Articles
A microprobe for parallel optical and electrical
recordings from single neurons in vivo
Yoan LeChasseur1,2, Suzie Dufour1,2,4, Guillaume Lavertu1,4, Cyril Bories1, Martin Deschênes1,3, Réal Vallée1,2 &
Yves De Koninck1–3
Recording electrical activity from identified neurons in intact
tissue is key to understanding their role in information
processing. Recent fluorescence labeling techniques have
© 2011 Nature America, Inc. All rights reserved.
opened new possibilities to combine electrophysiological
recording with optical detection of individual neurons deep
in brain tissue. For this purpose we developed dual-core
fiberoptics–based microprobes, with an optical core to locally
excite and collect fluorescence, and an electrolyte-filled
hollow core for extracellular single unit electrophysiology.
This design provides microprobes with tips <10 mm, enabling
analyses with single-cell optical resolution. We demonstrate
combined electrical and optical detection of single fluorescent
neurons in rats and mice. We combined electrical recordings
and optical Ca2+ measurements from single thalamic relay
neurons in rats, and achieved detection and activation of
single channelrhodopsin-expressing neurons in Thy1::ChR2-YFP
transgenic mice. The microprobe expands possibilities for in
vivo electrophysiological recording, providing parallel access to
single-cell optical monitoring and control.
The central nervous system is characterized by heterogeneous
populations of cells with highly specialized phenotypes defined
by their morphological, biochemical and physiological proper-
ties. Efficient characterization of each cell type in intact tissue has
remained challenging because the neurons of interest are often
dispersed in these heterogeneous populations. There are several
approaches that allow electrophysiologists to identify the cell types
they record from, but most of these approaches have drawbacks.
Antidromic activation of projection neurons is only applicable to
a small subset of cells and requires preservation of connections
between the recording and projection sites1,2. Labeling cells with
dyes at the time of recording allows a posteriori identification, but
this approach is inefficient when dealing with subpopulations that
represent a small proportion of the overall neuronal population in
an area3,4. Labeling with fluorescent markers allows for targeted
recording, independently of connectivity5–9 and has been very
instrumental for studies in central nervous system tissue slices10–12.
However, it cannot be fully exploited for electrophysiological
1Centre de recherche Université Laval Robert-Giffard, Québec, Canada. 2Centre d’optique, photonique et laser, Université Laval, Québec, Canada. 3Department of
Psychiatry and Neuroscience, Université Laval, Québec, Canada. 4These authors contributed equally to this work. Correspondence should be addressed to
Y.D.K. (yves.dekoninck@crulrg.ulaval.ca).
Received 15 July 2010; accepted 20 January 2011; published online 13 february 2011; doi:10.1038/nmeth.1572
investigations in live animals because light scattering limits optical
microscopy to surface measurements (500–1,000 µm)13,14. Micro-
endoscopes help overcome limitations for deep tissue imaging
but do not allow for combined electrical and optical monitoring,
especially for single unit recording15,16. The challenge remains
to conduct combined electrophysiological recording and optical
identification of individual neurons deep into central nervous
system tissue in live animals. Combination of both approaches is
important because purely optical techniques remain limited for
resolving single action potentials and for prolonged functional
measurements (>1 h).
Several strategies have been sought to combine microelec-
trode and guided optical recording. One had used distinct probes
brought together using two independent microdrives17,18. Such
an approach is not practical for in vivo measurements. Other
groups have relied on attaching large fibers (250 µm in diameter)
to an electrode19,20. Even with smaller optical fibers (25–35 µm)
inserted into a micropipette, the final tip diameter of the probe
is 35–45 µm, precluding combined optical and electrical record-
ing from a single neuron. Furthermore, this approach relies on
an external source of illumination, which prevents efficient light
delivery into deep tissue and produces tissue excitation over
wide areas21.
To overcome these limitations, we designed a new type of opti-
cal fiber, incorporating both an optical and a hollow core. We
show that this type of microprobe provides, to our knowledge
for the first time, sufficient spatial resolution to correlate electro-
physiological and fluorescence signals that emanate from single
fluorescently labeled neurons at a depth of >6,000 µm in the
intact central nervous system.
RESULTS
The microprobe
Our objective was to detect, in parallel, the electrical field and
the optical signal from single fluorescent neurons in vivo. We
designed an optical fiber composed of a hollow core and an opti-
cal core (Fig. 1a,b and Supplementary Fig. 1a). We filled the
hollow core with an electrolyte solution (1–3 M NaCl) to record
nature methods  |  VOL.8  NO.4  |  APRIL 2011  |  319
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Figure 1 | The microprobe. (a) Schematic of
the microprobe (not drawn to scale), which is
a Multimode
optical fiber
c Acquisition
system
based on a dual-core optical fiber: a graded
UV-light
index optical core and a hollow core. The optical curable Laser
core is connected at one end to a multimode adhesive
Neutral
optical fiber, and the other end is tapered. 50-µm density
wire filter Dichroic
(b) Schematic of the optical fiber preform Microprobe
Graded index Fiber mirror
design: a series of different-size silica rods are optical core launch
PMT
assembled to position the optical and hollow
cores side by side in an inclusion tube. Scale
bar, 2 mm. (c) Diagram of the experimental Hollow
Light insulation box
setup. Light delivery and collection as well as core
electrical recording are all achieved through the d Signal amplitude

Probe position
Shoulder
same fiber probe. PMT, photomultiplier tube.
(d) Illustration of the directionality of optical
recording and the nondirectionality of electrical Shank
recording, with intensity profiles along the
trajectory of the microprobe and interpeak
Tip
distance shown on the right.
b
electrical activity, and the optical core was Graded
© 2011 Nature America, Inc. All rights reserved.

