieties attached to
the cell membrane either at a fixed angle
0
with respect to the
cell membrane or modeled them as a Gaussian distribution of
angles , with a mean of
0
and an s.d. (), to consider protein
dynamics, conformational flexibility and nanoscopic membrane
roughness. Our models for single-photon (Fig. 1) and two-
photon (Supplementary Fig. 2) linearly polarized excitation
showed that
0
can have notable effects on both the appearance
of fluorescently labeled cells and on the amount of observed fluo-
rescence (Fig. 1a,b). We visualized differences in absorption of
light with distinct polarizations, or linear dichroism, by coloring
fluorescence generated with horizontal polarization (F
h
) magenta
and fluorescence generated with vertical polarization (F
v
) green
(Fig. 1c,d). Conveniently, the hue of a pixel then directly
expressed the dichroic ratio, r (r = F
h
/F
v
). We used r as a measure
of LD, owing to its simplicity, experimental accessibility with little
image processing and closeness of the log(r) distribution to a normal
distribution (allowing facile statistical analysis). A deviation of r
from 1 (and log(r) from 0) signified presence of LD.
Our model showed (Fig. 1e) that LD should be observ-
able under a variety of suboptimal circumstances (including
membrane roughness and protein confor-
mational flexibility). In fact, absence of LD
should be an exception, generally occur-
ring only for
0
= 54.7 for single-photon
and 52.0 for two-photon absorption
(so-called magic angle of fluorescence
anisotropy) and for very disordered fluoro-
phore orientations. Our model also showed
(Fig. 1f) that even if the distribution of
TDM tilt angles is wide ( = 20), a 1
change in
0
(the mean TDM tilt) should
cause a 24% change in r (12% changes in
both F
h
and F
v
, in opposite directions). Fluorescence of constructs
with TDM close to perpendicular to the cell membrane should
be particularly sensitive to changes in
0
. Results for two-
photon excitation (Supplementary Fig. 2) were similar to those
obtained for single-photon excitation, with more pronounced LD
apparent (typically about two times higher r and r/r). However,
reliability of our two-photon model is limited because of approxi-
mations that had to be made in virtually complete absence of
information on the nature of two-photon absorptivity tensors in
fluorescent proteins.
In summary, our mathematical model predicts that LD should
be widespread among fluorescently tagged membrane proteins,
and it should be observable by single-photon polarization micros-
copy and 2PPM. Even small changes in orientation of the fluo-
rescent moiety should lead to observable changes in LD. Owing
to the monotonic relationship between r and
0
, with knowledge
of fluorescent protein TDMs and other parameters, it should
be possible to use polarization microscopy to gain quantitative
insights into membrane protein structure and function. Thus,
polarization microscopy should allow observation of a range of
molecular processes taking place in living cells and rational design
of sensitive optical probes of these processes.
8
6
e
4
2
0
0 30

0
()
60 90
1/4
0

5
10
20
45
1/2
1
2
4
16
64
256
r
2
l
o
g
2
(
r
)
f
0 30

0
()
60 90
0
5
10
15
(

r
/
r
)
/

0

(
%
)
0

5
10
20
45
0
0.1
0.2
0.3

l
o
g
2
(
r
)
/

0
a
b
c
d
Figure | Mathematical models. (a,b) Simulated
images of a fluorescently labeled spherical cell
(a) and cylindrical cell (b) shown as projections
of a single-photon confocal z-dimension
stack, for fluorophore tilt angle
0
values 0,
22.5, 45, 67.5 and 90 (left to right).
(c,d) Simulated images for a spherical
cell (c) and cylindrical cell (d) showing
fluorescence excited by horizontally and
vertically polarized light (F
h
and F
v
) colored
magenta and green, respectively. Nongray
color (excess of magenta or green) indicates
presence of LD. Direction of polarization and
coloring of corresponding fluorescence is
indicated by double-headed arrows. Orientation
of the fluorophore with respect to the cell
membrane (tilt angle
0
) is indicated by the
schematics in bottom right corners of individual
images. (e) LD, expressed as r = F
h
/F
v
and
log
2
(r), as a function of mean fluorophore
tilt angle
0
, for different widths (described
by ) of distribution of , for the cylindrical
cell in d. (f) Fractional changes in dichroic
ratio (r/r) of the cylindrical cell in d and e
upon a change in mean tilt angle
0
by 1,
for a range of starting tilt angles
0

and tilt angle .

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Articles
Fluorescent protein linear dichroism in live cells
To test whether anisotropic effects predicted by our mathematical
model could be observed, we carried out measurements in living
cells (Fig. 2) on a construct termed doubly lipidated enhanced
GFP (dleGFP)
12
and constructs derived from it. DleGFP consists
of GFP and two lipophilic cell membrane targeting tags, thought to
anchor the fluorescent protein to the cell membrane in an almost
fixed orientation, with the fluorophore (and the single-photon
TDM) close to perpendicular to the cell membrane (Fig. 2a).
When we observed cells expressing dleGFP using a wide-field
fluorescence microscope with a polarizer placed in the excita-
tion path, we discerned only subtle differences between images
acquired with different polarizations (data not shown). When
we used a single-photon laser-scanning confocal microscope, we
observed anisotropic effects consistent with our mathematical
model (Fig. 2bf). These effects, however, were not particularly
pronounced: the maximum observed dichroic ratio, r
max
= (F
h
/
F
v
)
max
, was 2.5 (15 cells). In contrast, when we used two-photon
excitation (Supplementary Fig. 3) to observe dleGFP-expressing
cells, the observed LD was notable (r
max
> 15, 200 cells, Fig. 2gk,
Supplementary Fig. 4 and Supplementary Video 1), consistent
with the pseudo-TDM orientation close to perpendicular to the
cell membrane. In all cases, we also observed weak polarization
of the emitted fluorescence (data not shown). To confirm that it
was indeed the fixed orientation of the fluorophore that caused
the apparent LD, we created and examined, using 2PPM, two
constructs based on dleGFP: internally lipidated eGFP (ileGFP)
and C-terminally lipidated eGFP (cleGFP) (Fig. 2lo). Each of
these contructs contained only one of the two original membrane-
targeting tags, which presumably would not be sufficient to maintain
a fixed orientation of the GFP moiety. Whereas cleGFP absorption
appeared isotropic (r
max
< 1.1, 25 cells), ileGFP showed distinct
LD (r
max
= 6, 32 cells; two-photon pseudo-TDM close to perpen-
dicular to the cell membrane). Thus, the anisotropic phenomena
observed in dleGFP were indeed due to the orientation of the
fluorophore. Furthermore, the LD observed in ileGFP, with
fluorophore orientation likely only partly restricted because of
membrane linkage through a loop region, was consistent with
our mathematical model predicting that LD should be observ-
able even in such cases. Membrane attachment through a flexible
C terminus (in cleGFP) did not give rise to LD.
Our results indicate that the sectioning ability of single-photon
confocal and two-photon imaging is beneficial for observations
of LD. The observed difference between single-photon and two-
photon LD is likely due to nonlinearity of the two-photon exci-
tation process (suppressing excitation by sides of the focal area
containing unwanted polarizations), lower sensitivity of the two-
photon polarization setup to minor alignment errors and stricter
orientational requirements of two-photon excitation. Different
orientations of the single-photon TDM and two-photon pseudo-
TDM in the eGFP molecule are also a possibility. Thus, likely for
a combination of reasons, two-photon polarization fluorescence
microscopy with non-descanned detection appears to report
fluorescent protein LD with markedly higher sensitivity than single-
photon microscopy. Our observations of LD in living mammalian
cells using one-photon polarization microscopy and better using
2PPM validate our mathematical model.
imaging protein-protein interactions: G-protein complexes
To test whether 2PPM of fluorescently labeled membrane pro-
teins can report on protein-protein interactions in living cells,
we focused on processes involving heterotrimeric G proteins
(composed of G, G and G subunits). Because there are
functional, fluorescent proteinlabeled constructs available, we
performed most of our experiments on fluorescent protein
tagged G subunits, particularly of the G
i
-G
o
family. We
investigated 14 different G
i
and G
o
constructs (Fig. 3 and
Supplementary Table 1).
DleGFP
CleGFP IleGFP
r
2.5
1
2.5
r
1.5
1
1.5
r
6
1
6
r
10
1
10
a
b c d e f
g
h i
j
k
l m n o
Figure | Proof of principle. (a) Schematic
of the dleGFP construct. (bf) Single-photon
confocal images of a dleGFP-expressing cell.
Direction of polarization and coloring of
corresponding fluorescence is indicated as in
Figure . Shown are projections of z-dimension
stacks acquired with excitation light polarized
horizontally (b) and vertically (c); a composite
of images in b and c colored magenta and green,
respectively, without any color lookup table
(LUT) adjustment (d); a single confocal slice
of the same cell (e); and the same image as
in e, but after application of an LUT suitable
for displaying the range of dichroic ratio r in
the image (12.5; pixels exceeding this range
appear pure magenta or pure green; only a small
number of such pixels are visible, indicating that
r
max
= (F
h
/F
v
)
max
= ~2.5) (f). (gk) Images as in
bf but acquired using two-photon excitation.
(l) Schematic of the ileGFP construct. (m) A
two-photon section of an ileGFP-expressing cell,
processed as in k but with a color scale covering
a narrower range of values as indicated.
(n) Schematic of the cleGFP construct. (o) A
two-photon section of a cleGFP-expressing cell,
processed as in k but with a different color scale
as indicated. All scale bars, 5 m.

