Wide-field three-photon excitation in biological samples
Light: Science & Applications
Wide-field three-photon excitation in biological samples
Christopher J Rowlands
Oliver T Bruns
Kiryl D Piatkevich
Rakesh K Jain
Moungi G Bawendi
Edward S Boyden
Peter TC So
biophotonics; multiphoton microscopy; optogenetics; temporal focusing; three-photon
While two-photon microscopy is currently the method of choice for
exciting chromophores with close-to diffraction-limited resolution
hundreds of microns into biological tissue1, three-photon has several
advantages over conventional two-photon excitation. The longer
wavelengths used in three-photon microscopy undergo less scattering2;
also the cubic power dependence serves to reduce out-of-focus
excitation. This excitation is responsible for reducing contrast, and ultimately
limits the achievable penetration depth in multiphoton microscopy3.
Disadvantages of three-photon excitation include increased absorption
at longer infrared wavelengths2, the need for optical pulses with a higher
peak power compared with two-photon microscopy and, on occasion,
the requirement for custom-built light sources3.
These disadvantages have limited the use of three-photon excitation
to point-scanning of an excitation spot3,4, despite the fact that
pointscanning is either suboptimal or unsuitable for several optical
techniques, such as optogenetic excitation (where it is necessary to
excite as many opsins as possible in a short space of time)5,
highthroughput imaging6 (where being able to image many spots in
parallel is one means of increasing image throughput), stroboscopic
imaging of fast processes7 (where all points in the image must be
illuminated simultaneously), and targeted photodynamic therapy8
(in which pixel dwell times are necessarily long, and hence
pointscanning is an inefficient use of time), to name but a few.
In this paper, we report the use of commercially available light
sources to perform depth-resolved wide-field three-photon excitation
of chromophores in fixed and live biological samples, with an
excitation wavelength of 1300 nm. Although this type of instrument
cannot image at depths approaching that of point-scanning
multiphoton microscopy, due to scattering of the emission photons9, in
applications such as photodynamic therapy or optogenetic excitation,
penetration depths compete with, or potentially even exceed10,11, those
achievable by point-scanning.
Since this is the first example of wide-field three-photon excitation,
we first image quantum dots (QDs), demonstrating that they can be
excited in fixed biological samples, followed by in vivo imaging of the
cerebral vasculature in a mouse. To demonstrate biological utility, we
perform three-photon excitation of a channelrhodopsin, a class of
light-sensitive ion channels each containing a retinal chromophore.
These are known to be difficult to excite even under two-photon
conditions12, owing to the need to simultaneously excite many
chromophores over the surface of a cell.
MATERIALS AND METHODS
QD samples were prepared by dispersing a 4 ?L drop of QDs
suspended in water (525 nm emitting nanocrystals provided by QD
Vision) on a microscope slide. A drop of UV-curing adhesive
(Norland Products NOA 74) was placed on the dried QDs, a coverslip
placed on top, and the sample cured by exposure to a UV source.
HeLa cells on a glass coverslip were fixed at room temperature using
4% paraformaldehyde (Electron Microscopy Sciences 15742-10) for
10 min, permeablized for 2 ? 15 min using 0.25% Triton X-100
(Sigma Aldrich 93427) in phosphate-buffered saline, and then treated
with an endogenous biotin blocker (Life Technologies E21390). This
was followed by treatment with Biotin-XX Phalloidin (Life
Technologies B7474) for 20 min, then a 605-nm-emission QD-streptavidin
conjugate (Life Technologies Q10151MP) before sealing with a
coverslip using Mount Quick medium (Electron Microscopy Sciences
Mouse vasculature imaging
Intravital images were taken through a cranial window implanted into
a male nude mouse13,14. The mouse was anesthetized by
intraperitoneal injection of ketamine and xylazine. A tail vein catheter was
placed for injecting the QD solution during imaging, however the
injection was not performed until the mouse had been secured on the
The QD solution consisted of green-emitting QDs in polyethylene
glycol (PEG)-phospholipid micelles, synthesized as follows: QDs were
transferred into aqueous buffers using a previously reported
procedure15,16. Three milligrams (dry weight) QDs were mixed with
25 mg 18:1 PEG2000 PE
(1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-methoxy(polyethylene glycol)-2000; ammonium salt; Avanti
Polar Lipids; cat. no. 880130) in chloroform. After sonication for 10 s,
the solvent was removed under nitrogen flow and 2 mL isotonic saline
or water was added. To completely solubilize the QDs, the aqueous
solution was sonicated with a probe sonicator for 5 min, and filtered
through a 0.2 ?m filter. A total volume of 200 ?L of this solution was
injected during imaging.