index optical
a graded refractive index waveguide allow- core
Excitation light
ing efficient light delivery and collection Hollow
core Fluorescence
through a tapered tip. To achieve single- Electric field potential

cell resolution with the optical probe, we

pulled the fiber to tip sizes smaller than that of the neuron somata
under study, using a conventional laser micropipette puller. We
generated probes with end tip diameters of 6–20 µm (the small-
est probe tips had an optical core of 4 µm and a hollow core of
0.7 µm; Fig. 1a and Supplementary Fig. 1a). The all-glass dual-
core fiber could be pulled into a variety of shapes as standard
glass microelectrodes (Supplementary Fig. 1b). To link an optical
a 1
Intensity (a.u.)
b 1
setup to the biological preparation, we connected the optical core
to a multimode fiber (Fig. 1c). We injected light into the probe
for local delivery at the tip end and also used this tip to collect
fluorescence emanating from labeled cells.
With this design, fluorescence collection is asymmetrical,
with only light in front of the tip being collected. Therefore,
as the probe moves past a labeled neuron, the detected fluo-
rescence signal should follow a characteristic intensity profile:
Neuron diameter = 14 µm growing gradually when approaching a labeled cell and drop-
Probe diameter = 9 µm
ping abruptly when the probe moves beyond the cell (Fig. 1d).

0.8 0.8
0.6 0.6
In contrast, the electrical signal should grow and decline sym-
0.4 metrically as the probe moves past a neuron. The peaks of both
0.4
0.2 signals emanating from a cell are thus not necessarily aligned
0.2
0 along the trajectory of the probe: the optical signal should peak
–20 –10 0 10 20
0
0 10 20 30 40 50
when the probe is at nearest approach to the cell body, whereas
Transverse position (µm) Axial neuron-probe distance (µm) the electrical signal should peak near the maximal current sink
c Transverse d (typically the axon hillock22). The interpeak distance should
Axial
thus be equal to or smaller than the length of the cell body and
Intensity (a.u.)
0 0.5 1
0 Figure 2 | Microprobe optical resolution. (a,b) Fluorescence
intensity profile as a function of transverse (a) and axial (b) probe
Axial position (µm)

10
1 displacement around single Lucifer yellow–labeled neurons in brain
20
slices. Zero displacement in a corresponds to the center of the cell body,
Intensity (a.u.)

30
0.5
zero displacement in b is at nearest approach to the cell body. Shown
40 are seven superimposed detection profiles (thin black lines), average
50 detection profile (thick black line) and the expected detection profile
0 based on numerical simulations (red line). (c,d) Photomicrographs of
0 20 40 60 80 two adjacent Lucifer yellow–labeled fluorescent neurons in fixed brain
Transverse position (µm)
e 20
f 1 slices used for testing probe detection in transverse (c) and axial (d)
translation axes. Microprobes are outlined in white, and the arrow
Intensity (a.u.)
50% axial distance (µm)

R = –0.05 shows the probe trajectories. Scale bars, 10 µm. Detected fluorescence
15 0.5
P = 0.92 during probe displacement is plotted; zero displacement corresponds
10 to the start of the white arrows in the micrographs. (e) Rising distance
from 50% to peak fluorescence plotted versus laser power. Dashed line
0
5 Ax indicates the fitted regression. (f) Simulated fluorescence collection
ial
po 50 –10
sit
ion 100 10
0
n (µm
) field of a 9-µm-diameter probe in front of a 12-µm-diameter neuron
0 (µm p ositio
0 2 4 6 8 10 ) Transvers
e plotted against the axial and transverse positions of the probe. Color
Laser power (mW) coding is arbitrary.

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a c d 6 Fluorescence f* *
Spikes

Cumulative probability
100
4

Intensity (a.u.)
13 µm
50 2
100 µV
0
100 ms
0
5 10 15 20 25 30 35 40 45 –2 g
20 0 5 10 15 20
Time (s) 30

Number of spikes
Neuron count
Probe travel direction 100 µV
10 e 13 µm 20 0.5 ms
1
Fluorescence
Spike amplitude 10
0 0.8
b

Intensity (a.u.)
Recording 5 10 15 20 25 30 35 40 45
microprobe Neuron diameter (µm) 0.6 0
DiI injection 0 20 40 60 80 100
0.4 Interspike interval (ms)
1,000
0.2 h 15
Natural

Optical S/N
0 100

Number of neurons
stimulus
0 50 100 150
10
Axial relative position (µm)
© 2011 Nature America, Inc. All rights reserved.

10
R = –0.13
P = 0.23
Figure 3 | Single-unit recordings from neurons in vivo. (a) Photomicrograph of a spinal cord transverse 5 1
section in which spinothalamic tract neurons were retrograde-labeled with DiI. The overlaid drawing 0 5 10 15 20
Probe diameter (µm)
of a microprobe (gray) illustrates a typical descent (arrow). Inset, magnification of a stained neuron.
Scale bars, 1 mm and 20 µm in inset. (b) Schematic representation of the experiment. (c) Distribution 0
of labeled spinothalamic tract neuron diameters plotted as a cumulative probability (top) or a histogram 0 100 200 300 400 500 600
Optical S/N
(bottom). The dotted arrow indicates the distance between the optical and electrical signal maxima in e;
more than 90% of cells had a diameter >16 µm. (d) Fluorescence signal and spikes evoked from the stimulation of the receptive field as
the microprobe descended into the spinal cord at constant speed (6 µm s −1). (e) Plot of the data shown in d, with the time scale converted
to spatial position. (f) Data from d shown on an expanded time scale. Asterisks indicate digitally subtracted movement artifacts. (g) Distribution
of interspike intervals for cell analyzed in d–f. Inset, 212 superimposed individual spikes. (h) Distribution of optical signal-to-noise ratio
(S/N) obtained with 87 detected neurons (average S/N was 21; n = 87 cells). Inset, relationship between the optical S/N and microprobe
tip diameter.