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On the basis of LD, we could distinguish three distinct
categories of fluorescent proteinlabeled G
i
-G
o
constructs.
Constructs were named either as G subunit name followed
by a fluorescent protein insertion site location and protein
name or as N-terminal tag name (GAP43) followed by a fluores-
cent protein name and G subunit name. Two constructs (G
i2
-
Leu91-YFP
13
and G
o
-Gly92-CFP
14
) did not exhibit LD (r
max
< 1.1)
when overexpressed alone or when expressed together with
G
1
and G
2
. Another four constructs (G
i1
-Leu91-CFP
15
,
G
i1
-Leu91-YFP
16
, G
i3
-Leu91-YFP
13
and G
i1
-Ala114-YFP
17
)
showed pronounced LD (r
max
= 26) both when expressed
alone and when expressed together with G
1
and G
2
. A third
group of constructs (GAP43-CFP-G
i1
(ref. 18), GAP43-
CFP-G
i2
(ref. 18), GAP43-CFP-G
i3
(ref. 18) and G
o
-Leu91-
YFP
13
) showed little or no LD when overexpressed alone but
distinct LD (r
max
= 1.53) when expressed together with G
1

and G
2
.
These results illustrate structural differ-
ences between the investigated constructs,
even between constructs of similar overall
amino acid sequences (G
i1
-Leu91-YFP
16
,
G
i2
-Leu91-YFP
13
and G
o
-Leu91-YFP
13
).
These structural differences are consist-
ent with amino acid sequence diversity in
vicinity of the insertion site (the
a
-
b
loop
region) as well as with known
19
functional
differences within this group of G subu-
nits. Our results also support existence
of a physical interaction between at least
four of the overexpressed fluorescent pro-
teintagged G constructs and the overex-
pressed G and G subunits (presumably
forming a G complex), and suggest that
2PPM can be used to monitor protein-
protein interactions in live cells.
To ascertain whether the observed
interactions were physiological and not
an artifact owing to presence of multiple
proteins overexpressed at high levels, we
carried out a series of G-protein activation experiments. We
expressed four constructs together: a Gfluorescent protein,
G
1
, G
2
and a suitable G proteincoupled receptor (GPCR; typi-
cally, the
2a
adrenergic receptor tagged with YFP or CFP). We
then activated the overexpressed receptor with an agonist (nore-
pinephrine). Little or no changes in LD upon receptor activation
could be seen in G constructs lacking LD and in constructs
showing high LD both in absence and presence of G (data not
shown). In contrast, all four Gfluorescent protein constructs
that showed differing amounts of LD in absence and in presence
of overexpressed G exhibited changes in LD upon receptor
activation (>10 cells examined for each construct, >80% cells
showing responses; Fig. 4, Supplementary Videos 2 and 3
and data not shown). Changes in LD in fluorescent protein
tagged subunits could only be observed in cells in which expres-
sion of a receptor (fluorescently labeled) was detectable. The
G
o
-Leu91-YFP
13
construct showed complete disappearance
r
1.5
1
1.5
r
1.5
1
1.5
r
1.5
1
1.5
r
5
1
5
r
1.5
1
1.5
r
1.5
1
1.5
r
1.5
1
1.5
r
5
1
5
a b c d
e f
g
h
Figure | 2PPM imaging of G-protein complexes. (ad) Images of cells expressing fluorescently
tagged G subunits GAP43-CFP-G
i2
(a), G
i2
-Leu91-YFP (b), G
o
-Leu91-YFP (c) and G
i1
-Leu91-
YFP (d). (eh) Images of the same G subunits as in ad but expressed together with G
1
and G
2
.
Coloring is as in Figure . Scale bars, 5 m.
Figure | 2PPM imaging of G-protein
activation. (a) Cyan fluorescence of a
cell expressing GAP43-CFP-G
i2
, G
1
,
G
2
and
2a
-adrenergic receptor-YFP
before addition of norepinephrine (left),
after addition of norepinephrine (center)
and after removal of norepinephrine
(right). Coloring is as in Figures and .
(b) Plot of LD (expressed as r and log
2
(r)) of
the GAP43-CFP-G
i2
expressing cell in a, as a
function of time. Triangles and squares denote
data from the indicated horizontally and
vertically oriented sections of the membrane,
respectively. Dashed traces indicate s.e.m.,
n = 110160 pixels. The 10-s period of
presence of norepinephrine is indicated by
a bar (top left). (c) Yellow fluorescence of a
cell expressing G
o
-Leu91-YFP, G
1
, G
2
and

2a
adrenergic receptorCFP before addition
of norepinephrine (left), after addition of norepinephrine (center) and after removal of norepinephrine (right). (d) Plot as in b but for the
G
o
-Leu91-YFPexpressing cell in c (n = 90160 pixels). All scale bars, 5 m.
r
2
1
2
r
1.5
1
1.5
0.50
0.25
0
0.25
L
o
g
2
(
r
)
0.50
0 50
Time (s)
100 150
1/1.4
1/1.2
r
r
1
1.2
1.4
a
1
0.5
0
0.5
L
o
g
2
(
r
)
1
0 50
Time (s)
100 150
1/2
1/1.5
1
1.5
2
b
d c
Horizontal
Vertical

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of LD, consistent with dissociation of the G-G-G complex.
In contrast, the fluorescent proteintagged G
i
subunits typically
showed only a decrease, not complete disappearance of LD
upon activation, consistent either with incomplete (~80%)
activation (Supplementary Note) or with rearrangement
16