Animal experiments were conducted in accordance with approved
institutional protocols of MGH and MIT.
A regenerative amplifier (Coherent Legend Elite) produced 130 fs
pulses at 10 kHz, with 600 ?J pulse energy and 800 nm center
wavelength. 3.2 W average power was used to pump an optical
parametric amplifier (OPA; Coherent, Opera Solo with second
harmonic option) that converted the 800 nm pulses to 1300 nm,
with average power up to 450 mW (up to 14% conversion
efficiency). After an upgrade, 5.5 W average power was used to
pump the OPA, yielding up to 960 mW at 1300 nm (17%
The light from the OPA was attenuated either by altering the
regenerative amplifier?s pulse compressor, by using a half waveplate
(Thor Labs AHWP05M-1600) and polarizer (Newport 10GL08AR.18),
or by placing a large tilted coverslip in the beam. The light then passed
through a shutter (Vincent Associates VS25S2ZM1R3, suspended from
an overhead gantry to avoid vibrational coupling to the table) and an
850 nm long-pass filter (Thor Labs FELH0850) to a variable beam
expander (capable of ? 1, ? 2.5, ? 3.5 and ? 5 beam expansion).
It then reflected off a silver mirror (Thor Labs PFSQ20-03-P01) onto a
diffraction grating (Custom, Spectrogon 715.706.410 G 0750 NIR).
The -1 order from the grating was imaged onto the microscope image
plane through a ? 0.25 telescope consisting of a 200-mm focal length,
75 mm diameter lens (Edmund Optics 86-923) and a 50 mm focal
length compound lens made from two 100 mm focal length 30 mm
diameter lenses (Edmund Optics 67-572). The silver mirror was
placed as close to the 75 mm lens as possible, to operate the grating in
a near-Littrow condition for efficiency.
After the telescope, excitation light entering the microscope was
collimated by a tube lens (Zeiss 425308-0000-000), reflected off a
1200 nm short-pass dichroic mirror (Edmund Optics 86-699) and
imaged onto the sample through the ? 25 1.0 NA near-IR microscope
objective (Olympus XLPN25XSVMP) mounted on a 400-?m travel
focusing drive (Piezosystem Jena MIPOS 500 SG). The median
axial full-width half-maximum (FWHM) was found to be ~ 3.0 ?m
(Supplementary Fig. 2).
The two-photon excitation path was very similar; light from the
regenerative amplifier was expanded using a ? 5 Galilean cylindrical
telescope constructed from a -15 mm focal length lens (Thor Labs,
LK1006L1-B) and 75 mm focal length lens (Edmund Optics, 69-762).
The beam struck a grating (Richardson Gratings, 53006BK02-540R) at
an angle of 74?, such that the -1 order diffracted beam propagated
along the microscope?s optical axis. The beam was demagnified using a
? 1.75 telescope before being imaged onto the intermediate image
plane as before; this telescope consisted of a 3? diameter 190 mm focal
length compound lens made from two singlets (Edmund Optics
86-920 and Newport KPX232AR.16), and the same 30 mm diameter
50 mm focal length lens used for three-photon excitation. Other than
a change of filter cube to a 750 nm dichroic (Semrock,
FF750-SDi0225 ? 36) and 775 nm short-pass emission filter (Semrock, FF01-775/
SP-25), the microscope was identical to the three-photon
configuration. The median axial FWHM was found to be ~ 18 ?m
(Supplementary Fig. 3).
For imaging experiments, fluorescence emission passed through
the objective, dichroic, emission filter (1200 nm short pass,
Edmund Optics 86-693) and microscope tube lens, before being
detected on an EMCCD camera (Andor iXon 885k). The optical
layout can be seen in Supplementary Fig. 1. For the HeLa cells, the
imaging parameters were 10 s integration time, ? 100 EM gain on
the EMCCD, 80 mW incident power and ? 5 beam expander.