could serve as a criterion to determine whether the electrical
and optical signals emanate from the same neuron (Fig. 1d).
Optical resolution and single cell detection
We estimated the resolution of the microprobe assuming a fluo-
rescence point source, as is commonly used for calculation of
point spread functions in microscopy (Supplementary Data). For
probe tip diameters of 10 µm (6 µm optical core) or 6 µm (4 µm
optical core), the calculated full width half maximum (FWHM) of
the detected signal distribution was 13 µm or 12 µm in the axial
direction and 6 µm or 4 µm in the transverse plane, respectively.
Our calculations revealed that numerical aperture (NA) values
of 0.2–0.3 yielded an optimal compromise between axial and
transverse resolution (Supplementary Data and Supplementary
Fig. 2a,b). We then estimated numerically (Supplementary Data)
the fluorescence decay profiles as a function of both fiber and cell
diameters. With optical cores smaller than the cells, the decay
profile of the detected signal in the transverse axis was sensitive
to cell size but insensitive to probe tip size. Conversely, in the axial
direction, the decay profile was sensitive to fiber diameter but not
cell size, indicating that controlling tip size is important for axial
resolution of single cells (Supplementary Fig. 2c,f).
To test the applicability of these resolution estimates based on
physical parameters of the fibers (Supplementary Fig. 2a–f), we
performed measurements in fixed brain slices with cortical neu-
rons labeled using intracellular injection of Lucifer yellow. First
we examined slices under epifluorescence to locate labeled cells.
Then we turned off the external light source and moved the probe
in the transverse and axial directions across the labeled cells. The
detected signal FWHM using a 9-µm-diameter probe tip was 15 ±
5 µm transversely and 11 ± 4 µm axially for 14 ± 1 µm-diameter
neurons (mean ± s.d.; n = 6 cells; Fig. 2a,b), consistent with our
predictions (Supplementary Fig. 2c–f). Using this tip diameter, we
achieved sufficient resolution to distinguish adjacent cells in both
transverse and axial directions (Fig. 2c,d). Although the detected
fluorescence peak intensity varied from cell to cell, as expected, the
axial resolution remained constant and independent of excitation
power for a given microprobe (Fig. 2e,f). We confirmed this by
repeated measurements with different excitation powers (Fig. 2e).
Recording from single retrograde-labeled neurons in vivo
To test the optical and electrical performance of the microprobe
in vivo, we first used it to detect spinothalamic tract neurons that
we retrograde-labeled via injections in the thalamus of the fluores-
cent dye DiI (Fig. 3a,b). As estimated from histological sections,
spinothalamic tract neurons in spinal laminae III–VI had soma
diameters of 24 ± 6 µm (mean ± s.d.; n = 153 cells; Fig. 3c).
We used microprobes with 14 ± 3 µm tip diameter to record
from spinothalamic tract neurons down to 2,200 µm in the tis-
sue. The electrical channel of the probes had 6–26 MΩ resistance,
yielding spike recordings with a signal-to-noise ratio of 7.6 ± 3.7
(n = 155 cells; Supplementary Fig. 3) and stable recordings >> 1 h
(Fig. 3d–g). The probes’ recording capabilities and stability were
comparable to those of conventional micropipettes.

nature methods  |  VOL.8  NO.4  |  APRIL 2011  |  321

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Figure 4 | Optical and electrical signal profiles of single cells in vivo.
a –50 µm 50 µm b –50 µm 150 µm
(a,b) Schematics (top) show microprobe displacement (arrow) relative to
neuron position. Spike amplitudes (a) as the microprobe passed by a neuron
Neuron Microprobe Neuron Microprobe
were detected (curve indicates the average) and normalized spike amplitude

Fluorescence intensity (a.u.)

500 was plotted as a function of probe travel distance (bottom, average in
25 a.u. blue, n = 15 cells). Typical fluorescence detection patterns (b; middle)
when the microprobe passed by four fluorescently labeled neurons and
25 µm 300
normalized fluorescence profiles (bottom), with the average (thick black
line) (n = 14) and the expected profile from simulations (red line) shown.
100
Dotted lines indicate value at 50% to peak. (c) Successive units detected
by fluorescence and/or electrical signals during a 225-µm, bidirectional
–50 0 50 100 150
50% Axial relative position (µm) probe excursion. E1 and E2 represent two electrically recorded neurons
1 and O1 and O2 represent two optically recorded ones. Insets, examples of
50% 50%
Spike amplitude (a.u.)

Fluorescence intensity (a.u.)

0.8 1 1 spikes recorded in E1 and E2. (d) Photomicrograph of a GAD-GFP mouse

brain section. The overlaid drawing of a microprobe (gray) illustrates a
0.6
typical descent (white arrow) into the reticular thalamic nucleus. Inset,
0.4 0.5 0.5
high-magnification photomicrograph of GAD-GFP neurons. Scale bar, 1 mm
0.2 (20 µm in inset). (e) Magnitude of optical signal and spikes as the probe
0 0
passed by a reticular thalamic GFP-expressing neuron (bottom). Zero value
0
–100 –50 0 50 100 –50 0 0 50 100 on the x axis is aligned with the peak of the fluorescence curve. Continuous
Axial relative position (µm) Axial relative position (µm) electrophysiological signal as the probe traveled through the tissue (top).
c (f) Distribution of interspike intervals. Inset, consecutive superimposed
© 2011 Nature America, Inc. All rights reserved.

