Articles

Scanless two-photon excitation of channelrhodopsin-2

Eirini Papagiakoumou1,5, Francesca Anselmi1,5, Aurélien Bègue1,5, Vincent de Sars1, Jesper Glückstad2,
Ehud Y Isacoff 3,4 & Valentina Emiliani1

Light-gated ion channels and pumps have made it possible
to probe intact neural circuits by manipulating the activity
of groups of genetically similar neurons. What is needed
now is a method for precisely aiming the stimulating light
© 2010 Nature America, Inc. All rights reserved.
fibers7–9 or by miniaturized LEDs10. The limitations imposed
by the low penetration depth of blue light and the lack of optical
sectioning inherent for single-photon excitation are partly miti-
gated by the low excitation levels necessary for photoactivation
(~1 mW mm−2)7 and by genetic targeting of ChR2 expression,

at single neuronal processes, neurons or groups of neurons.

We developed a method that combines generalized phase
contrast with temporal focusing (TF-GPC) to shape two-photon
excitation for this purpose. The illumination patterns are
generated automatically from fluorescence images of neurons
and shaped to cover the cell body or dendrites, or distributed
groups of cells. The TF-GPC two-photon excitation patterns
generated large photocurrents in Channelrhodopsin-2–
expressing cultured cells and neurons and in mouse acute
cortical slices. The amplitudes of the photocurrents can be
precisely modulated by controlling the size and shape of the
excitation volume and, thereby, be used to trigger single action
potentials or trains of action potentials.
Since early studies1, an important component of what we know
about the brain has come from electrical stimulation or local
drug application, but these approaches have poor spatial resolu-
tion, require physical contact or slow exchange and cannot target
specific cell types. Advanced microscopy and optogenetics have
recently greatly advanced the precision of these manipulations.
Optical stimulation can be less invasive, allows superior spatial
and temporal resolution as well as specificity for cell type and
quick reversibility. The most widely used optogenetic tool is
Channelrhodopsin-2 (ChR2)2, a cation-selective channel that,
like other genetically encoded pumps and channels, can be func-
tionally expressed in mammalian neurons under the control of
cell-specific promoters. Upon illumination with blue light, the
cation flux generated through ChR2 produces rapid membrane
depolarization, which can evoke reliable trains of action poten-
tials at frequencies up to 200 Hz (ref. 3).
Several methods have been used to activate ChR2, including
widefield lamp illumination4, laser-scanning illumination5 or
illumination with a micro-LED array6. For in vivo applications,
ChR2 is usually stimulated by light sources coupled to optical
permitting applications in freely moving mice, including the
optical control of whisker movement7, locomotion8, the prob-
ing of Parkinsonian neuronal circuits9 and the restoration of
visual function in retinal degeneration11. The precision of these
manipulations would be considerably enhanced if two-photon
excitation could be used to confine the stimulation in three
dimensions to select subsets of neurons that cannot be distin-
guished based on cell-specific promoters or distinct subcellular
compartments in a neuron.
Several factors make it challenging to use two-photon excita-
tion to stimulate ChR2. First, ChR2 has a low conductance (~80
femtosiemens)12, making it difficult to drive action potentials by
photoactivation with the standard small two-photon excitation
volume (~2–5 μm3). Increasing excitation density would not help,
as saturation of excited ChR2 channels is quickly reached owing
to the high two-photon absorption cross-section of ChR2 (~260
Goeppert-Mayer units at 920 nm) and the long lifetime of the
conducting excited states (~10 ms) as demonstrated in the first
paper, to our knowledge, reporting action-potential ­generation
by two-photon ChR2 photoactivation13. Increasing the fraction of
the cell membrane stimulated by two-photon excitation by under-
filling the objective back aperture13 or fast scanning of the laser
beam through multiple positions13,14 suffers from substantial loss
in axial (z axis) or temporal resolution.
The above considerations suggest that the optimal illumination
for ChR2 stimulation in two-photon excitation experiments is
with low excitation density, large excitation area, and millisec-
ond and microscale resolution. We recently proposed a solution
that could satisfy these requirements: a method for generating
two-photon light patterns by combining digital holography15–17
with a dispersive optical setup for temporal focusing18 (TF-DH),
in which large two-dimensional areas are excited rapidly with a
depth resolution of <6 μm19,20.

1Wavefront-Engineering Microscopy Group, Neurophysiology and New Microscopies Laboratory, Centre National de la Recherche Scientifique, Unité Mixte de Recherche

8154, Institut National de la Santé et de la Recherche Médicale U603, Paris Descartes University, Paris, France. 2Department of Photonics Engineering, Technical
University of Denmark (Fotonik), Lyngby, Denmark. 3Department of Molecular and Cell Biology and Helen Wills Neuroscience Institute, University of California,
Berkeley, Berkeley, California, USA. 4Material Science Division and Physical Bioscience Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA.
5These authors contributed equally to this work. Correspondence should be addressed to V.E. (valentina.emiliani@parisdescartes.fr).

Received 5 February; accepted 20 August; published online 19 september 2010; doi:10.1038/nmeth.1505