Flatfield correction was applied to reduce the effect of non-uniform
To determine the power scaling, pure QD samples were imaged
with varying excitation intensities (controlled by a tilted coverslip
to minimize dispersion). Images were captured on the camera with
a 1 s integration time for the 525 nm QDs, and 0.1 s for the 605 nm
QDs. The excitation region was cropped, the background
subtracted and the average fluorescence intensity plotted as a function
of incident power.
For electrophysiology experiments, a Faraday cage was built around
microscope, and an illumination system constructed, consisting of
a red (Thor Labs M660L3, 660 nm center wavelength) and blue
(Thor Labs M470L2, 470 nm center wavelength) LED combined using
a dichroic mirror (make and model unknown; taken from a Green
Fluorescent Protein filter cube) and focused onto the sample using a
microscope objective (Olympus PlanN ? 10/0.25). The red LED was
used to locate the cells by bright-field illumination without triggering
an action potential, and the blue LED was used to both excite
fluorescence (in order to locate cells with high expression of CoChR)
and to excite an action potential when necessary. Incident power
required to excite an action potential was found to be between ~ 0.13
and 1.8 mW mm ?2. For experiments establishing the maximum
achievable frequency due to one-photon absorption, the intensity
was optimized until the highest frequency could be achieved; these are
the data reported.
To investigate the primary damage mechanism, the focal plane was
placed ~ 180 ?m from the cell surface (equal to one turn of the coarse
focus knob on the microscope). Maximum laser power was used to
ensure operation well above the damage threshold; this power
averaged 720 mW at 1300 nm, corresponding to ~ 144 mW at the
sample. The lowest power used was 640 mW, corresponding to
~ 128 mW at the sample. Four exposures of 300 ms were used as
before, in addition to one exposure of 3 s.
Penetration depth in fixed slices
An epifluorescence microscope was constructed above the sample
stage of the previously described three-photon instrument,
consisting of an objective (Zeiss 421452-9880-000), tube lens (Thor Labs
AC508-150-A-ML) and camera (PCO Edge 5.5). A dichroic mirror
(make and model unknown; taken from a Green Fluorescent
Protein filter cube) in the beam path reflected light from a 470 nm
LED (Thor Labs M470L2) onto the sample to aid in locating the
Animal experiments were conducted in accordance with approved
institutional protocols of MIT and MGH. Brain slices were obtained
from a male C57BL/6J mouse; it was anesthetized and the tissues
were fixed with 4% paraformaldehyde through cardiac perfusion.
The fixed brain was extracted and kept in 4% paraformaldehyde,
followed by embedding in low-melting agarose. It was then sliced to
varying thicknesses using a vibratome (Leica VT1000S). The slices
were placed in the well of a glass-bottomed dish (MatTek
P50G-0-14F) and covered with distilled water. A 4 ?L drop of QDs suspended in
water (525 nm emitting nanocrystals provided by QD Vision) was
placed on a 30 mm diameter coverslip and allowed to dry; this
coverslip was then placed on top of the brain slice with the QDs on the
inside, the excess water wiped away and the coverslip sealed using clear
The sample was mounted on the three-photon instrument, and the
upper ?imaging? microscope brought into focus. The lower ?excitation?
microscope was also focussed on an unobscured region of the QDs, to
ensure the two microscopes were co-aligned. A 20 lp mm 1 Ronchi
ruling (Edmund Optics 58-777) was placed at the image plane of the
excitation microscope; the pattern projected onto the sample had a
period of 2.2 ?m. The brain slice was then translated towards the
illumination region until the fluorescence from the QDs was not
observable on the excitation microscope, even with 1 s exposure and
? 10 electron multiplying gain. For thinner samples, green
fluorescence was observable even when the brain slice completely overlapped
the excitation region; in these cases, the brain slice was translated until
it filled the field of view of the camera. Finally, the excitation light was
refocused to maximize signal in the imaging microscope.
For comparison with two-photon excitation, 6 W of average power
was available at 800 nm, so a larger area was illuminated. Total
power at the sample was 50 mW with an illuminated area of
~ 600 ?m ? 600 ?m, however the increased cross-section of
twophoton excitation relative to three-photon excitation resulted in
similar emission intensities in the absence of scattering. Excitation
was performed by routing light from the regenerative amplifier
through a separate beam path described above.
Imaging was performed with a 2 s integration time on the sCMOS
camera; to compensate for vibration in the imaging microscope setup
(an unavoidable consequence of having to mount the microscope so
far above the table surface) 16 frames were captured and the frame
exhibiting the least vibration-induced blurring was taken.