E1 O2 E2 E2 O2 E1
E1 spikes E2 spikes
action potentials (n = 26).
1 1
0.1 mV
0.1 mV

4 ms 4 ms
a non­symmetrical fashion, regardless of the probe travel direction
Intensity (a.u.)

O1
O1
0.5 0.5
(up or down), indicating no hysteresis (Fig. 4b,c). In 87 optical
recordings, the mean fluorescence signal-to-noise ratio was 21. As
the probe approached the cell, the fluorescence rose from 50% to
peak over a distance of 21 ± 8 µm; it decayed from peak to 50% over
0
0 50 100 150 200
0
–200 –150 –100 –50 0
7 ± 3 µm when the probe passed by the cell (n = 14 cells; Fig. 4b).
Displacement forward (µm) Displacement backward (µm) These values were consistent with our simulated predictions and
200 measurements in slices (Supplementary Fig. 2b,d). The asym-
d e µV f metry between fluorescence rise and decay was consistent with
1s
7 µm our working model (Fig. 1d). Based on results obtained for many
Signal intensity (a.u.)

1 200 µV descents, the criterion we used to identify a neuron optically was

Number of spikes

10 0.5 ms that the fluorescence signal had to rise to a maximum within the
0.5
distance predicted from the probe characteristics (Supplementary
5
Optical Fig. 2a,b,d) and drop back to baseline within less than half of the
Electrical
0
–10 0 10 20
0
0 2 4 6 8 10
rising distance with a signal-to-noise ratio >9.
Axial relative position (µm) Time (ms)

Combined optical and electrical detection criterion

We lowered the microprobe at constant speed into the spinal To determine whether the optical and electrical signals originated
cord (4–8 µm s−1), maintaining the excitation light on throughout from the same cell, we measured the distance between the axial
the descent. We observed one of the following: (i) a gradual rise of positions of the two signal peaks (Fig. 3e). We first considered
fluorescence followed by a decay over several tens of micrometers that both signals arose from the same neuron when the interpeak
without any clear spiking activity; (ii) spikes increasing gradually in distance was less than the mean neuron diameter according to our
amplitude followed by a decrease without noticeable change in flu- working model (Fig. 1d). In 16 spinothalamic tract neurons, the dis-
orescence; or (iii) a parallel growth and decay of fluorescence and tance between the optical and electrical signal peaks was < 24 µm,
spike amplitude (Fig. 3d,e). In the vast majority of cases, changes which corresponded to the mean spinothalamic tract neuron soma
in optical and electrical signals occurred without signs of neuron diameter. As a more stringent criterion, we used an interpeak
damage. Peak optical signal-to-noise ratio was not correlated with distance below 90% of these soma diameters (16 µm; Fig. 3c);
tip diameter (Fig. 3h) indicating that there was no disadvantage in we considered the two signals to originate from the same cells in
using smaller probes to increase optical resolution. 12/16 cases. This likely increased the number of false negatives
We analyzed quantitatively how the amplitude of the electri- but minimized false positives, which is more critical.
cal and fluorescence signals changed as a function of the axial
position of the probe. We performed this analysis in well-isolated Recording from single eGFP-expressing GABAergic neurons
single units as assessed by waveform analysis and the absence We next targeted reticular thalamic neurons expressing
of short interspike intervals (<2 ms) in spike trains (Fig. 3f,g). enhanced GFP (eGFP) under the control of the glutamic acid
Under these conditions, the spike amplitude rose and decayed decarboxylase promoter (isoform 65; GAD65) in transgenic
symmetrically with a 48 ± 19 µm FWHM (n = 15 cells; Fig. 4a). mice. These cells’ average soma diameter was 12.4 ± 2.3 µm
In contrast, the fluorescence signal intensity rose and decayed in (mean ± s.d.; n = 41 cells; Fig. 4d and Supplementary Fig. 4a).

322  |  VOL.8  NO.4  |  APRIL 2011  |  nature methods

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Figure 5 | Monitoring Ca2+ fluctuations confirm optical and electrical
a b
Spike Fluorescence

5% 0.28 recording from the same neuron in vivo. (a,b) Spontaneous spiking and

Ca2+ response (a.u.)

signal

∆F/F
0.24 Ca2+ fluctuations in a thalamic relay neuron (recorded at a depth of 6 mm
in the brain). The fluorescence signal and spike bursts (a), and Ca2+
0.20 response (quantified as the area under the curve) plotted as a function
bursts

0.16 R = 0.92
of the number of spikes in each burst (b) for the same cell. The two
P < 0.0001 parameters were linearly correlated (n = 24 bursts). ∆F/F = (F – F0)/F0,
0.12 in which F is the fluorescence and F0 = F in the stained region – F outside
0 10 20 30
200 µV Number of spikes the stained region. (c) Schematic representation of the whisker deflection
250 ms 5% using a piezoelectric mechanical stimulator. Inset, superposition of
c ∆F/F d 5 events
three spiking and calcium responses following a whisker deflection.
1% ∆F/F
Ca2+ response 0° (d) Peristimulus time histograms of spiking responses to 20 whisker
deflections (four directions indicated in degrees) and the associated
Spiking
90° averaged Ca2+ response over the same 20 stimuli for the same neuron.
The duration of whisker deflection is represented in gray. (e) The
0° 100 µV
90° 50 ms
180° angular preferences of spike response latency plotted against angular
preference for Ca2+ response (n = 8 cells). Inset, polar representation
270° Piezo
180°
270° of the normalized inverted spike latency after stimulation (black)
and the normalized inverted latency of the Ca2+ response (green).
0°
100 ms (f) Comparison between Ca2+ responses (top) and spiking responses
e f 5% (middle and bottom) of a neuron-to-whisker deflection. Black traces
indicate two representative successful responses, and gray traces indicate
© 2011 Nature America, Inc. All rights reserved.