(rather than dissociation
17,20
) of the G-protein complex upon
activation. Notably, a constitutively active mutant
21
(Q204L) of
G
i1
-Leu91-YFP
16
showed significantly higher LD (P < 0.001)
when expressed together with G and G (r
max
= 2.8,
0
> 52,
25 cells) than when expressed alone (r
max
= 2.0,
0
> 52, 30 cells),
providing strong evidence for existence of a G-protein trimer in
activated G
i
subunits.
Our results show that it is possible to use 2PPM to visualize
physical interactions between subunits of heterotrimeric G proteins.
The observed molecular interactions are physiological: that is,
in presence of a suitable GPCR (and only in its presence) they
respond to presence of a receptor agonist, in a fashion consistent
with current knowledge. The size of the responses observed during
G-protein activation is remarkable: F/F of up to 50% in a particular
channel. In comparison, only ~2% changes in donor fluorescence
and 10% in acceptor fluorescence have been reported
16
for FRET
between Gfluorescent protein and Gfluorescent protein sub-
units. Sensitivity of 2PPM is such that it should allow distinguish-
ing between resting and activated states of the current G
i
-G
o

constructs within 200 s (Supplementary Discussion). Thus,
2PPM permits visualization of protein-protein interactions in liv-
ing cells, yielding insights into molecular mechanisms of G-protein
activation and allowing monitoring the process of activation of
GPCRs in live cells, in real time, with very high sensitivity.
imaging changes in protein conformation: calcium imaging
To test the ability of 2PPM to report conformational changes in
membrane proteins, we used 2PPM to image changes in intracel-
lular calcium concentration. Several lines of genetically encoded
calcium indicators have become commonly used
22
. Two of them
(Cameleon-based
23
and troponin-based
24
) rely on conformational
changes reported by FRET. One of the cameleon sensors, lynD-
3cpV
25
, is membrane-tethered. It consists of a calcium-sensing
domain sandwiched between two fluorescent proteins (CFP and
circular permuted (cp)Venus). About 30% changes in both donor
and acceptor fluorescence have been reported for lynD3cpV upon
an intracellular calcium concentration increase
25
.
Our 2PPM lynD3cpV observations (Fig. 5 and Supplementary
Table 2) showed that at low calcium concentrations, the CFP
moiety is in a fairly well-defined orientation with respect to
the cell membrane (r
max
= 1.5; Fig. 5a). In contrast, the cp
Venus moiety showed little LD (r
max
= 1.07) in resting state cells.
Upon an increase in intracellular calcium concentration through
stimulation by ATP, LD in cpVenus increased (r
max
= 1.2).
Upon removal of ATP, cpVenus LD returned to original values
(Fig. 5b,c and Supplementary Video 4). LD of CFP remained
constant (data not shown). Calibration of cpVenus LD by
controlling intracellular calcium concentration allowed char-
acterization of the construct (saturated cpVenus r
max
= 1.42,
dissociation constant (K
d
) = 0.64 M, Hill coefficient of 3.0;
Supplementary Note) and determination of calcium concen-
trations during ATP stimulation (0.40.6 M in different cells;
mean = 0.495 M; r
max
= 1.151.20) (Fig. 5d). These values
are in very good agreement with results we obtained by FRET
(K
d
= 0.71 M, Hill coefficient of 2.6, ATP-induced calcium
concentrations 0.40.6 M, with mean of 0.505 M), although
the confidence intervals for calcium concentration values
determined in individual cells were considerably larger in
2PPM experiments than in FRET experiments (Fig. 5d and
Supplementary Discussion).
Our results are consistent with the lynD3cpV design and
demonstrate that even small changes in LD can be reproducibly
observed, quantified and used to infer both structural and func-
tional information. During ATP stimulation, cpVenus r
max
/r
max
=
~0.15, corresponding to F/F of 7% in both the F
h
and F
v

channels, in opposite directions. Both F
h
and F
v
are measured
virtually simultaneously, so although photobleaching adversely
affects both values, it has little effect on the F
h
/F
v
ratio. Even the
modest observed response size should (lynD3cpV response
rate permitting) allow 2PPM observations of calcium spikes of
13 ms (Supplementary Discussion).
0.2
0.1
0
0.1
L
o
g
2
(
r
)
0.2
0.6
0.4
0.2 L
o
g
2
(
r
m
a
x
)
L
o
g
2
(
F
c
p
V
e
n
u
s
/
F
C
F
P
)
0
0.5
1.5
2.5
0 50
Time (s)
100 150 0.001 0.01 0.1
[Ca
2+
] (M)
[
C
a
2
+
]

(

M
)
1 10 100
1/1.1
r
1
1.1
0 150
0
0.5
FRET
LD
Time (s)
0.4
0.4
0.5
0.6
0.5 0.6
[Ca
2+
]
FRET
(M)
[
C
a
2
+
]
2
P
P
M

(

M
) c d
r
1.3
1.3
1
r
1.15
1.15
1
a b
Figure | 2PPM imaging of intracellular
calcium concentration through conformational
changes in the calcium sensor lynD3cpV.
(a) CFP signal of lynD3cpV. (b) cpVenus
fluorescence before application of ATP (left),
during application of ATP (center) and after
ATP removal (right). Coloring is as in Figures .
All scale bars, 5 m. (c) Plot of cpVenus LD
(expressed as r and log
2
(r)) of the outlined
sections (inset) of the cell shown in a and b,
as a function of time. Triangles and squares
denote data from the indicated horizontally and
vertically oriented sections of the membrane,
respectively. Dashed traces indicate s.e.m.,
n = 160200 pixels. The 40-s period of
presence of ATP is indicated by a bar. (d) LD
of cpVenus and FRET of lynD3cpV as a function
of intracellular calcium concentration. Error
bars, s.e.m.; n = 1530 cells. The curve shown
is a prediction for intermediate values of K
d
(0.68 M) and Hill coefficient (2.8) obtained from LD and FRET measurements. Top inset, intracellular calcium
concentrations for six typical cells stimulated by ATP, determined by FRET (horizontal axis) and 2PPM (vertical axis). Error bars, s.e.m., n = 60400 pixels with
|| < 3 and n = 10,00013,000 pixels used for FRET measurements. Bottom inset, experiment in c, interpreted in terms of intracellular calcium concentration.