848  |  VOL.7  NO.10  |  OCTOBER 2010  |  nature methods


However, the TF-DH approach has two limitations intrinsic to
digital holography. First, the light distribution of the illumination
spots has notable spatial intensity fluctuations, called speckles
(~50% in the two-photon excitation mode19). Simple solutions
for eliminating the speckles, such as averaging over many ran-
domized speckle patterns by introducing a rapidly rotating dif-
fuser after the dispersive grating19 or by projecting a sequence of
shifted holograms21, are not practical because they deteriorate
depth resolution, lengthen exposure time and, in the case of the
diffuser, cause power loss. Second, the rapid phase variations,
typical of holographic wavefronts, interfere with the geometrical
dispersion of the grating, which is the basis of temporal focusing,
causing a broadening of the axial resolution19,20.
To overcome these limitations, we developed a method for
optically confining two-photon excitation patterns in three
dimensions by combining temporal focusing and generalized
phase contrast (TF-GPC)22–24 (Supplementary Note 1). We used
this sculpted two-photon illumination to activate ChR2 in mouse
cultured neurons and cortical slices with sufficient efficacy to
© 2010 Nature America, Inc. All rights reserved.
Articles
were homogeneous and had sharp edges (Supplementary Fig. 1),
and the generated wavefront had a spatially smooth output phase
(Supplementary Fig. 2), providing the optimal conditions for
attaining the limiting axial resolution of temporal focusing20. To
characterize the effect of temporal focusing, we measured beam
propagation with the diffraction grating replaced by a mirror
(Fig. 1c,d). We observed an almost cylindrical excitation shape
with light modulation resulting from the interference between the
diffraction component generated by the sharp edges of the circular
phase profile and the light from the circular pattern. We compared
these results with the measured z-axis distribution when we placed
the grating for the temporal focusing at the output mapping plane
of the GPC (Fig. 1e). The range of focus, b, defined as the full
width at half maximum (FWHM) of the axial integrated inten-
sity, was extracted from the integrated intensity for the different
planes of the optical stack shown in Figure 1e (Fig. 1f) and had
a value of ~3 μm. This value is a twofold better confinement in
the z axis than one obtains with TF-DH19,20 and corresponds to
the axial resolution of a conventional line-scanning two-photon

reliably fire action potentials with millisecond temporal resolu-
tion and low excitation power when the light was shaped over
the cell body, one or more dendritic subdomains or multiple
cells simultaneously.
RESULTS
Spatiotemporal light patterning by TF-GPC
The optical path combining the GPC light mapping scheme with
temporal focusing is schematized in Figure 1a. We illustrate three
examples of GPC illumination visualized by exciting a thin fluo-
rescent layer (Fig. 1b): a circular spot, an excitation shape tailored
to the geometry of a fluorescence image of a dendrite and an exci-
tation shape tailored to multiple cell bodies. The intensity profiles
microscope, that is, the limiting axial resolution achievable with
temporally focused two-dimensional patterns18 (Fig. 1f). This
axial resolution was pattern-independent (data not shown).
Shaped two-photon excitation of ChR2-expressing HEK 293 cells
We used whole-cell patch-clamp recordings from HEK 293 cells
transfected with a plasmid encoding GFP-tagged mutant of ChR2
(ChR2(H134R)-GFP) to measure ionic currents through ChR2
channels in response to two-photon illumination. We used wide-
field fluorescence imaging to visualize cells expressing ChR2. We
used the TF-GPC system to modulate photocurrent amplitude by
generating excitation spots of variable size and shape (Fig. 2a).
The average current amplitude for the illumination of the whole

Figure 1 | TF-GPC design. (a) Output of Ti: a Beam SLM Intensity

b
sapphire laser is expanded to illuminate an Attenuator expander
LCOS-SLM that is located at the front focal Phase
plane of a 4-f (f is focal length) imaging setup x
Ti:sapphire laser �/2 Polarizer
(L1 and L2 are lenses with focal lengths of
L1 y
400 mm and 300 mm, respectively), with a CCD x
Intensity
phase-contrast filter (PCF) at the confocal plane PCF camera
between the lenses. For temporal focusing, a L2 Dichroic y
Phase mirror
blazed reflectance grating (830 lines mm−1)
L Intensity x
is placed at the output mapping plane of the Objective
Diffraction
GPC system. L, lens with focal length of 500 mm. grating
Beam Patch y
dump pipet Phase
Intensity and phase distributions at the SLM,
Sample
grating and sample planes are indicated.
CCD, charge-coupled device. (b) Images of c d e f
circular spot (20 μm diameter; left) and TF-GPC
shaped patterns (right) created by two-photon TF-DH
Intensity (a.u.)

Theory
excitation of a thin (~1 μm) fluorescent layer
(λexc = 780 nm; objective 60×, 0.9 numerical FWHM = 3 µm
aperture (NA)). Shaped patterns were based
on a confocal image of a Purkinje cell (center,
z CCD
top) and widefield fluorescence image of CA1 z z
–20 –10 0 10 20
hippocampal neurons loaded with Oregon Green
y y y z-axis position (µm)
Bapta (center, bottom), in selected regions of
interest (yellow outlines). (c,d) Theoretical (c)
and experimental (d) y-z section axial propagation of the 20 μm spot without temporal focusing. (e) Experimental y-z section axial propagation of the
spot in d with temporal focusing (left). Experimental propagation measured on double microscope setup 16,19 (right). Scale bars, 10 μm. (f) Axial profile
of fluorescence intensity in e (TF-GPC) compared to 20 μm circular spot generated by TF-DH and theoretical curve for the axial integrated intensity in
line-scanning two-photon microscopy (theory), where I ≈ (1 + (z / zR)2)−0.5, with I being the integrated light intensity, z the actual axial position and zR
the Rayleigh range for a focused Gaussian beam in our experimental conditions, that is, zR = 0.8 μm.

nature methods  |  VOL.7  NO.10  |  OCTOBER 2010  |  849

 Articles

a b c Measured

Normalized integrated current

Antishape Shape

–2 Intensity (a.u.)
1 Simulation

0
0

10
20
–1
0
z-axis position (µm)

50 pA
100 pA
50 ms
50 ms Antishape 0
Shape –30 –20 –10 0 10 20 30
z-axis position (µm)

Figure 2 | Two-photon photoactivation of ChR2 by TF-GPC in HEK 293 cells. (a) Fluorescence images (λexc = 488 nm, bandwidth 10 nm) of HEK 293
cells transfected with plasmids encoding ChR2(H134R)-GFP with superimposed excitation patterns (red) of increasing size (top; left to right spots have
5, 8, 10, 12 and 14 μm diameter and whole cell). Whole-cell photocurrents (bottom) evoked by 10-ms laser pulses (red bars) of excitation spots in
respective images above (λexc = 850 nm, 0.45 mW μm−2). (b) Widefield epi-fluorescence image of an HEK 293 cell transfected with plasmids encoding
ChR2(H134R)-GFP and superimposed excitation patterns illuminating around the cell (antishape; top left) or covering the whole cell (shape; top right).
Whole-cell photocurrents (bottom; 0.38 mW μm−2). Scale bars, 20 μm. (c) Normalized integrated currents elicited by moving shaped excitation pattern
shown in b along the z axis by steps of 2 μm (dots) (0.17 mW μm−2) and simulation with cell modeled as a parallelepiped of size x = 15 μm, y = 26 μm,
z = 10 μm, corresponding to measured (x, y) and estimated (z, distance between the two experimental peaks) cellular dimensions. Inset, excitation
volume represented as infinite sheet of light with axial distribution given by the experimental curve, measured by scanning the 40×, 0.8 NA objective,
through a fluorescent layer and integrating the light collected by the CCD camera (red line is a guide for the eye). λexc = 850 nm.
© 2010 Nature America, Inc. All rights reserved.