∆F/F
300° 90°
two representative failures to respond in the same cell.
Spiking angular
preference

150°

R = 0.98 In these experiments, we identified single GAD65-eGFP neurons

100 µV

0° P < 0.001
in a dense nucleus of inhibitory interneurons (Fig. 4d–f), which
0° 150° 300° were not spatially distributed in any identifiable way; our tech-
Ca2+ angular preference Whisker 100 ms
nique was particularly useful under such conditions.

We thus used microprobes with 6–10 µm tips. Applying the Parallel recording and Ca2+ measurements in single neurons
stringent criteria described above, combined detection from To test whether optical and electrophysiological recordings with
the same cell required that the optical and electrical signal our microprobe originated from the same cell, we combined
peaks were ≤10 µm apart (Supplementary Fig. 4a). For this Ca2+ measurements with electrophysiological recording. We
neuron population, the optical signal rose from 50% to peak first recorded spontaneous activity in thalamic barreloids after
over a distance of 12 µm and decayed from peak to 50% over loading neurons with Oregon Green BAPTA1 acetoxymethyl
6 µm (Supplementary Fig. 4b). Of 11 cases with overlapping (AM) ester via local injections in vivo in adult rats. Single unit
electrical and optical signals, seven fell
within the combined detection criterion
(Fig. 4e and Supplementary Fig. 4a). a b 5 µm c Light stimulation

0.9
Intensity (a.u.)

0.6
Figure 6 | Detection and activation of
single ChR2-expressing neurons in vivo.
0.3
(a) Photomicrograph of a sagittal brain section
of a Thy1::ChR2-EYFP mouse. White boxes 0
200 µV
highlight recording sites. Scale bar, 1 mm. –10 0 10
(b) Magnitude of optical and electrical signals Relative position (µm) 40 ms
of a ChR2-EYFP–expressing neuron as a function d 80 e 0.4
f 1.2
of probe position. Zero position was aligned to
Response (spikes s–1)

Spike amplitude (mV)

peak fluorescence value. (c) Response of the 1 2

Delay s.d. (ms)

3 0.8
neuron in b to light stimulation through the 40
probe. Arrowheads point to the depolarization 0.2
200 µV

and repolarization of the cells at the pulse onset 0.4

and offset. (d) Response of an individual neuron 0 1 2 3 1 ms
versus light intensity. Individual responses 0 0
are shown in insets. (e) Spike amplitude as a 0.1 1 10 0.1 1 10 Th MC
function of light intensity at probe tip (same Light intensity (mW mm–2)

cell as in d). Each inset represents seven g Micro-whisker Light stimulation Both combined

superimposed spikes. (f) Mean ± s.d. of the

response delay to light stimulus for motor
cortical neurons (MC) (n = 15) and thalamic
neurons (Th) (n = 9). (g) Responses of a thalamic
100 µV
ChR2-expressing neuron to micro-whisker 50 ms
deflection, light stimulation or both.