All procedures involving animals were in accordance with the US
National Institutes of Health Guide for the Care and Use of Laboratory
Animals and approved by the MIT Committee on Animal Care.
Hippocampal neuron cultures were prepared from postnatal day 0 or
1 Swiss Webster (Taconic) mice as previously described17,18 but with
the following modifications: dissected hippocampal tissue was digested
with 50 units of papain (Worthington Biochem) for 6?8 min, and the
digestion was stopped with ovomucoid trypsin inhibitor (Worthington
Biochem). Cells were plated at a density of 20,000?50,000 per glass
coverslip coated with Matrigel (BD Biosciences). Neurons were seeded
in 100 ?L Plating Medium containing MEM (Life Technologies),
glucose (33 mM, Sigma), transferrin (0.01%, Sigma), Hepes (10 mM),
Glutagro (2 mM, Corning), Insulin (0.13%, Millipore), B27
supplement (2%, Gibco), heat inactivated fetal bovine serum (7.5%,
Corning). After cell adhesion, additional Plating Medium was added.
AraC (0.002 mM, Sigma) was added when glia density was
50?70%. Neurons were grown at 37 ?C and 5% CO2 in a humidified
atmosphere. Cultured neurons were induced at 4 days in vitro (DIV)
with 1.0 ?L of rAAV2/8-Synapsin-CoChR-GFP (titer: 3.8 ? 1012
particles per mL) per well. AAV particles were produced by the
University of North Carolina Chapel Hill Vector Core.
Coverslips supporting the prepared neuronal cultures were
mounted on glass-bottomed dishes (MatTek P50G-0-14-F) at 14-20
DIV and immersed in Tyrode solution containing 125 mM NaCl,
2 mM KCl, 3 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 30 mM
glucose, 0.01 mM NBQX and 0.01 mM GABAzine. The pH was 7.3.
Patch clamping was performed using a micromanipulator system
(Sutter Instruments MP285 micromanipulator, MPC-200 controller
and Axon Instruments CV-7B headstage). Signals from the headstage
were recorded using an amplifier (Molecular Devices MultiClamp
700B) and data acquisition system (Molecular Devices Digidata
1440a), controlled using pCLAMP 10 software. Exposure was
controlled by the shutter, which was controlled in turn by TTL input from
the data acquisition system.
Patching was performed using borosilicate glass pipettes
(Warner Instruments) with an outer diameter of 1.2 mm and a
wall thickness of 0.255 mm. These were pulled to a resistance of
5?10 M? with a P-97 Flaming/Brown micropipette puller (Sutter
Instruments) and filled with a solution containing 135 mM
K-gluconate, 8 mM NaCl, 0.1 mM CaCl2, 0.6 mM MgCl2, 1 mM
EGTA, 10 mM HEPES, 4 mM Mg-ATP, and 0.4 mM Na-GTP, and
with pH 7.3 and 290 mOsm. To ensure accurate measurements,
cells were used with access resistance between 5 and 35 M?.
Before all experiments, the holding current was adjusted such that
the measured potential was between 60 and 65 mV; holding
currents were within ? 100 pA in all cases.
In some cases a 60 Hz signal was present in the data; this was
computationally removed from the presented data by fitting a 60 Hz
sine wave and subtracting the fit.
RESULTS AND DISCUSSION
Wide-field three-photon excitation was achieved using temporal
focusing, a technique in which a high-peak-power ultrafast pulse is
dispersed using a grating, before being imaged onto the sample19,20.
This dispersion results in temporal broadening outside of the focal
plane, and since multiphoton excitation is sensitive to the reciprocal
of the pulse duration (raised to the power of the number of photons
in the excitation process minus one1), efficient multiphoton excitation
only occurs within a few microns of this plane.
To achieve efficient multiphoton excitation, the optimal strategy is
to increase the pulse energy until the excitation spot is saturated
(that is, any further increase in pulse energy results in unacceptable
deterioration of the point-spread function21) and then to increase
either the degree of parallelization (that is, increase the number of
illuminated spots) or the laser repetition rate until the limit on the
acceptable average power in the sample is reached. Previously,
researchers have optimized the repetition rate3, however we argue
for an increase in parallelization, using a commercially available Ti:
Sapphire regenerative amplifier pumping an OPA.