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| ADVANCEONLINEPUBLICATION | nAture methods
Articles
discussion
The 2PPM method is applicable to many protein targets and
fluorescent proteins (Supplementary Tables 1 and 2) in vari-
ous cell types and organisms (including Saccharomyces cerevisiae,
Caenorhabditis elegans and Drosophila melanogaster;
Supplementary Fig. 5). Apart from membrane proteins, the
method is also applicable to cytoskeletal proteins and poten-
tially even to cytoplasmic proteins (after anchoring to the cell
membrane or cytoskeleton, or after photoselection). The tech-
nique can be very sensitive (potentially allowing observations
of cellular processes with sub-millisecond temporal resolution,
Supplementary Discussion), allows multiplexing and is resilient
to bleaching artifacts. As 2PPM requires only one fluorescent
tag (unlike FRET), many existing fluorescent proteintagged
constructs are likely to act as 2PPM probes (similarly to the
constructs used in this study). The information 2PPM provides
(fluorophore orientation and its changes) is largely complemen-
tary to that provided by other methods, such as FRET. The 2PPM
approach can be used in rational development of both 2PPM and
FRET-based probes.
In a basic form, 2PPM can easily be implemented on any
two-photon microscope using a simple polarization modula-
tor (Supplementary Fig. 3b). Such a setup allows observing LD
in constructs with r
max
> ~1.5. More sophisticated equipment
(Supplementary Fig. 3c) allows detection of LD in most fluores-
cent proteintagged membrane proteins, and observation and
monitoring of even very rapid processes.
Unlike other techniques, 2PPM relies on the shape and orienta-
tion of the observed cell. Thus, cells with long sections of outline
oriented horizontally or vertically are more suitable for measure-
ments than others. We have not found this to be a substantial
drawback (Supplementary Fig. 4), and in the future it is likely to
be mitigated by advances in polarization modulation and software.
Quantification of 2PPM data does require accounting for cell
shape, but our results show that our procedure (Supplementary
Fig. 6) allows accurate quantification of biophysical properties
(Fig. 5d and Supplementary Discussion).
In principle, 2PPM is also capable of providing quantitative
information about membrane-protein structure, including
determining the orientation, with respect to the cell mem-
brane or cytoskeleton, of the fluorescent protein (or another
fluorescent moiety) attached to the studied protein. Such
determinations are currently not possible because of our
limited knowledge of the micro- and nanoscopic geometry
and dynamics of the cell membrane of living cells, the detailed
optical parameters of our imaging system and the nature of
two-photon absorptivity tensors of fluorescent proteins. Until
these parameters are determined, information presented
in Supplementary Figure 2 can serve as a crude guide to
structural interpretation of 2PPM results.
Owing to its conceptual and experimental simplicity, robust-
ness, wide applicability, availability of probes and high value
of information it can provide, we believe that 2PPM could
become an important method for visualization and analysis
of molecular processes in living cells. Many phenomena that
are at present out of reach of imaging techniques, such as the
investigation of G proteinGPCR interactions and observing
individual action potentials in neurons, may soon become
observable using 2PPM.
methods
Methods and any associated references are available in the online
version of the paper at http://www.nature.com/naturemethods/.
Note: Supplementary information is available on the Nature Methods website.
AcknowledGments
We thank L. Nedbal, Z. Benedikty, R. Uhl, M. Buenemann, Z. Peterlin and
J. Leps for discussions; C. Seebacher and A. Reshak for assistance with
imaging; T. Bergmann, K. Tosnerova and members of the Institute of Physical
Biology cell culture facility for technical assistance; R. Axel for inspiration;
and G. Miesenboeck (Oxford University), M. Buenemann (Philipps University
Marburg), A. Tinker (University College London), R. Tsien (University of
California, San Diego), M. Asahina-Jindrova (Institute of Parasitology,
Academy of Sciences of the Czech Republic), C. Berlot (Geisinger Clinic),
J. Blahos (Institute of Molecular Genetics, Academy of Sciences of the Czech
Republic), K. Deisseroth (Stanford University), S. Engelhardt (Technical
University Munich), N. Gautam (Washington University in St. Louis), A. Gilman
(University of Texas, Dallas), S. Ikeda (US National Institute on Alcohol Abuse
and Alcoholism), M. Jindra (Institute of Entomology, Academy of Sciences
of the Czech Republic), T. Knopfel (RIKEN Brain Science Institute), Y. Kubo
(National Institute for Physiological Sciences, Japan), J. Ludwig (University of
South Bohemia), R. Miller (Northwestern University), M. Rasenick (University
of Illinois at Chicago), T. Montgomery, H. Sitte and T. Steinkellner (Medical
University of Vienna) and M. Wildwater (Utrecht University) for constructs,
cells and animals. The research was supported by the European Commission
(FP7 Marie Curie International Reintegration grant PIRG-GA-2007-209789
MemSensors (J.L.), FP6-2005-Health project LSHG-CT-2007-037897
Autoscreen (J.L.)), Columbia University Science Fellowship to J.L., McKnight
Innovation in Neuroscience Award (S.J.F. and J.L.), Czech government
institutional grants MSM6007665808, MSM6007665801 and AVOZ60870520
(J.L.), EU Structural Funds grant CZ.1.07/2.3.00/09.0203 (J.L. and S.T.),
University of South Bohemia fellowship (A.B.) and J.L.s personal savings.
Author contriButions
J.L. conceived the idea, carried out mathematical modeling and analyses,
performed initial microscopy experiments, developed image-processing software,
directed the project and wrote the manuscript. A.B. performed microscopy
experiments, prepared constructs, analyzed data and devised experimental
strategies. S.T. developed software for quantitative analysis. S.J.F. contributed
inspiration, consultations and funding.
comPetinG FinAnciAl interests
The authors declare competing financial interests: details accompany the
full-text HTML version of the paper at http://www.nature.com/naturemethods/.
Published online at http://www.nature.com/naturemethods/.
reprints and permissions information is available online at http://www.nature.
com/reprints/index.html.
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online methods
Mathematical modeling. We developed a mathematical model of
a spherical and a cylindrical cell (Supplementary Fig. 1) based on
geometrical optics and similar to published models
10,11
. Briefly,
the surface of the cell contained idealized fluorophore molecules,
each possessing a single absorption and emission TDM with iden-
tical orientations. Angles and (spherical cell), or an angle
and a y coordinate (cylindrical cell) defined the position of a
fluorescent molecule on the surface of the model cell. We defined
the orientation of the TDM for each fluorophore molecule by an
angle between the TDM and a normal to the cell membrane,
and an angle describing rotation along an axis normal to the
cell membrane. We approximated excitation polarization by a
single, linear polarization in the focal plane. The model took into
account collection of fluorescence by a high-numerical-aperture
lens. We carried out modeling for a range of discreet values of ,
for Gaussian distributions of described by a mean value of
0