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 Articles
recordings from thalamic relay neurons revealed low-frequency
(2–3 Hz) bursts, which coincided with Ca2+ transients (Fig. 5a).
We found that over several minutes of recording in five different
neurons, virtually all spike bursts coincided with a rise in Ca2+
and, inversely, Ca2+ fluctuations were always associated with spik-
ing (Fig. 5a). Furthermore, the magnitude of the Ca2+ responses
was highly correlated to the number of spikes per burst (Fig. 5b).
We found the same linear relation for the five neurons tested
(P < 0.01). Because population activity varies much less than indi-
vidual neuron activity, this correlation indicates that, in these
cases, the two signals most likely arose from the same cell.
Once we achieved single-unit recording of spontaneous activity
in the stained area, we studied evoked responses to deflection of
single whiskers (Fig. 5c). We recorded concurrent robust evoked
electrophysiological and Ca2+ responses in 47 neurons. Thalamic
relay cells in a barreloid respond differently to the direction of
whisker deflection, and this angular preference is cell-­dependent4.
Thus, to correlate both signals we determined the angular pre­
ference of recorded cells using the Ca2+ response latency and
© 2011 Nature America, Inc. All rights reserved.
spikes elicited by moving the whiskers in four distinct directions
(Fig. 5d)23. We found a linear relationship with a slope of 1
between the angular preferences for both measurements, again
indicating that both signals emanated from the same cells
(Fig. 5e; n = 8 cells; P < 0.001).
Finally, we analyzed spike ‘failures’ in response to whisker
deflection. Using stimuli of constant repetition rate and strength,
we found that whenever we elicited spikes in thalamic neurons,
this caused a Ca2+ response (Fig. 5f). In contrast, when spike
failures occurred, we recorded no significant Ca2+ response
(amplitude decreased by 94 ± 8%; P > 0.5; 10 spikes and 10 spike
failures in three cells). As spike failures are characteristics of indi-
vidual neurons, as opposed to field (population) responses, coin-
cidence of spiking and Ca2+ response failures provided further
evidence that both signals were unitary and originated from the
same neuron.
Optogenetic activation of single cells
We performed experiments on Thy1::ChR2-eYFP transgenic
mice24, which express the light-gated channel ChR2 in various
subpopulations of neurons, including in the thalamus and the
cortical pyramidal layer 5 (Fig. 6a)25. We tested the ability of the
microprobe to activate and record from individual neurons and
thus confirm that they expressed ChR2. We used a 488-nm laser
to simultaneously stimulate ChR2 and eYFP with brief (5–500 ms)
pulses of light. We optically detected 27 cells in the motor ­cortex
(layer 5) and the thalamus in six mice. Of the 27 cells, 22 satis­
fied the combined detection criteria, and of these 19 showed
clear excitatory responses to light pulses (Fig. 6b). We recorded
spikes of constant amplitude and shape in response to local light
pulses at different intensity, confirming single unit recordings
(Fig. 6c–e). At the light pulse onset and offset, we detected small
deflections with opposite polarities reflecting depolarization and
repolarization at the beginning and end of the pulse (Fig. 6c).
These deflections were neither present at subthreshold light
intensities (Fig. 6d) nor in front of nonresponsive neurons
(data not shown). The light intensity activation threshold (1.3 ±
0.3 mW mm−2; n = 13 neurons) was consistent with that pre­viously
reported26 (Fig. 6d). Response delay to light stimulation showed
minimal jitter (Fig. 6f). These observations indicated direct cell
324  |  VOL.8  NO.4  |  APRIL 2011  |  nature methods
depolarization resulting from ChR2 activation in the target cell
and not indirect activation via a neighboring neuron. Finally,
because light stimulation could be used to directly drive spiking,
we could observe postsynaptic inhibition (Fig. 6g; for ­example,
microwhisker deflection silenced the cell during light-driven
activity). These results indicate that the microprobe can be used
to deliver sufficient light for local stimulation in deep brain areas
to control activity from identified single neurons.
DISCUSSION
Our technique is particularly useful for analyzing sparsely dis-
tributed cells that are not otherwise identifiable based on spatial
distribution (for example, inhibitory interneurons dispersed in a
nucleus). With densely packed neurons such as pyramidal neu-
rons in the hippocampal CA1 layer, the ability to unequivocally
identify single neurons would be more challenging. If a specific
phenotypic property distinguished a subset of these pyramidal
neurons, however, they would probably be more sparsely distrib-
uted and hence discernable by the microprobe, for instance, upon
engineering to express a fluorescent protein.
The optical search protocol described here involves local light
delivery and does not require prolonged light exposure; the system
could also be adapted to further minimize excitation cycles using
pulsed illumination. Optical probing allows localization of cells with-
out electrically induced search stimuli such as antidromic or afferent
pathway stimulation, minimizing potential physiological alterations
of neural circuits. An important asset of the probe is that it comprises
only glass; it can be easily shaped and hence can be tailored to dif-
ferent approaches (for example, shorter or longer shanks, tips and
so forth) and can also be used for microinjection (iontophoresis or
pressure ejection) into an optically identified targeted area.
The simple optical design thus yields a versatile system adapt-
able for multiple applications. It will allow exploitation of the
expanding array of tools that use fluorescent sensors and light-
activated channels24–27 and could be used for spectroscopic
detection of intrinsic signals. Exploiting the Brainbow mouse 28,
it would be possible to identify each cell by adapting the system
for multicolor recording. The combined optical and electrical
capabilities of the microprobe described here vastly expand pos-
sibilities for conventional in vivo electrophysiology with access
to single-cell molecular technologies.
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
This work was supported by the Canadian Institutes for Health Research.
Y.D.K. was funded by a Chercheur National award from the Fonds de la recherche en
santé du Québec. Y.L. and S.D. received a Studentship from the Canadian Institutes
for Health Research Neurophysics Training program grant. We thank A. Proulx and
A. Croteau for the collaboration on the dual-core fiber design and fabrication;
D. Côté and A. Castonguay for helpful comments on the manuscript; and S.L. Côté,
K. Bachand, K. Vandal and M. Demers for expert technical assistance.
AUTHOR CONTRIBUTIONS
Y.L., R.V. and Y.D.K. designed the microprobe. Y.L., S.D., G.L., C.B., M.D. and
Y.D.K. designed the experiments. Y.L., S.D., G.L. and Y.D.K. analyzed data.
Y.L., S.D., G.L. and C.B. performed experiments. S.D. performed Ca2+ measurement
and photostimulation experiments. Y.L. and S.D. performed numerical simulations.
Y.L., S.D., G.L., C.B., M.D., R.V. and Y.D.K. contributed to writing the manuscript.

COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
Published online at http://www.nature.com/naturemethods/. 16. Levene, M.J., Dombeck, D.A., Kasischke, K.A., Molloy, R.P. & Webb, W.W.
Reprints and permissions information is available online at http://npg.nature.
com/reprintsandpermissions/.
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nature methods  |  VOL.8  NO.4  |  APRIL 2011  |  325
 ONLINE METHODS
Microprobe fabrication. We designed dual-core optical fibers
in collaboration with the Institut National d’Optique, where the
fibers were fabricated. The fiber preform was assembled through
a procedure inspired from the stack-and-draw method previously
reported in the literature for the fabrication of microstructured
optical fibers29,30. The goal was to position a graded refractive
index optical core and a silica capillary hole side by side in a
silica inclusion tube (Fig. 1b). The optical core was created by
modified chemical vapor deposition using GeO2 dopant31. The
preform was pulled to obtain an optical fiber with a 900-µm exter-
nal diameter, a 550-µm optical core (NA = 0.22) and a 95–105-µm
hollow core. The fiber was cleaved by a diamond pen into
5–7-cm-long rods. Then the rods were heated and pulled with
a standard laser puller (P-2000 laser-based micropipette puller,
required for this type of glass; Sutter). Probes were generated with
2–3-mm-long shoulders, 3–8-mm-long shanks (Fig. 1a) with a
tip pulled to sub-micrometer diameter and grinded back at 90°
to the desired diameter on a rotating diamond abrasive plate
© 2011 Nature America, Inc. All rights reserved.
(Fig. 1b; BV-10 micropipette beveler; plate 104C; Sutter). A wet-
ting agent (Kodak Photo-Flo 200 solution) was added on the abra-
sive plate to prevent small glass residues from blocking the hollow
core. The hollow core was filled with 1–3 M NaCl solution using
negative pressure. Then, a 50 µm-diameter tungsten or stainless
steel wire was introduced into the large extremity of the fiber
to make electric contact with the tip as with conventional glass
microelectrodes. A 2-m-long multimode step index fiber (BFL22-
550, core = 550 µm, cladding = 600 µm, NA = 0.22; Thorlabs) was
aligned with the graded index core and fixed with a UV curing
adhesive (NOA81, Norland products and Curing system UV75
light, Thorlabs) inside a borosilicate glass capillary (1.5 cm long,
1.5 mm inner diameter).
Optical setup. A laser diode was used as excitation light source
(Lambda pro 50 mW 532 nm DPSS laser, Alltech Solutions
or FCD488 20 mW 488 nm, JDS Uniphase). The light passed
through a neutral density filter set (N.D. 1-3, NEK01, Thorlabs)
and was reflected on a dichroic beam splitter (z543rdc or T495LP,
Chroma). The beam was injected into a 10× microscope objec-
tive (UIS-2 Plan-N, NA = 0.25; Olympus) fixed on a fiber launch
system (KT110; Thorlabs) mounted on an xyz translation stage
(PT3; Thorlabs). The laser beam was injected into the probe and
propagated to the tip. The emitted fluorescence was collected
through the microprobe tip, propagated back and collimated by
the 10× microscope objective, passed through the dichroic beam
splitter and entered in a 5.08-cm-long light isolator tube (SM1L30,
Thorlabs). The fluorescent light was then filtered by two succes-
sive band-pass filters (ET605/70M or ET525/50M, Chroma) and
focused by a plano-convex lens (focal length = 50 mm, LA1131-A,
Thorlabs) on a photomultiplier tube (PMT) detector (photosensor
module H6780-20, Hamamatsu) mounted on an xyz translation
stage. The entire optical system was enclosed in a light isolator
box. The PMT output was amplified (gain = 0.5 to 10), filtered
(30 Hz low pass, model 440, Brown lee precision), digitized and
stored on a disk (Fig. 1c).
Brain-slice preparations. Brain slices were prepared from adult
male Sprague-Dawley rats and bacterial artificial chromosome
(BAC) transgenic mice expressing eGFP under the control of the
GAD65 promoter32. All procedures were performed according to
the guidelines of the Canadian Council on Animal Care and were
approved by the Animal Care Committee at Laval University. Mice
and rats were deeply anaesthetized with ketamine and xylazine,
and were decapitated. The brains were removed quickly (<60 s)
and placed in a ice-cold solution containing 210 mM sucrose,
3 mM KCl, 0.75 mM CaCl2, 3 mM MgSO4, 1 mM NaH2PO4,
26 mM NaHCO3 and 10 mM glucose, saturated with 95% O2–5%
CO2. Coronal slices, including the prelimbic area, were cut at
400 µm on a vibrating tissue slicer (VT 1000s, Leica) and kept into
artificial cerebral spinal fluid (ACSF) containing 124 mM NaCl,
3 mM KCl, 1.5 mM CaCl2, 1.3 mM MgSO4, 1 mM NaH2PO4,
26 mM NaHCO3 and 20 mM glucose, saturated with 95% O2–5%
CO2 at room temperature (21–23 °C). A slice was transferred to
a submerge-type chamber where it was continuously exposed to
ACSF at 30–32 °C saturated with 95% O2–5% CO2 and perfused
at a rate of 2.0–2.5 ml min−1. The slice was viewed first with a
10× objective and the prelimbic area of the prefrontal cortex was
localized as the area between the forceps minor corpus callosum
and the midline. Neurons of the prelimbic area were viewed under
near infrared illumination with a 40× water-immersion objec-
tive (IR-ACHROPLAN, 40×, 0.80 W, Zeiss) and a charge-cou-
pled device (CCD) camera (CCD-KP-M1AN, Hitachi). Labeled
cells were identified by epifluorescence. The microprobes were
mounted and moved with a MPC-200 microelectrode manipula-
tor (Sutter Instrument).
Single-cell labeling in fixed tissue. In a subset of experiments,
Lucifer yellow was injected in single neurons in fixed brain slices
to control the distance between labeled cells. Briefly, the frontal
part of a rat brain was fixed overnight in 4% paraformaldehyde
(PFA) in 0.1 M phosphate buffer (PB) and then stored in 0.1 M
PB at 4 °C. Coronal slices (400-µm-thick) were cut on a vibratome
(Leica VT-1200S) and then placed in a patch-clamp recording
chamber in 0.1 M PB for Lucifer yellow injection. Several pairs
of pyramidal cell were thus injected, under visual control, with