cell was −365 ± 166 pA (n = 3 cells). This corresponded to ~40%
of the peak current generated by illumination of the whole cell
(that is, including the bottom and top cell membranes) with blue
light (midpoint wavelength of excitation, λexc = 470 nm, bandwidth
17 nm, 2.5 mW μm−2). Illumination of an area just outside the edge
of the cell (antishape) reduced the evoked current by ~87% compared
to the current evoked by illuminating the whole soma (−27 ± 10 pA
for antishape; −205 ± 137 pA (± s.e.m.; n = 5 cells) for shape covering
the whole cell) (Fig. 2b). Some of the off-target current could be due
to the excitation of thin processes that were not visible.
Because a key advantage of two-photon excitation is the ability
to confine the excitation narrowly along the z axis, we tested the
axial resolution by moving the whole cell–shaped excitation in
z-axis steps of 2 μm. The photocurrent dropped off sharply at focal
points above and below the cell (Fig. 2c; FWHM = 13.5 ± 4.0 μm
a We used TF-GPC illumination patterns to photostimulate dissociated
20 mV
(± s.e.m.; n = 7 cells)). In some cells (4 of 7), the ­photocurrents
reached a maximum near the top cell membrane, decreased near
the cell center and increased again to a second maximum at the
bottom cell membrane. This was not evident in all cells, perhaps
because of differences in cell flatness. The experimental results
could be simulated by convolving the geometry of the cell mem-
brane, approximated with a parallelepiped, with the excitation
volume represented by an infinite sheet of light with an axial dis-
tribution given by the experimental curve (Fig. 2c).
These results demonstrate that TF-GPC permits a precise two-
­photon activation of ChR2 channels at nonsaturating ­excitation den-
sity, and that this stimulation evokes photocurrents that approach those
achieved with blue-light, single-photon ­widefield illumination but that
afford the axial resolution advantage of two-photon excitation.
Shaped two-photon excitation in cultured cortical neurons
mouse cultured cortical neurons transfected with plasmids encod-
ing ChR2(H134R)-GFP. We used widefield illumination to find the
ChR2-expressing neurons. We used fluorescence images of the cells to
generate local excitation patterns of different sizes and shapes (Fig. 3a).
In current clamp, a large illumination shape covering the whole cell
evoked action potentials (16 of 19 cells, 50 ms pulse). Action poten-

tials could also be evoked with an excitation area selectively shaped

30 ms
onto the dendrites (Supplementary Fig. 3). Finally, we generated
action-­potential trains by shaped excitation that covered the whole cell
b c body (Fig. 3b) in response to a burst of light pulses at 5, 10 and 15 Hz
(Fig. 3c). Excitation with a sustained 1-s light pulse generated action-
potential trains with a firing frequency of up to 17 Hz (Fig. 3d).

Figure 3 | Action potential generation by two-photon TF-GPC in primary

50 mV

neuronal culture. (a) Excitation spots of increasing coverage of cell

100 ms body superimposed on the fluorescence image of a neuron (top). Current
clamp recordings (bottom) corresponding to the condition above. Action
potentials were generated for an excitation area covering about one-third
of the surface of the cell body (0.60 mW μm−2, 30 ms pulse duration).
(b) Excitation pattern used for action-potential train experiments.
d (c,d) Light-activated trains of action potentials at 5, 10 and 15 Hz
(0.5 mW µm−2; c) or by a 1-s pulse (0.6 mW µm−2; d). Scale bars, 20 μm.
λexc = 920 nm, 40×, 0.8 NA objective.

850  |  VOL.7  NO.10  |  OCTOBER 2010  |  nature methods

 Articles
Figure 4 | Two-photon photoactivation by TF-GPC a b c 3 µm
7 µm
in cortical brain slices. (a) Widefield fluorescence 10 µm
image of a layer V pyramidal neuron positive 400 15 µm 40

Current (–pA)

Latency (ms)
for ChR2-YFP (λexc = 488 nm, 5 nm bandwidth).

4 mV
300 30
(b) Plot of the peak current (inward currents
200 10 ms 20
indicated in negative picoamperes) (n = 6 cells)
as a function of excitation spot diameter (average 10
100
on three trials in all cases, 0.52 mW μm−2, 10 ms 8 9 10 1112 13 14 15 10 11 12 13 14 15
pulse). (c) Voltage responses to photoexcitation Spot diameter (µm) Spot diameter (µm)

with spots of increasing size (left; 3, 7, 10 and

40 mV
20 mV
15 μm in diameter; 0.52 mW μm−2; 10 ms pulse). d
100 ms 50 ms
Action potential latency as a function of the 15 µm
spot 10 Hz
excitation spot diameter (right; n = 7 cells). Error
bars, s.e.m. Average of three trials is shown in 10 µm
spot
all cases. (d) Widefield epi-fluorescence image 20 Hz
of cell body loaded through the patch pipet with 7 µm
the fluorescent indicator Alexa Fluor 594 (left; λexc spot

= 590 nm, bandwidth 10 nm). Action potential 5 µm 30 Hz

trains evoked by 1-s light pulse with increasing spot
excitation spot size (middle). Average frequencies
were 11.8 ± 0.8 Hz (6 trials) for a 15 μm spot, 8.7 ± 0.3 Hz (3 trials) for a 10 μm spot, 7.7 ± 0.3 Hz (3 trials) for a 7 μm spot and 4.8 Hz ± 0.3 (4 trials) for a
© 2010 Nature America, Inc. All rights reserved.