and an s.d., , and for other distributions. We performed some
modeling of individual confocal or two-photon slices, but we pre-
ferred to model z-dimension stack projections, to avoid effects
of different cell sizes. For two-photon excitation, we assumed a
cosine to the fourth power relationship of the rate of absorption
on the angle between the electric field vector of the excitation light
and the two-photon pseudo-TDM. The models were implemented
using Mathematica and Perl. Images were generated using the
PerlMagick Perl module.
Constructs. DleGFP
12
was a gift from G. Miesenboeck. From
dleGFP we derived the cleGFP and ileGFP constructs through
removal of one of the membrane targeting tags by PCR (Phusion
polymerase, New England Biolabs (NEB)) using suitable primers
(cleGFP_F and cleGFP_R for cleGFP, and ileGFP_F and ileGFP_R
for ileGFP; Supplementary Table 3). We purified PCR products
by agarose gel electrophoresis, extracted the DNA from the gel
(QIAexII gel extraction kit, Qiagen), phosphorylated the DNA
(T4 polynucleotide kinase, NEB), circularized it (T4 DNA ligase,
NEB) and transformed it into Escherichia coli (DH5, Invitrogen),
using standard or manufacturer-recommended procedures. For
each construct, we grew two bacterial colonies in 5 ml of LB
medium with 100 mg l
1
ampicillin. We isolated the plasmids
(QIAquick Spin kit, Qiagen) and verified sequences of the inserts
by DNA sequencing (Agowa).
Other constructs were gifts from M. Buenemann (Gi1-Leu91-
YFP, Gi1-Leu91-CFP, Gi2-Leu91-YFP, Gi3-Leu91-YFP,
Go-Leu91-YFP, Gs-Gly72-YFP, 2aAR-YFP, 2aAR-CFP,
a2A adenosine receptorYFP, 2AR-CFP and 2AR-YFP),
A. Tinker (GAP43-CFP-Gi1, GAP43-CFP-Gi2, GAP43-
CFP-Gi3, GAP43-CFP-Go and GAP43-YFP-Go), Y. Kubo
(mGluR1-i1-YFP, mGluR1-i2-YFP and mGluR1-C-tail-
YFP), R. Miller (G1-YFP), S. Ikeda (G1, G2 and G2-CFP),
M. Rasenick (Gs-D71-GFP), A. Gilman (Gi1-Ala114-YFP
and Gi2-Ala114-YFP), N. Gautam (Gi2-Leu91-CFP and Go-
Gly92-CFP), C. Berlot (Gq-Phe124-GFP, Gs-Gly72-CFP and
PKC-DsRed), J. Blahos (mGluR2-GFP), S. Engelhardt (1AR-
Cer/YFP), T. Knopfel (VSFP 3.1, Addgene plasmid 18951),
K. Deisseroth (opto-1-AR-YFP and opto-2-AR-YFP; Addgene
plasmids 20947 and 20948), E. Boyden (FCK-ChR2-GFP and
FCK-Halo-GFP; Addgene plasmids 15814 and 14750, respec-
tively) and R. Tsien (SuperGluSnFR and lynD3cpV).
Mammalian cell culture. We cultured HEK293 cells at 37 C
under an atmosphere of 95% air, 5% CO
2
, in Dulbeccos modified
Eagles medium with Glutamax I and high glucose (Invitrogen),
supplemented with 10% fetal bovine serum. Before observa-
tion, we typically plated cells on 8-chamber microscopy slides
(-Slides, Ibidi) and transfected them using Lipofectamine 2000
(Invitrogen), according to the manufacturers protocol. We per-
formed microscopy experiments 2448 h after transfection. We
performed G-protein activation and calcium-imaging experiments
in flow chambers (-Slide I
0.8
Luer slides, Ibidi), using a peristaltic
pump (Minipuls3, Gilson). We washed cells with HEPES-buffered
Hanks balanced salt solution (pH 7.4) and stimulated them with
norepinephrine (()-norepinephrine (+)-bitartrate salt; Sigma)
at a final concentration of 1 M for G-protein activation, or
with ATP (Sigma) at a final concentration of 10 M for calcium
imaging. To calibrate lynD3cpV responses, we applied calcium
chlorideEGTA buffers containing ionomycin (Sigma, 5 M) and
1 nM to 39 M of free calcium for 30 min before imaging.
Polarization fluorescence microscopy. Polarization microscopy
was performed on a customized laser-scanning confocal micro-
scope iMic (Till Photonics) equipped with a Yanus beam scan-
ner (Till Photonics), a 488-nm argon laser (LGK 7812-1, Zeiss)
for single-photon confocal imaging and a tunable pulsed tita-
nium:sapphire laser (Chameleon Ultra II with GVD compensa-
tion, Coherent) operated at 800 nm (CFP) or 960 nm (GFP, YFP
and DsRed) for two-photon imaging. We used a UApoPlan/IR
60, numerical aperture (NA) 1.2 water-immersion objective
lens (Olympus). For single-photon confocal imaging, we used a
combination of a long-pass dichroic beam splitter (FF495-Di02,
Semrock) and a Brightline 500/24 (Semrock) emission filter. For
two-photon imaging (with non-descanned detection), we used a
long-pass dichroic (FF705-Di01, Semrock) and a suitable emis-
sion filter (Brightline 479/40 for CFP, Brightline 500/24 for GFP
and Brightline 542/27 for YFP; all Semrock), combined with an
infrared-blocking filter (HQ700SP-2P, Chroma). Fluorescence was
detected by a photomultiplier (R6357, Hamamatsu Photonics),
operated at 700900 V, providing 16-bit output.
A polarization modulator (Supplementary Fig. 3) allowed
rotating polarization of the excitation beam. In our initial experi-
ments (Figs. 2 and 3, Supplementary Fig. 4 and Supplementary
Video 1) we used a simple, manually operated polarization modu-
lator (Supplementary Fig. 3b) consisting of a Glan-laser polar-
izing beam splitter (CVI Laser) and a rotatable half-wave plate
(488 nm zero-order, or 6901,080 nm achromatic; Thorlabs).
We acquired, successively, two images of the same cell, with the
polarization of the excitation beam oriented horizontally and ver-
tically in the reference frame of the acquired image. All images
were acquired at 100200 nm pixel size (typically, 100 nm) and
10 s pixel
1
acquisition time.
In our later experiments (Figs. 45, Supplementary Figs. 56,
and Supplementary Videos 24) we used a rapid polarization
modulator (RPM) (Supplementary Fig. 3c) custom-made by
BME Bergmann. The RPM consisted of a Pockels cell (RTP-3-20-
AR800-1000, Leysop) and a high-voltage driver synchronized with
the microscope, so that polarization of the excitation beam would
alternate (between horizontal and vertical) between acquisition
of subsequent pixels. Typically, we acquired an image at 50 nm
100 nm pixel size and 10 s pixel dwell time. We split the resulting

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nAture methods
image into two images, one consisting of odd-numbered pixels of
the original image (acquired with horizontal polarization) and the
other consisting of even-numbered pixels of the original image
(vertical polarization). We imaged and quantitatively analyzed at
least 15 cells for each construct or combination of constructs.
Image processing. We performed basic image processing with
ImageJ, using standard ImageJ tools and in-housedeveloped
macros. We subtracted background of acquired images and
adjusted brightness (but not contrast). We colored correspond-
ing images acquired with horizontal and vertical polarization
magenta (equal intensity blue and red) and green, respectively,
and merged them into a composite image. When desired, the
resulting image was colored using a color LUT designed to show
a suitable range of F
h
/F
v
ratios while keeping the overall bright-
ness constant. This procedure allowed simultaneous visualization
of LD (of any size) and total fluorescence intensity. For small
values of LD (r < 1.5), we applied a small (<10%) correction to
compensate for differences in beam intensity and group velocity
dispersion between the two polarizations. In one case (Fig. 5b),
the images shown are averages of three subsequent frames of a
time series (Supplementary Video 4). To create the graphs shown
in Figures 4b,d and 5c, we thresholded acquired images to remove
nonmembrane-localized fluorescence, and calculated the F
h
/F
v