sharp electrode filled with 0.5% Lucifer yellow33.
In vivo extracellular single unit recording. Retrograde labeling
of spinothalamic tract neurons was achieved using four injections
of 1 µl DiI (2.5%, Invitrogen) under stereotaxic guidance in the
thalamus of male Sprague-Dawley rats (200–250 g) under isoflu-
rane anesthesia. Rats were anesthetized with isofluorane 7–10 d
later and spinal segments L5–S1 were exposed by a laminaectomy
and stabilized with two lateral clamps on the closest vertebra. A
recording chamber was created on the back of the rat using agar
(3%), and this chamber was perfused continually with buffered
saline heated at 37 °C. A tracheotomy was performed, and the
jugular vein was canulated. Rats were paralyzed with pancuro-
nium bromide (Sigma, 5 mg ml−1, 0.05 ml h−1, intravenously)
and ventilated to reduce breathing movements. Expiratory CO2
was maintained at 4% and body temperature at 37.5 °C using a
heating blanket. The microprobe was mounted on a microman-
ipulator (Burleigh, 6000). The extracellular signal was amplified
on an ER-1 extracellular amplifier (Cygnus technology), filtered
(bandwidth, 0.1–3.0 kHz), digitized and stored on disk simultane-
ously with the PMT output signal.
In vivo experiments were also conducted on the GAD-eGFP
BAC transgenic mice under ketamine (100 mg kg−1) and xylazine
doi:10.1038/nmeth.1572
 (10 mg kg−1) anesthesia. The mice were placed in a stereotaxic
apparatus. The mice breathed freely and body temperature was
maintained at 37.5 °C with a heating pad controlled thermostati-
cally. Throughout the experiment, a deep level of anesthesia was
maintained (stage III-3) using additional doses of anesthetics
given at 1-h intervals (20 mg kg−1 ketamine plus 0.3 mg kg−1 xyla-
zine intra muscularly) The microprobe was mounted on a micro-
manipulator (Burleigh 8200, Inchworm) and descended into the
brain with a 30° angle to reach GFP-expressing thalamic reticular
neurons (2,200–2,500 µm deep). The extracellular electrophysio­
logical signal was amplified (Neurodata IR183, Cygnus technol-
ogy), filtered (band pass, 300–3,000 Hz, model 440, Brownlee
Precision), digitized and stored on disk simultaneously with the
PMT output signal.
Single-cell Ca2+ measurements. Ca2+ measurements were carried
out in adult rats anesthetized as described above. A craniotomy
was performed over the VPM region, and the dura matter was
resected. In some experiments, another craniotomy was per-
© 2011 Nature America, Inc. All rights reserved.
formed over the barrel cortex and the exposed tissue was lesioned
with silver nitrate to abolish rhythmic thalamic activity induced by
corticothalamic inputs. Vibrissae were cut at 5 mm from the skin.
Before stereotaxic injection of Ca2+ indicators, the coordinates of
the whisker responsive neurons were established by electrophysio-
logical recording. The Ca2+ indicator Oregon Green BAPTA-1 AM
(OGB-1; Invitrogen) was dissolved in dimethyl sulfoxide (DMSO)
containing 20% pluronic (Invitrogen). This solution was then
diluted (1/20) in 0.9% saline to obtain the final staining solution,
which was loaded into 30-µm-tip injection micropipettes (Fischer
Scientific, Drummond wiretrol II calibrated micropipettes). The
injection micro-pipette was lowered to coordinates established
by prior electrophysiological recording, and 200 nl of the OGB-1
AM solution was injected. Three to four whisker responsive sites
were localized in the VPM and were labeled in each rat. When
recording from single units, the whisker corresponding to the
receptive field of the cell was deflected for 70 ms in four different
directions by means of a piezoelectric stimulator.
Photostimulation in Thy1::ChR2-EYFP transgenic mice.
Channelrhodopsin-2 transgenic mice were purchased from the
Jackson Labs (B6.Cg-Tg(Thy1øCOP4/EYFP)18Gfng/J). In these
experiments a craniotomy was performed over the motor cortex
or over the VPM region of the thalamus. The microprobe was
descended into the brain (800–4,000 µm deep). A 488-nm laser
(FCD488 20 mW 488 nm, JDS Uniphase Corporation) was used
to detect and activate ChR2-expressing cells. A set of different
doi:10.1038/nmeth.1572
neutral density filters ((ND) 0.1–4, Thorlabs) was used to con-
trol the light stimulus strength and a mechanical shutter (LS3,
Uniblitz) was placed in front of the laser beam to control stimulus
duration. The light intensity at the microprobe tip (Itip) is given
by (Pi × TND × Tfiber × Tp)/(πr2), where Pi is the laser power,
TND is the transmission coefficient of the neutral density filters at
488 nm, Tfiber is the transmission coefficient after the injection,
propagation in the relay fiber and across the optical junction with
the probe, Tp is the transmission of the probe and r is the optical
core radius at the tip. Tfiber and Tp were determined empirically
to be 72% and 0.1%, respectively.
Histology. Brain and spinal cords were extracted from mice and
rats under deep anesthesia. The entire mouse brain and the rat
spinal segments L5–S1 were immersed in 4% paraformaldehyde
and rinsed with a phosphate buffer solution. Tissue was cut in
80–100-µm-thick sections and mounted on glass slides. DiI- or
GFP-labeled cell body diameters were measured as the average of
two perpendicular diameters for each cell. Only cell bodies with
visible nuclei in the section were used for the quantification.
Simulations and data presentation. Theoretical calculations and
numerical simulations used to estimate the microprobe trans-
verse and axial resolutions as well as its excitation and collection
dynamics in tissue are described in Supplementary Data.
Statistical analysis. Correlation analysis was performed to ana-
lyze relationship between FWHM and laser power, optical signal-
to-noise ratio and probe diameter as well as Ca2+ responses and
number of spikes discharged by the neurons. Regression analysis
(ANOVA, F-test) was conducted to analyze the angular prefer-
ence to test whether the relationship was linear and whether the
slope was equal to 1.
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