5 μm spot (0.4 mW μm−2). Example of action potential firing after light stimulation at 10 Hz (5/5 trials), 20 Hz (5/5 trials) and 30 Hz (4/11 trials) (0.40 mW
μm−2; 10 ms pulse; 15 μm excitation spot; right). Scale bars, 20 μm. λexc = 920 nm, 40×, 0.8 NA objective, excitation depth of 50–70 μm.

From these results we conclude that two-photon TF-GPC gen-
erates sufficiently large ChR2 photocurrents to reliably evoke
action potentials in primary neuronal cultures at excitation den-
sities well below the damage threshold25.
Shaped two-photon excitation in cortical brain slices
To explore the efficiency of TF-GPC in brain slices, we used coro-
nal slices of somatosensory cortex from Thy1-ChR2-YFP trans-
genic mice, in which wild-type ChR2 is expressed in pyramidal
neurons of cortical layer V (ref. 5). Using widefield imaging we
searched for YFP-ChR2–positive cells and used the fluorescence
or transmission images to design and locate the excitation spot
(Fig. 4a). Most tested neurons (13/14) responded with an action
potential to a 10-μm excitation spot (0.30–0.52 mW μm−2;
10 ms pulse). To define the exact threshold for action-potential
generation, we stimulated neurons with excitation spots of ­various
a Shaped Antishaped c larger than that evoked by the antishaped excitation in two
diameters: the average threshold was 9.8 ± 0.8 μm (± s.e.m.;
n = 9 cells). The amplitude of the photocurrents increased, and
the latency to the action potential decreased as the diameter of the
excitation spot increased (Fig. 4b,c). Similar trends occurred with
increasing excitation density (Supplementary Fig. 4).
Increasing the illumination area during a sustained (1 s) light
pulse increased action-potential firing (Fig. 4d), reaching a spik-
ing rate of 15 Hz (11.8 ± 0.8 Hz; 6 trials). Stimulation with a train
of brief light pulses evoked action-potential trains at 10 Hz or
greater (of eight cells, five reached 10 Hz, three reached 20 Hz
and two reached 30 Hz; Fig. 4d).
Finally, we tested the ability of TF-GPC to maintain the
two-photon excitation confinement in the greater depth and
scattering medium of the cortical slice. To test the lateral
­precision, we compared (Fig. 5a) the photocurrent evoked by
an excitation shape covering the whole cell to the one evoked
by an illumination area covering the space surrounding the cell
(­antishape). The shape-evoked current was about sevenfold
different cells (ratio between antishape- and shape-evoked cur-
rents was 7.7- and 7.1-fold, respectively). This demonstrated
an x-y axis contrast almost as sharp as what we observed in
monolayer cultures (Fig. 2b).

z = –5 µm
Shaped Figure 5 | TF-GPC provides lateral and axial precision in ChR2 activation
Antishaped
10 pA in brain slices. (a) Widefield fluorescence images of a ChR2-YFP positive
20 ms z = –3 µm
neuron filled with Alexa Fluor 594 and superimposed excitation patterns
(red) with shaped and anti-shaped profiles (top). Photocurrents evoked
b z = –2 µm
by shaped and antishaped excitation (bottom; 10 ms laser pulses,
∆z = 5 µm

3 0.24 mW μm−2). (b) Integrated photocurrent (area under inward current

Total charge (–pC)

z=0 measured in picocoulombs and shown as negative picocoloumbs) evoked

2 by a 10 μm excitation spot centered on the cell body when displaced
z = 3 µm along the z axis in a ChR2-YFP–positive neuron (0.30 mW μm−2).
1 (c) Fluorescence image of a ChR2-YFP positive neuron filled with Alexa
Fluor 594 with superimposed shaped excitation profile covering the apical
z = 4 µm
0 dendrite (red; top). Photo-depolarizations evoked by the excitation shape
40 mV
–45 –30 –15 0 15 30 45 at different z-axis positions (bottom; 10 ms pulse, 0.30 mW μm−2).
z-axis position (µm) z = 6 µm 20 ms
Scale bars, 20 μm. λexc = 920 nm, 40×, 0.8 NA objective.