ratios and log ratios for the selected area.
Quantitation of linear dichroism. For accurate quantitation
of LD, both for individual optical sections and for z-dimension
stacks of images, we used a combination of available software
and in-housedeveloped Matlab (Mathworks) scripts (accessi-
ble at http://www.usbe.cas.cz/people/lazar/celler/). Briefly, in two
dimensions (a single, approximately equatorial optical section
of a cell, Supplementary Fig. 6a), we defined pixels belonging
to the cell outline by a combination of rolling ball background
subtraction and thresholding, and, if needed, by manual removal
of obviously extraneous areas. We then approximated the cell
outline by a spline, using ImageJ and A 3D editing plugin
26
. We
determined the direction of the spline (angle , Supplementary
Fig. 1a) in each point by calculating the first derivative of the
spline function. For each pixel judged to belong to the cell outline,
we then set the orientation of the cell outline to be the orientation
of the nearest point on the spline. Application of this procedure
produced a value of angle (cell outline orientation) for each pixel
of the cell outline. Combining values of with LD data yielded an
empirical relationship between LD and cell-surface orientation
(LD-CSO relationship, Supplementary Fig. 6b). For quantitation,
we used solitary cells, and we took r
max
(or log
2
(r
max
)) to be the
mean value of r (or log
2
(r
max
)) for = 0 3.
We developed an analogous procedure for processing vertical
stacks of images. Briefly, we created a three-dimensional (3D)
model of a cell, in the form of a triangular mesh, using Amira
software (Visage Imaging). For each triangle, we calculated (from
vertex coordinates) a normal vector, and described it by angles
and (Supplementary Fig. 6c). For each optical section, we
identified pixels belonging to the cell surface as described for
LD quantitation in two dimensions. For each pixel judged to be
part of the cell surface, we set the cell surface orientation (angles
and ) to be that of the nearest point on the triangular mesh.
Associating data on cell surface orientation with information on
LD yielded an empirical LD-CSO relationship (Supplementary
Fig. 6d). Owing to similarity of results obtained by 2D and 3D
processing, and comparative ease of 2D processing, we generally
used the 2D procedure for LD quantitation.
Quantitation of molecular interactions from 2PPM measure-
ments. We calculated the extent of G-protein activation by using
equation 12 in the Supplementary Note. We determined the
parameters of lynD3cpV/calcium interaction (K
d
, Hill coefficient
n) by combining equation (12) (Supplementary Note) with Hill
equation (x
A
= [Ca
2+
]
n
/ (K
d
n
+ [Ca
2+
]
n
)), and fitting the result-
ing expression to values of r
max
measured at intracellular calcium
concentrations ranging from 1 nM to 39 M (n = 1030 cells for
different concentrations).
FRET imaging and data analysis. We used the same microscope
and optics for FRET imaging as we did for 2PPM imaging. We
used a decolimated 405 nm laser (50 mW; PowerLaser) for wide-
field epi-illumination. To minimize photobleaching, we attenuated
the laser output to 1 mW, and we only applied illumination during
image acquisition. We acquired CFP and cpVenus images sequen-
tially, using a Brightline 458 nm long-pass dichroic beamsplitter,
a Brightline 472/30 (CFP) or 540/25 (cpVenus) emission filter
(all from Semrock) and an Imago QE (Till Photonics) camera,
with exposure time set to 500 ms. We used background-subtracted
images to calculate log ratios of cpVenus and CFP fluorescence.
We used a standard mathematical model of ratiometric FRET
experiments
27
to describe our data, and a value of S
f2
/S
b2
= 2.6.
We obtained the parameters of the molecular interaction (K
d
, Hill
coefficient n) by fitting the experimental data by the chosen math-
ematical model using the Solver tool of Microsoft Excel.
26. Cardona, A., Hartenstein, V. & Romero, R. Early embryogenesis of planaria:
a cryptic larva feeding on maternal resources. Dev. Genes Evol. ,
667681 (2006).
27. Grynkiewicz, G., Poenie, M. & Tsien, R.Y. A new generation of Ca2+
indicators with greatly improved fuorescence properties. J. Biol. Chem. 0,
34403450 (1985).
Nature Methods
Two-photon polarization microscopy reveals protein structure
and function
Josef Lazar
14
, Alexey Bondar
1,2
, Stepan Timr
5
& Stuart J Firestein
4


Supplementary Figure 1 Mathematical models.
Supplementary Figure 2 Results of mathematical modelling for two-photon excitation.
Supplementary Figure 3 Setup for two-photon polarization microscopy.
Supplementary Figure 4 A 2PPM image of HEK293 cells expressing dleGFP.
Supplementary Figure 5 Examples of cells and constructs showing linear dichroism.
Supplementary Figure 6 Quantitation of linear dichroism.
Supplementary Table 1 G-protein constructs
Supplementary Table 2 NonG-protein constructs
Supplementary Table 3 Primers
Supplementary Note Linear dichroism of a two-state system.
Supplementary Discussion Signal-to-noise analysis.

Note: Supplementary Videos 14 are available on the Nature Methods website.
Nature Methods: doi:10.1038/nmeth.1643

x
z
y

x
z
y

a b c
Supplementary Figure 1: Mathematical models.
(a) Model of a spherical cell. Position of the fluorophore on the surface of the cell is described
by angles and . (b) Model of a cylindrical cell. Position of the fluorophore on the surface
of the cell is described by coordinates and y. (c) Definition of variables describing the orienta-
tion of the fluorophore (transition dipole moment): mean tilt angle
0
, standard deviation , and
rotational angle . (d) Behavior of a linearly polarized laser beam used for single-photon excita-
tion, passing through an objective lens (geometrical optics approximation). Polarization of the
beam remains perpendicular to the direction of light propagation. In the focal area, the present
directions of polarization add up in a vector fashion, restoring the original polarization of the
laser beam. Fluorescence excitation occurs throughout the light double cone created by the
objective lens. Observed area can be restricted by use of a confocal pinhole. (e) Behavior of a
linearly polarized laser beam used for two-photon excitation, passing through an objective lens
(geometrical optics approximation). Similar to d, but with excitation occurring invariably only in
the focal area.
Focal point:
Elsewhere:
=
Focal point:
Elsewhere:
=
d e
Nature Methods: doi:10.1038/nmeth.1643
0
5
10
20
45

0 30 60 90
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0
10%
20%
30%
40%

0
()