Increasing parallelization has many advantages; changing the
illuminated area in a temporal focusing microscope involves merely
changing the size of the spot on the grating, and hence can be trivially
adapted depending on the chromophore. Changing the repetition rate
of a laser, on the other hand, is often difficult and the tunable range is
frequently limited. The second advantage applies to chromophores
with a long excited-state lifetime, or equivalent. In the case of a
phosphorescent probe, for example, lifetimes of hundreds of
nanoseconds are not unusual22. The excited-state lifetime of a QD can also be
very long; up to several microseconds in some cases23. Other more
unusual chromophores, such as opsins, have a photocycle turnover
time (equivalent to the excited state lifetime for the purpose of this
analysis) on the order of 10?20 ms24, hence increasing the repetition
rate is an extremely inefficient means of optimizing the excitation.
Excessive repetition rate may even contribute to reduced quantum
yield and increased photobleaching, via excited-state absorption and
intersystem crossing to triplet states. In contrast, by keeping the
repetition rate low and increasing the degree of parallelization, these
chromophores can be efficiently excited.
The final advantage is practical; obtaining a desired wavelength with
high peak power at an optimal repetition rate (~1 MHz) is difficult,
whereas a commercially available OPA system can tune to many
different wavelengths from the ultraviolet to the mid-infrared.
To demonstrate efficient wide-field three-photon excitation at
1300 nm, images of a layer of 525 nm QDs were taken with several
different beam expanders (Figure 1a?1c). To further validate the
capability of this system for wide-field excitation, fixed HeLa cells
stained with a 605 nm QD-streptavidin conjugate were imaged. A large
field of view of up to ~ 150 ?m diameter can be observed (Figure 1d).
The microscope?s axial resolution was determined to be 3.0 ?m
median FWHM (Supplementary Fig. 2), comparable to the theoretical
value for two-photon microscopy25 of ~ 1.8 ?m, for a 1.0 NA lens,
1300 nm excitation and refractive index of 1.333. Compromises in
axial resolution are therefore not necessary in order to employ this
Figure 1e plots the absorption and emission of the 525 nm QDs. In
particular, it should be noted that two-photon absorption at 1300 nm
is extremely unlikely, as there are no absorption features in the
onephoton spectrum near 650 nm. A similar absorption profile was
obtained from the manufacturer of the 605 nm QD-streptavidin
conjugates. As an additional precaution, an 850 nm long-pass filter
was used to avoid one- and two-photon excitation by residual
frequency-converted light from the OPA. Further confirmation of
the three-photon nature of the excitation can be found in Figure 1f;
the fluorescence intensity for both 525 and 605 nm QDs scales as the
laser power to the power 3.31. The fact that the power scaling is not an
integer indicates a contribution from four-photon excitation,
consistent with the absorption cross-section of a QD which increases
significantly at shorter wavelengths.
Efficient three-photon excitation can therefore be performed over a
field of view sufficient to encompass several cells. Integration times of
less than one second were possible with a spot size of ~ 100 ?m
diameter. Owing to the cubic dependence on excitation power,
increased excitation can be achieved with modest reduction in
illuminated area or increase in incident power.
Compatibility with in vivo excitation
To demonstrate compatibility of three-photon wide-field excitation
with biological specimens, QDs were injected into a mouse and
imaged. Supplementary Videos 1 and 2 show focal stacks of
vasculature in a live mouse brain. QDs were injected into the tail
vein and allowed to circulate. Microvessels were subsequently imaged
using the three-photon instrument through a glass window implanted
in the skull. The 5 s integration time and ~ 250 ?m diameter field of
view are competitive with existing three-photon excitation methods3,
and the 166 mW (Supplementary Video 1)/138 mW (Supplementary
Video 2) incident power (with ? 2.5 beam expander) caused no
observable damage to the mouse.
A map of the maximum irradiance for 166 mW incident power can
be seen in Supplementary Fig. 4. The aberrated mode that was caused
by a misalignment in the OPA was compensated by the extra power
available from the OPA; peak irradiance was ~ 15 W mm ?2. After
imaging, the mouse was allowed to recover from anesthesia, and when
observed after an hour, no evidence of any neurological damage was
No attempt was made to maximize penetration depth, as tissue
scattering of the emission light would limit the achievable depth to
approximately the scattering mean-free path in tissue. Optimization
was instead made for field-of-view and integration time. Nevertheless,
for the broader use-case of three-photon excitation, an exploration of
the penetration depth follows.