nature methods  |  VOL.7  NO.10  |  OCTOBER 2010  |  851

 Articles
a b c 400
300
Current (–pA)
20 mV 200
10 ms
100
d four cells or five spots of 11 μm diameter in one cell). (d) Transmission
e f
Stimulation Record Stimulation
A A
A A
B C
© 2010 Nature America, Inc. All rights reserved.
A A
B C
B C
A A
A+B+C A+B+C
B C
To evaluate the capability for optical sectioning in brain slices,
we created an excitation spot on the cell body and moved it axi-
ally in 2-μm steps (Fig. 5b). The photocurrent dropped off at focal
positions above and below the cell body (FWHM = 25.8 ± 2.6 μm
(± s.e.m.; n = 5)). The drop-off was less steep than in monolayer
cultures (Fig. 2c), perhaps owing to light scattering or to the excita-
tion of dendritic or axonal processes located above or below the cell
body, which were not resolved in the widefield imaging. Even so, the
results showed that the peak energy could be focused over a depth
that approximately corresponds to the diameter of a cell body.
The micrometer axial resolution of TF-GPC allows targeting
a single cell process so that we could evoke an action potential
with an illumination shape generated to selectively excite one or
few dendritic segments (Fig. 5c and Supplementary Fig. 5). We
observed dendritic selectivity by moving the focal plane of the
illumination shape below and above the dendritic segment. In
the example shown in Figure 5c, the ability to evoke an action
potential was restricted to an axial distance of 5 μm. To test the
lateral precision, we compared the photocurrent evoked by an
excitation shape covering a dendritic segment to the one evoked
by an illumination area that covered the space surrounding the
dendrite (antishape) in two different cells. The antishape-evoked
currents were 20% and 22% of the size of the shape-evoked currents.
Thus, TF-GPC can be used to target two-photon excitation of
ChR2 with sufficient three-dimensional precision and intensity to
excite neurons with short pulses of light in intact brain slices, even
when stimulating only subregions of the cell body or dendrite.
Simultaneous excitation of multiple cells and cell processes
One of the attractions of TF-GPC light patterning is the ability to
rapidly and automatically create excitation shapes for simultane-
ous excitation of multiple cells and cell processes. We designed
the TF-GPC setup to maintain the excitation density at a constant
value, independent of the size of the excitation area or the number
852  |  VOL.7  NO.10  |  OCTOBER 2010  |  nature methods
Figure 6 | Multispot photoactivation in cortical slices. (a) Transmission
images of layer V pyramidal neurons expressing ChR2-YFP. One spot (red)
was placed over the recorded cell (top) or this spot was combined with
four identical spots placed elsewhere (bottom). White line indicates
the excitation field of 60 μm in diameter. Scale bars, 10 μm. (b) Action
potential evoked by single-spot (top) and multispot (bottom) illumination
(red bars) (0.29–0.34 mW μm−2; λexc = 920 nm). (c) Averaged (three trials per
Single-spot Multispot cell) amplitudes of photocurrents measured under voltage clamp in response
to single or multispot illumination (three spots of 12 μm diameter for
B A image of three ChR2-YFP–positive neurons (A, B and C) with 15 μm
excitation spots (red) superimposed. Scale bar, 10 μm. (e) Neurons A and
B were simultaneously patch clamped. A spot over neuron A triggered
C an action potential only in A. A spot over neuron B triggered an action
potential only in neuron B. When the three cells were illuminated
simultaneously both A and B fired an action potential. (f) After recordings
50 mV
shown in e, the patch pipet was removed from B and a patch recording
Record
50 ms was established in C. A spot over neuron A triggered an action potential
only in A. A spot over C triggered an action potential in C and a small
depolarization in A. When the three cells were illuminated simultaneously
both A and C fired an action potential (0.25 mW μm−2; λexc = 850 nm, 40×,
0.8 NA objective). Recordings were collected in 10 μM NBQX.
of spots (Online Methods and Supplementary Fig. 6). This avoids
an attenuation of the intensity with the addition of spots and
maintains the potency of each of the multispot to stimulate a
photocurrent of sufficient magnitude to fire action potentials.
To test the efficacy of TF-GPC for the simultaneous excitation of
different cells in cortical slices, we compared stimulation with a
single excitation spot over a patch-clamped neuron with stimula-
tion with the same spot accompanied by additional identical spots
placed elsewhere in the slice at locations that, themselves, evoked
no response in the cell (Fig. 6a–c; 5 cells).
We compared excitation with a single spot to excitation with
three simultaneous spots of 12 μm diameter (4 cells) or five simul-
taneous spots of 11 μm diameter (1 cell). In keeping with the uni-
form excitation density design, photo-depolarization and action
potential–triggering were the same whether the spot over the cell
was excited alone, or along with two or four additional spots at the
same time (5 of 5 cells; 3/3 trials for each cell; Fig. 6b). Similarly,
the amount of photocurrent recorded in the single cell did not
differ between single spot and multispot illumination (P = 0.24;
n = 5; Fig. 6c).
In additional experiments, we performed double recordings
in which two ChR2-positive neurons in the cortical slice were
simultaneously patch-clamped (4 pairs). In each case, we could
stimulate the neurons separately with a pulse of light and simul-
taneously when we applied the two light spots at the same time. In
one case, we illuminated three ChR2-positive neurons with spots
and two of them were patch clamped and fired simultaneously
(Fig. 6d,e). We then removed the patch recording from one of the
patch-clamped cells and switched it to the third cell, and it also
fired in response to the three-spot illumination (Fig. 6f).
Finally, we asked whether we could use multispot illumination
to evoke graded increases in dendritic excitation of a neuron,
to mimic summed excitation by multiple presynaptic inputs.
Pyramidal cells were patch clamped in voltage clamp mode and
optically stimulated with shaped spots placed on their thin basal
dendrites or on their apical dendrite. In all cases (5/5 neurons),
the photostimulation of an additional dendrite increased the size
of the response (Supplementary Fig. 5c).