l
o
g
2
(
r
)
/

0 (

r
/
r
)
/

0
0
5
10
20
45

0 30 60 90
-2
0
2
4
6
8
10
12
14
16
1/4
1
4
16
64
256
1024
4096
16384

0
()
l
o
g
2
(
r
)
r
d c
b
a
Supplementary Figure 2: Results of mathematical modeling for two-photon excitation.
(a) Simulated images of a fluorescently labeled spherical cell a projection of a z-stack, for
TDM tilt angle
0
values 0, 22.5, 45, 67.5, and 90. Direction of polarization and coloring of
corresponding fluorescence is indicated by double-headed arrows. Linear dichroism (non-gray
color) is present for all shown
0
values. (b) Same as in a, but for a cylindrical cell. (c) Graph
of observed LD expressed as r (r = F
h
/F
v
) and log
2
(r), as a function of
0
and for experimental
arrangement in b. LD (r 1) is generally present, except when
0
= 50-55 or is very high
(45). (d) Graph of expected fractional changes in dichroic ratio (r/r) upon a change in mean
tilt angle
0
, for experimental arrangement in b. The graph, in effect, shows the percentage
change in r upon a change in the mean tilt angle
0
by 1, for different starting values of
0
(x-
axis) and for different standard deviations () of the Gaussian distribution of tilt angles. Even for
wide distributions of ( = 20), a 1 change in
0
typically leads to a sizeable (2-4%) change
in r.
Nature Methods: doi:10.1038/nmeth.1643
laser
polarization
modulator
detector
scanning mirrors
dichroic mirror
objective lens
a
b c
Supplementary Figure 3: Setup for two-photon polarization microscopy.
(a) Schematic diagram of a two-photon polarization microscope. (b) A simple polarization
modulator composed of a Glan-laser polarization beamsplitter and a manually rotatable half-
wave plate. (c) A rapid polarization modulator (RPM) based on a Pockels cell, driven in syn-
chrony with scanning of the microscope.
Nature Methods: doi:10.1038/nmeth.1643
Supplementary Figure 4: A 2PPM image of HEK293 cells expressing dleGFP.
The image was created by merging images acquired with horizontal and vertical polarization
(colored magenta and green, respectively). Polarization of the excitation beam was rotated
manually between acquisition of individual images. No background subtraction, brightness
or contrast adjustments were applied. Scale bar: 10 m.
Nature Methods: doi:10.1038/nmeth.1643
1.3
1.3
1
r
1.3
1.3
1
r
1.3
1.3
1
r
6
6
1
r
10
10
1
r
1.3
1.3
1
r
1.3
1.3
1
r
1.3
1.3
1
r
1.3
1.3
1
r
1.3
1.3
1
r
f g h i j
a b c d e
Supplementary Figure 5: Examples of cells and constructs showing linear dichroism.
Coloring as in Figs. 2-5. Scale bars: 5 m. a-f, HEK293 cells. (a) Protein kinase C-DsRed (C-
terminal fusion)
1
. The top, non-activated cell shows cytoplasmic localization of fluorescence and
no LD, unlike the bottom, activated cell (r
max
= 1.5,
0
< 52). (b) a2A-adenosine receptor-YFP (C-
terminal fusion)
2
; r
max
= 1.5,
0
> 52. (c) Metabotrapic glutamate receptor mGluR1-YFP
(intracellular loop 1 insertion)
3
; r
max
= 1.5,
0
>> 52. (d) 2a-adrenergic receptor-CFP (C-terminal
fusion)
4
, r
max
= 1.5,
0
> 52. (e) VSFP3.1, an engineered sensor of membrane voltage
5
, r
max
=
1.5,
0
> 52. (f) Microtubule associated protein tau-GFP (C-terminal fusion)
6
. LD is visible both in
cytoplasmic and in membrane associated microtubules (r
max
= 1.5,
0
> 52). (g) DleGFP7
expressed in a rat hippocampal neuron (r
max
= 1.5,
0
< 52). (h) Yeast S. cerevisiae expressing
potassium channel TOK1-GFP (r
max
= 1.5,
0
< 52)
8
. (i) An epithelial seam cell of a live C.
elegans worm, expressing pleckstrin-homology domain-GFP (r
max
= 1.5,
0
< 52). (j) Epithelium
of fruitfly D. melanogaster expressing E-cadherin-GFP (r
max
= 1.5,
0
> 52)
9
.
Nature Methods: doi:10.1038/nmeth.1643
d b
a
r
5
3
1

1 -1 0 0.5 -0.5 1.5 -1.5

r
0
3
2
1

0
1
2
-2
-1

-2
-1
0
1
2
0
4
0
/2
-/2

-/2 0 /2
0
/2
-/2

c
Supplementary Figure 6: Quantitation of linear dichroism.
(a) Analysis of geometry of an ileGFP expressing cell, a single optical section. From left to
right: an image used for construct characterization, processed as in Fig. 2; pixels judged to be
part of the cell outline; a spline approximating the cell outline; the same spline, colored accord-
ing to orientation (angle ); pixels of the cell outline colored according to the orientation (angle
) of the closest point on the spline, with color bar shown. (b) Graph of LD as a function of cell
outline orientation (LD/CSO relationship), for the cell in a. The data was fitted by a curve
predicted for a particular combination of fluorophore tilt angle
0
and tilt angle distribution
width (
0
= 0.7, = 0.1). (c) Three-dimensional reconstruction of an ileGFP expressing cell,
shown as a triangular mesh, and colored according to cell surface orientation (angles , ),
with color bar shown. (d) Graph of LD as a function of cell surface orientation, for the cell in
c. The data was fitted by a surface predicted for the same combination of
0
and as in b.
Nature Methods: doi:10.1038/nmeth.1643
Supplementary Table 1: G-protein constructs
When expressed alone
When co-expressed
with G|1, G2
G-protein construct
Design /
fluorescent
protein
insertion site
LD r
max
o
0
n LD r
max
o
0
n
GAP43-CFP-Goi1
10

20-AA tag
CFP Goi1
- < 1.05 N/A 20 + 2.0 < 52 25
Goi1-Leu91-YFP
11
91-92 + 2.8 > 52 38 + 3.1 > 52 33
Q204L-Goi1-Leu91-
YFP
11

91-92 + 2.1 > 52 25 + 2.9 > 52 30
Goi1-Leu91-CFP
12
91-92 + 2.0 > 52 24 + 3.1 > 52 20
Goi1-Ala114-YFP
13
114-115 + 2.1 > 52 30 + 3.2 > 52 30
GAP43-CFP-Goi2
10

20-AA tag
CFP Goi2
- < 1.05 N/A 35 + 2.1 < 52 37
Goi2-Leu91-YFP
14
91-92 - < 1.05 N/A 30 - < 1.05 N/A 25
Goi2-Ala114- YFP
13
114-115 + 1.1 > 52 16 + 1.1 > 52 15
GAP43-CFP-Goi3
10

20-AA tag
CFP Goi3
- < 1.05 N/A 22 + 2.1 < 52 18
Goi3-Leu91-YFP
14
91-92 + 2.0 > 52 17 + 3.2 > 52 18
GAP43-CFP-Goo
10

20-AA tag
CFP Goo
- < 1.05 N/A 15 + 1.6 < 52 15
GAP43-YFP-Goo
10

20-AA tag
YFP Goo
- < 1.05 N/A 20 + 1.6 < 52 22
Goo-Leu91-YFP
14
91-92 - < 1.05 N/A 52 + 1.7 < 52 62
Goo-Gly92-CFP
15
92-93 - < 1.05 N/A 15 - < 1.05 N/A 15
Gos-Asp71-GFP
16
71-82 - < 1.1 N/A 15 + 1.3 < 52 15
Gos-Gly72-CFP
17
72-85 - < 1.05 N/A 20 + 1.8 < 52 25
Gos-Gly72-YFP
2
72-85 - < 1.1 N/A 15 - < 1.1 N/A 15
Goq-Phe124-GFP
1
124-125 + 1.8 < 52 25 + 1.8 < 52 25
G|1-YFP
18
N-terminal - < 1.05 N/A 15 + 1.4 > 52 20
Nature Methods: doi:10.1038/nmeth.1643
Supplementary Table 2: Non-G-protein constructs


Construct LD r
max
o
0
n
o2aAR-CFP
4
+ 1.7 < 52 35
o2aAR-YFP
4
+ 1.3 < 52 30
|2AR-YFP
4
- <1.05 N/A 20
|2AR-CFP
4
- <1.05 N/A 15
a2A adenosine receptor-YFP
2
+ 1.4 < 52 25
mGluR2-GFP - <1.05 N/A 15
mGluR1o-i1-YFP
3
+ 6.0 < 52 25
mGluR1o-i2-YFP
3
- <1.05 N/A 20
mGluR1o-C-tail-YFP
3
+ 1.6 < 52 20
1AR Cer/YFP
19

-
-
<1.05 (Cerulean)
<1.05 (YFP)
N/A 15
Opto-o1AR-YFP
20
+ 1.2 < 52 15
Opto-|2AR-YFP
20
+ 1.2 < 52 15
LynD3cpV
21

+
+
1.4 (CFP)
1.07-1.42 (cpVenus)
> 52 (CFP)
< 52 (cpVenus)
> 100
SuperGluSnFR
22