These results demonstrate that shaped TF-GPC two-­photon
excitation enabled efficient in-depth photoactivation of ChR2
enabling, to our knowledge for the first time, the excitation of
­multiple neurons or multiple neuronal compartments separately
or together.
DISCUSSION
We performed the experiments in brain slices at a maximum
depth of ~60 μm. The main factor limiting the working depth was
the quality of fluorescence imaging in widefield epi-­fluorescence.
An experimental setup with two independent laser sources
combining two-photon TF-GPC photoactivation with two-­photon
imaging would overcome this limitation and permit much deeper
in vivo photoactivation.
GPC can be implemented with binary half-wave modulat-
ing input phase patterns without compromising light efficiency
(Supplementary Fig. 7 and Supplementary Note 1), permitting
use of low phase stroke devices, such as ferroelectric high refresh
rate (kilohertz) spatial modulating devices. The use of high frame
© 2010 Nature America, Inc. All rights reserved.
rate devices is of particular relevance for dynamically patterned
excitation. In this case, sequential excitation can be performed
by precalculating the phase profiles corresponding to a defined
series of excitation patterns and sequentially addressing them on
the spatial light modulator (SLM) with a frequency limited only
by the refresh rate of the spatial light modulator in use.
TF-GPC compares favorably with three other approaches that
have been used recently for patterned photoactivation: the digital
micromirror-based device26,27 (DMD), fast scanning devices and
large spots. A DMD spreads the exciting light over a mirror array
and patterns the light by redirecting out of the field of view light
pointing toward the regions one wants to be dark. This approach
has the advantage of simplicity and fast switching rates and pre-
serves a flat incident beam wavefront, permitting optimal axial
resolution to be obtained if combined with temporal focusing.
However, the DMD suffers from a low diffraction efficiency
inherently attributed to amplitude modulation (Supplementary
Fig. 7 and Supplementary Note 1) and requires a fine adjust-
ment of the optical alignment for each modification of excita-
tion wavelengths.
Fast scanning devices, such as galvanometric mirrors13,14 or
acousto-optic deflectors28–30, can move a single spot using most
of the laser intensity over a large excitation area. However, the
need for a minimal residence time limits the maximum area that
can be excited within the ChR2 decay time (~10 ms) and makes
excitation of multiple cells extremely difficult. The residence time
can be reduced (and, thus, the temporal resolution improved)
by increasing the excitation power, but this results in a stark (up
to tenfold) deterioration of lateral and axial resolution owing to
the strong contribution of the out-of-focus light to the evoked
responses14. Fast scanning with an acousto-optic deflector has the
additional drawback of requiring compensation for pulse broad-
ening and chromatic aberrations, effects that are negligible for a
beam reflected by a liquid crystal on silicon–spatial light modula-
tor (LCOS-SLM), as used in our TF-GPC method (the laser pulse
duration after reflection on the LCOS-SLM was 140 fs, measured
with an autocorrelator19). Finally, a Gaussian beam focused as a
large spot straight onto the diffraction grating can produce effi-
cient two-photon activation with the optimal axial resolution of
temporal focusing14. However, this approach requires that the
Articles
optical path be changed every time one adjusts the excitation spot
size and is limited to a fixed circularly shaped excitation area.
TF-GPC is uniquely powerful for shaping two-photon excita-
tion in three dimensions for the stimulation of ChR2 and, by
extension, other light-gated proteins. The shapes can be hand-
drawn or automatically generated from fluorescent images of
light-gated protein expressing cells. The stimulated areas can
be small segments of dendrites, groups of dendrites, an entire
cell or a group of cells, which can be stimulated individually or
simultaneously. This feature will permit simultaneous ­excitation
of genetically related cells to test their circuit interaction. In
the future, this technology will make it possible to analyze how
­circuits of neurons change their interactions depending on the
timing of their activity resulting from learning rules defined by
spike timing dependent plasticity.
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 I. Perch-Nielsen for the phase-contrast filter layout design, E. Schwartz
for genotyping ChR2-YFP mice, S. Wiese, Z. Fu and M. Viesel for generating cDNA
constructs, A. Triller, T. Gally, K. Spence, A. Burgo and K. Zylbersztejn for cell
culture preparation, all members of the Neurophysiology and New Microscopy
Laboratory for comments and technical help, D. Oron, D. Palima, S. Dieudonné,
M. Diana and G. Fortin for helpful discussions, J. Feldmann for critical reading of
the paper, Spectra-Physics, Inc. for loan of the high-power laser, and Phasics S.A.
for providing the phase-analyzer software. V.E. was supported by the European
Science Foundation and the Centre National de la Recherche Scientifique through
the European Young Investigator program and by the European Network of
Neuroscience Institutes (LSHM-CT-2005-19063). E.P. and V.E. were supported by
the European Commission FP6 Specific Targeted Project Photolysis (LSHM-CT-2007-
037765). E.P. was supported by the Fondation pour la Recherche Médicale. F.A. was
supported by the European doctoral school Frontières du Vivant. A.B. was supported
by Paris School of Neuroscience. J.G. was supported by the Danish Technical
Scientific Research Councils (09-060742), E.Y.I. was supported by the US National
Institutes of Health Nanomedicine Development Center for the Optical Control of
Biological Function (PN2EY018241) and the Paris School of Neuroscience. E.Y.I. and
V.E. were supported by Human Frontier Science Program (RGP0013/2010).
AUTHOR CONTRIBUTIONS
E.P. set up and characterized the optical properties of the TF-GPC microscope;
F.A. and A.B. implemented the optical microscope with electrophysiological
recording; E.P., F.A. and A.B. performed the experiments on cell cultures and
brain slices; F.A. and A.B. analyzed the experiments on cell cultures and brain
slices; V.d.S. developed the software; J.G. contributed to the set up of the
GPC microscope; E.Y.I. contributed in conceiving the experiments in cultured
cells and brain slices and discussed the results; E.Y.I. and V.E. prepared the
manuscript; and V.E. conceived and supervised the project.
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
Published online at http://www.nature.com/naturemethods/.
Reprints and permissions information is available online at http://npg.nature.
com/reprintsandpermissions/.
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 ONLINE METHODS
GPC-TF of ultrashort pulses for photoactivation. The optical
setup for photoactivation experiments (Fig. 1a) was built around
a commercial epifluorescence upright microscope (Olympus
BX50WI). A mode-locked Ti:sapphire laser was used as the light
source, which in the case of photoactivation of ChR2 in HEK
293 cells and double patch experiments was a Tsunami oscillator
pumped by a 5 W Millennia Pro s-Series CW laser (λ = 532 nm)
(Spectra-Physics, Inc.), operating at 850 nm (Δλ = 11 nm, out-
put power = 700 mW), and in the other cases a Mai Tai laser
operating at λ = 920 nm (Δλ = 11 nm, output power = 1.85 W),
kindly provided by Spectra-Physics, Inc. for a short period dur-
ing the needs of the experiment. In all cases the output beam
power was controlled by a liquid crystal variable phase retarder
(Meadowlark Optics, LRC-200-IR1) combined with a polarizer
cube (Meadowlark Optics, BB-050-IR1).
Two-photon arbitrary excitation patterns were generated by
using the GPC method22 via the use of a reconfigurable liquid
crystal on silicon spatial light modulator (LCOS-SLM; Hamamatsu
© 2010 Nature America, Inc. All rights reserved.
Photonics X10468-02), illuminated at oblique incidence by the
expanded laser beam (5×). The device was controlled by a cus-
tom-designed software16 that, given a target intensity distribution
at the focal plane of the microscope objective converted (in ~30
ms) the intensity map, A(x,y), into a binary phase map, Aφ(x,y)SLM
and addressed (in ~15 ms) the output profile to the SLM. The tar-
get intensity distribution is typically obtained by selecting on the
transmission or fluorescence picture a region of interest (ROI) and
automatically generated by the software using threshold detection
in the selected ROI. For excitation patterns precisely shaped on
cell or dendritic morphology, we increased contrast and defini-
tion in primary fluorescence images by filling individual neurons
with Alexa Fluor 594 (Invitrogen Molecular Probes) through the
patch pipet and imaging in wide-field (λexc = 590 nm, bandwidth
10 nm). The total time for acquiring the fluorescence image and
generating the ROI was approximately 1 s. This time would not
slow the experiment, because a series of ROIs can be made during
setting up the experiment, in advance of recording.
The beam reflected from the SLM was separated in its Fourier
components by a 400 mm focal length achromatic lens (L1) and
focused on the PCF, positioned at the Fourier plane of the lens. An
iris was placed in front of the SLM, whose radius (Rc = 5 mm) was
adapted to optimize the contrast of the output intensity distribu-
tion. The on-axis, low-spatial-frequency components were shifted
in phase by the PCF and then, the second 300 mm achromatic
lens (L2) recombined the high (signal wave) and low (synthetic
reference wave) spatial frequency components. The introduced
phase shift caused the different components to interfere and
produced an intensity distribution according to the spatial phase
information carried by the higher spatial frequencies. The lens L
(f = 500 mm) (Fig. 1a) and the microscope objective (Olympus,
LUMPLFL60XW/IR 60×, NA = 0.9 or LUMPLFL40XW/IR; 40×,
NA = 0.8) formed the second 4-f lens system that scaled the inten-
sity distribution (~1/110) on the sample plane, via a dichroic mir-
ror (Chroma Technology 640DCSPXR).
The PCF is selected from a patterned phase mask, fabricated by
depositing a photoresist on a glass optical flat (DanChip) contain-
ing circular pits of variable diameter (4–100 μm) arranged in a
squared array of 10 × 10 filters, which provide a half-wave phase
shift in the wavelength range 850–950 nm. The radius, R1, of the
doi:10.1038/nmeth.1505
filter was chosen to obtain optimal phase contrast together with
a reasonable excitation field. In our experimental conditions the
radius R2 of the main lobe of the Airy profile of the ­synthetic
reference wave focused at the PCF plane (measured with a wave-
front analyzer CCD camera; SID4-028, PHASICS S.A.) was
45 μm and 50 μm at 850 nm and 920 nm, respectively. Therefore,
with a PCF of 27.5 μm we had a value for the η term, which
relates the radius R1 of the PCF to R2 (ref. 31), of η = 0.61 or 0.55.
These η values gave in each case a good contrast with a circular
excitation field of 60 μm in diameter (Supplementary Fig. 7 and
Supplementary Note 1).
To have optimal contrast conditions in GPC, for binary 0/π-
phase input modulation, the phase-shifting area, Aπ, on the SLM
should cover around one-quarter of the illuminated surface of the
SLM, ASLM, that is F = Aπ / (ASLM) = Aπ / (Aπ + A0) = 0.25 (ref. 32),
where Aπ and A0 are the numbers of pixels at the SLM addressed
with phase π and 0, respectively. To always fulfill this condition, no
matter of the selected excitation shaped area at the sample plane,
a ring of area A(ring), surrounding the desired shape, was added
by the software to the LCOS-SLM phase pattern (Supplementary
Fig. 6a–c). The thickness of the ring was adjusted so that the
total phase-shifting area Aπ(tot) = Aπ(spot) + Aπ(ring) was always
around one-quarter of the illuminated surface of the SLM, ASLM.
The external diameter of the ring was adjusted to the size of the
circular aperture placed in front of the SLM. As a result, the laser
power at the output plane was distributed between the ring and
the excitation shaped area in such a way, that excitation density
in the excitation spot had approximately the same value, indepen­
dently of its size.
For temporal focusing, a blazed reflectance grating (830 lines
per mm) was placed at the focal plane of the L2 lens, to geometri-
cally disperse the frequencies of the ultrashort laser pulses and
temporally focus the beam on the sample plane. An illumination
angle of 40.5° was chosen, such that the central frequency of the
+1 order beam diffracted by the blazed grating (~80% of the inci-
dent beam) was directed along the optical axis of the microscope
while the zero order beam was blocked (for details concerning the
alignment of the grating, see reference 19). The tilted illumina-
tion of the grating caused a horizontal stretching of the original
intensity pattern by a factor of cos (40.5°). To compensate for the
tilt, the phase pattern was equally preshrunk along the x direction,
at the SLM’s plane (Supplementary Fig. 6a–c). To block the ring
added to balance the filling factor of the SLM from the excitation
field, an iris was placed right after the grating (not shown on the
layout for simplicity).
The duration of the laser pulses for photoactivation was control-
led by a mechanical shutter (Uniblitz, VCM-D1 Shutter Driver)
controlled either by the electrophysiology amplifier (Axon 200B)