-
+
<1.05 (CFP)
1.2 (YFP)
N/A (CFP)
> 52 (YFP)
15
PKC-dsRed
1
+ 1.5 > 52 15
VSFP3.1
5
+ 1.6 < 52 15
tau-GFP
6
+ 1.2 < 52 25
Channelrhodopsin2-GFP
23
+ 1.3 < 52 15
Halorhodopsin-GFP
1
+ 1.2 < 52 10
Prestin-YFP + 1.2 < 52 25
Nature Methods: doi:10.1038/nmeth.1643
Supplementary Table 3: Primers

Primer name Sequence
cleGFP_F TTTAATCTGTGTTGTAACTC
cleGFP_R GATGGAGGCGTTCAACTAG
ileGFP_F ACGACCCTAATGTGTACCGATTCT
ileGFP_R CCGCTTCCCTTTAGTGAG



Nature Methods: doi:10.1038/nmeth.1643
SUPPLEMENTARY NOTE

LD of a two-state system
The composition of a mixture (AB) of two states (A, B) can be inferred from the dichroic
ratio of the mixture (r
AB
), if the dichroic ratios of the individual components (r
A
, r
B
) of
the mixture are known. If x
A
, x
B
are the fractions of the protein present in the form A and
B, respectively, and we assume that the total amount of fluorescence (F = F
A
+ F
B
) is
approximately independent of x
A
and x
B
(F
A
= x
A
. F; F
B
= x
B
. F), we can write:

vA
A
F
r =
hA
F
, and
vB
B
F
r =
hB
F
. (Eq. 1, 2)
Thus,
A A A
vA
r r r
F
vA A vA A hA
F F x F F F
=

= = , (Eq. 3)
and therefore F
r
F
A
vA
1 +
=
x
A
(Eq. 4)
Similarly, it can be shown that
F
r
F
B
vB
1 +
=
x
A
1
, F
r
F
A
hA
1 +
=
x r
A A
, and F
r
F
B
hB
1 +
=
x r
A B
) 1 (
. (Eq. 5, 6, 7)
Thus, the dichroic ratio of a two-component system is:
) 1 )( 1 ( ) 1 (
) 1 )( 1 ( ) 1 (
1
) 1 (
1
1 1
+ + +
+ + +
=
+

+
+
+
+
+
=
+
+
=
A A B A
B A A B A A
B
A
A
A
B A
vB vA
hB hA
AB
r x r x
r r x r r x
F
r
x
F
r
x
F
r
F
r
F F
F F
r
) 1 (
B A A A
r x r x
(Eq. 8)
) ( 1
B A A A
AB
r r x r
r
+
=
) ( ) 1 (
B A A A B
r r x r r + +
(Eq. 9)
Nature Methods: doi:10.1038/nmeth.1643
) ( 1
B A A A
AB
r r x r
r
+
) ( ) ( ) ( ) 1 (
B A A B A A B B A A B A B
r r x r r x r r r x r r r + + +
= (Eq.10)
) ( 1 ) ( 1
B A A A
B
B A A A
B AB
r r x r
r
r r x r
r r
+
) 1 )( ( ) ( ) (
B B A A B A A B A A B
r r r x r r x r r x r
+ =
+
+ +
+ = (Eq.11)
The fraction of the component A can then be determined:

) 1 )( ( +
) 1 )( (
=
AB B A
A
r r r
x
+
A B AB
r r r
) (
B A A B AB
r r x r r
(Eq. 12)
For values of r
A
and r
B
similar to each other [specifically, for (r
A
-r
B
) considerably smaller
than (1+r
A
)], Eq. 11 can be approximated by a simple linear relationship,
+ = (Eq. 13)
and the fraction of component A then is:

) (
) (
B A
B AB
A
r r
r r
x

= (Eq. 14)
Apart from determinations of composition of mixtures, the above equations can
also be used for determining K
d
, by substituting for x
A
in Eq. 12 from a suitable
equilibrium equation [such as the Hill equation we used for determining the K
d
of
lynD3cpV, x
A
= [Ca
2+
]
n
/ (K
d
n
+ [Ca
2+
]
n
) ]. Alternatively, K
d
values can be determined
by applying a standard mathematical description developed for ratiometric FRET
imaging
24
to ratiometric 2PPM data (F
h
/F
v
). For a first order equilibrium reaction we can
write:
[Ca
2+
]
|
|
.

\
|
|
.

\

=
vA
vB
AB A
B AB
d
F r r
K
| | | | F r r
(Eq. 15)
The term F
vB
/F
vA
is not easily experimentally accessible. However, if we assume (as we
Nature Methods: doi:10.1038/nmeth.1643
did in Eqs. 1-14) that the total fluorescence intensity (F
h
+ F
v
) is the same for forms A
and B, we can approximate F
vB
/F
vA
by (r
A
+ 1)/(r
B
+ 1). For a system described by a Hill
equation we can then write:
[Ca
2+
]
n
|
|
.

\
+
|
|
.

\

=
1
) (
B
A
AB A
B AB n
d
r r r
K
| | + | | 1 r r r
(Eq. 16)
Eq. 16 is equivalent to a combination of Eq. 12 and the Hill equation.
Nature Methods: doi:10.1038/nmeth.1643
SUPPLEMENTARY DISCUSSION
Signal-to-noise analysis
Calibration of photomultiplier detector response through Poisson noise analysis
has allowed us to interpret pixel intensities in terms of photon counts. Typically, during
the pixel dwell time (10 us) used by our setup, 100-200 photons were detected in each
pixel within the cell outline. The 95% confidence interval (95%CI) for a single
measurement and Poisson distribution of 150 events is 127-177. Thus, a single pixel
allows measurement of fluorescence intensity with ~20% accuracy. A Poisson
distribution of the mean value of 150, being far from 0, can be approximated by a normal
distribution with a mean of 150 and variance of 150 (thus, with both the standard
deviation and standard error equal to \150 = ~12). For values of r = F
h
/F
v
close to 1 we
can estimate the standard deviation of r to be \2 standard deviation of individual
fluorescence measurement/mean fluorescence intensity = 1.41 12 / 150 = 0.11.
Therefore, from a single pixel we know the value of r within ~22%, with 95% confidence.
A short stretch (0.5 um) of the cell outline contains about 100 pixels, allowing
measurement of r to 2.2% accuracy with 95% confidence. The acquisition time for 100
pixels with two polarization states is 2 ms, indicating that 2PPM is capable of
distinguishing between two states differing in r by ~5% in 2 ms. In molecular systems
displaying a larger change in r, the temporal resolution of 2PPM can be well below 1 ms.
Our experimental results (Figs. 4, 5) are in excellent agreement with this analysis.

Nature Methods: doi:10.1038/nmeth.1643
For r
max
determination, apart from the photon noise issues analyzed above, noise
and errors stemming from geometrical approximation of cell shape need to be considered.
Our data (Fig. 5d) indicates that the 95%CI for r
max
in a single cell is typically 8-12% of
the mean (including 2-3% caused by photon noise). Averaging of several cells allows
narrowing the CI. While the 95%CIs for intracellular calcium concentrations determined
by FRET are typically ~40 smaller than for concentrations determined by 2PPM, this
needs to be considered in context. The FRET determinations used 6,000 longer
illumination time and ~50,000 larger cell area from which quantitative data was being
acquired. Improvements in polarization modulation and image processing software
(allowing using larger parts of cells to be used for r
max
determination), and other
enhancements should allow very fast and accurate determinations of r
max
and other
biophysical properties in individual cells.

Nature Methods: doi:10.1038/nmeth.1643
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Nature Methods: doi:10.1038/nmeth.1643