or by a home-made electronic circuit, based on a microcontrol-
ler (PIC 18F4550, Microchip), driven by a PC interface through
a USB connection. In the last case the circuit synchronized the
shutter, based on a trigger pulse sent by the Axon 200B amplifier.
The shutter’s rise time was 1.9 ms (Supplementary Fig. 8).
The theoretical calculation of the axial propagation of the exci-
tation beams around the objective focal plane was carried out by
using the formalism described previously16.
The experimental propagation was measured with a ‘double
microscope’ described in previous work16,17,19 using an upper
objective 60× (Olympus, LUMPLFL60XW/IR; NA 0.9). The axial
nature methods
 resolution of TF-GPC was determined each day of experiment
with a fluorescent layer and we always found the same value.
For fluorescence imaging we used a 75-W Xenon lamp pass-
ing through a monochromator (Optoscan, Cairn Research).
Fluorescence images were collected with a cooled (−30 °C)
12-bit CCD camera (CoolSNAP HQ2, Roper Scientific) using a
HQ535/50M filter or a HQ640/40M filter (Chroma Technology)
as emitting filters.
Optical sectioning and image acquisition have been performed
using the Metamorph software (version 7.1, Molecular Devices).
Acquisition time was 300–500 ms for fluorescence images and
200 ms for transmission images. Images superposition has been
realized by using Photofiltre software (version 6.4.0).
Cell cultures and brain slices preparation. All experiments fol-
lowed European Union and institutional guidelines of the care
and use of laboratory animals (Council directive 86/609 EEC).
HEK 293 cells were plated at approximately 105 cells per 12 mm
glass coverslip coated with poly(l-lysine) and maintained in DMEM
© 2010 Nature America, Inc. All rights reserved.
with 5% fetal bovine serum and 0.5% penicillin-streptomycin. Cells
were transfected with 2 μg of plasmids encoding ChR2(H134R)-
GFP33 using ExGen 500 (Euromedex). All recordings were per-
formed 24–48 h after transfection, at room temperature (22 °C).
Dissociated embryonic mouse cortical neurons (E16-17) were plated
on poly(l-lysine)-coated glass coverslips at a density of 1.5 × 105 cells.
Neurons were maintained in MEM supplemented with 5% FBS, serum
extender (BD Biosciences) and 0.5% penicillin-streptomycin. Ara-C
(4 μM) was added after DIV6. Cells were transfected by the calcium
phosphate method using 1 μg of plasmids encoding ChR2(H134R)-
GFP per 12 mm coverslip. Recordings were performed 24 h after trans-
fection, at room temperature.
Coronal cortical slices (250–280 μm) were obtained from wild
type and Thy1-ChR2-YFP mice34 older than postnatal day 30
purchased from the Jackson Laboratory (B6.Cg-Tg(Thy1-COP4/
EYFP)18Gfng/J strain).
Somatosensory slices were prepared in an ice-cold slicing
­solution35 containing 130 mM K-gluconate, 15 mM KCl, 0.2 mM
EGTA, 20 mM HEPES, 25 mM glucose, 50 μM D-APV and 50 nM
minocycline (pH 7.4) and then transferred for 30 s to a ­recovery
solution containing 225 mM d-mannitol, 2.5 mM KCl, 1.25 mM
NaH2PO4, 26 mM NaHCO3, 25 mM glucose, 0.8 mM CaCl2,
8 mM MgCl2, 50 μM D-APV and 50 nM minocycline (95% O2 and
5% CO2). Before recording slices were incubated for at least 30 min
at 33 °C in external solution containing 125 mM NaCl, 3.5 mM
KCl, 1.25 mM NaH2PO4, 26 mM NaHCO3, 25 mM glucose, 2 mM
CaCl2, 1 mM MgCl2 (95% O2 and 5% CO2).
Recordings were performed at room temperature.
Electrophysiology and data analysis. Patch-clamp pipets (4–8 MΩ
for neurons and 2–4 MΩ for HEK 293 cells) were back-filled with
intracellular solutions containing 145 mM CsCl, 10 mM HEPES,
0.5 mM CaCl2, 1 mM MgCl2, 5 mM EGTA (pH 7.2) for HEK
293 cells; 131 mM K-gluconate, 10 mM NaCl, 10 mM HEPES,
2 mM MgCl2, 2 mM MgATP, 1 mM EGTA (pH 7.4) for neurons
in culture; and 137 mM K-MeSO4, 6 mM NaCl, 5 mM MgCl2,
0.05 mM EGTA, 10 mM HEPES, 4 mM K2ATP and 0.4 mM
GTP-Na2 (pH 7.3) for brain slices. In some recordings, 10 μM
nature methods
Alexa Fluor 594 (Invitrogen Molecular Probes) were added to the
patch-clamp internal solution for morphological reconstruction.
External solutions contained 135 mM NaCl, 5.4 mM KCl, 10 mM
HEPES, 1.8 mM CaCl2, 0.9 mM MgCl2, 10 mM glucose (pH 7.6)
for HEK 293 cells; and 138 mM NaCl, 3 mM KCl, 5 mM HEPES,
2.5 mM CaCl2, 1.2 mM MgCl2 and 10 mM glucose (pH 7.4) for
neuronal primary cultures. For slices, the same external solution as
the one described above for incubation was used for recording.
HEK cells were maintained at −40 mV and hyperpolarized to
−60 mV just before recording light-induced currents. Cultured
neurons and pyramidal neurons in brain slices were clamped at
−60 mV and −77 mV, respectively. Current clamp recordings
were adjusted so that the resting potential was around −60 mV
(cultured neurons) or −67 mV (neurons in slices) with current
injections inferior of 100 pA. All presented data were already
corrected for liquid junction potentials of −10 mV (K gluconate
solution) and −7 mV (KMeSO4 solution) for cultured neurons
and brain slices, respectively. Currents were recorded with an
Axon 200B amplifier, filtered at 10 kHz, and digitized at 10 kHz
using a Digidata board (Axon instruments) and Clampex software
(Axon Instruments).
D-APV was purchased from Tocris Bioscience, and 2,3-dioxo-
6-nitro-1,2,3,4-tetrahydrobenzo[f ]quinoxaline-7-sulfonamide
disodium salt (NBQX) was purchased from Ascent Scientific.
Stock was prepared in water and dissolved in extracellular solu-
tion just before experiments. All other chemicals were purchased
from Sigma.
Data analysis was performed using either the Clampfit analysis
software (Axon Instruments) and custom routines within the Igor
Pro environment (Wavemetrics) or in Origin 8.0 (OriginLab).
Statistical data are reported as mean ± s.e.m. The significance
was calculated using paired Student’s t test.
Photodamage assessment protocols. In slices, photostimulation
of pyramidal neurons with twenty 10-Hz trains of 50 ms pulses
every 10 s (0.44 mW μm−2) or with thirty 400-ms pulses at the
maximum excitation power (0.53 mW μm−2) did not induce vis-
ible cell damage. Neurons were still alive and able to fire action
potentials at the end of each protocol (data not shown).
In the same preparation, illumination protocols similar to
those used in Figures 3 and 4 elicited no response in ChR2-
positive neurons if a block was inserted in the excitation path
preventing the laser light from reaching the preparation or in
ChR2-negative neurons.
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of Caenorhabditis elegans triggers rapid behavioral responses. Curr. Biol.
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transgenic mice expressing channelrhodopsin-2. Neuron 54, 205–218
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doi:10.1038/nmeth.1505