Penetration through fixed brain slices
Since one purpose of three-photon excitation is to penetrate deeper
into tissue than two-photon excitation, experiments were performed
to establish the achievable penetration depth of the excitation light.
A Ronchi ruling with a period of 20 line-pairs per millimeter was
placed at the intermediate image plane of the microscope, such that an
image of the ruling was projected onto the sample. In addition, a
microscope was constructed on top of the existing one, such that the
far side of the sample could be observed (Figure 2e). Fixed brain slices
of varying thickness were then placed on the microscope, and a layer
of QDs placed atop them, opposite the excitation objective and in the
focal plane of the imaging objective. By varying the thickness of the
brain slices and observing whether the modulation pattern was still
present, a measure of the minimum achievable penetration depth
could be determined.
The projected pattern had a period of 2.2 ?m at the sample; this was
deemed suitable for resolving cellular features. These images can be
seen in Figure 2, along with a diagram of the experimental
configuration. The experiment was repeated using two-photon excitation at
800 nm to provide a performance baseline; these results indicate that
the pattern is all but unobservable by 400 ?m (Figure 2b), consistent
with the 250 ?m penetration depth into fixed tissue achieved by
Papagiakoumou et al.11. In contrast, three-photon excitation at
1300 nm achieved a penetration depth of up to 800 ?m, albeit with
significantly reduced excitation power. By 900 ?m, the spot was
unobservable, hence it was impossible to determine whether the
pattern was successfully projected or not.
To confirm that arbitrary patterns could also be projected, an MIT
logo was used as a photomask (Figure 2d). Careful observers will note
that there is a small amount of astigmatism present in the image; this
is discussed in the Supplementary Information.
Figure 2c plots the fraction of intensity in the modulated spatial
frequency over the intensity in the whole image. It was calculated by
subtracting the mean value of each image and taking a 2D Fourier
transform; the intensity of the two peaks corresponding to the
modulation frequency were summed, then divided by the sum over
all frequencies. For uniform illumination with a superimposed
sinusoidal pattern and infinite extent, this value tends to 1; lower
values indicate a loss of contrast at the spatial frequency of interest, or
alternatively, increased contributions from other spatial frequencies
caused by either non-uniform excitation or fluorophore distribution.
This metric is illustrated in Supplementary Fig. 5, and examples of
different images and their corresponding values can be seen in
Supplementary Fig. 6.
Care was taken to ensure that the occluding region of the brain
tissue was located within ~ 100?300 ?m of the cortex surface, to
maximize the relevance of the data to conventional imaging. For
comparison, the experiment was repeated with the focus as far below
the surface of the brain as possible, and it was still possible to resolve
the grating up to 500 ?m into the sample, despite the clear increase in
scattering of the deeper tissue (Supplementary Fig. 7). In addition,
since fixation typically increases the scattering coefficient of tissue26, it
is expected that this penetration depth represents a lower bound; live
tissues will have lower scattering coefficients and larger penetration
depths, as demonstrated by Begue et al.27 who managed to project
two-photon temporal focusing patterns through a 550-?m-thick live
brain slice, albeit using a longer wavelength (950 nm) and coarser
features (on the order of 10 ?m) than tested here. If the increase in
performance due to the use of live tissue and coarser features applies
equally for three-photon patterning as it does for two-photon
patterning, the grating pattern could be preserved up to twice as far
as for two-photon excitation, provided sufficient power is available.
The earliest example of multiphoton excitation for optogenetics was
performed by Mohanty et al.28, who focussed a single point onto a
neuron to excite calcium activity in cell culture and hippocampal brain
slices. Other researchers have found that, while two-photon
singlepoint scanning can trigger an action potential in an opsin-expressing
neuron29?31, two-photon wide-field excitation is more effective at
triggering the action potential5,32?34. In a report using a similar laser
configuration to our high-power femtosecond OPA system, a
homebuilt three-stage OPA system was used to illuminate an entire fly
brain, without spatial selectivity, to demonstrate that two-photon
excitation of opsins is not limited to small numbers of neurons?
potentially the entire brain of an animal can be exposed
Demonstrating three-photon wide-field excitation of a neuron
therefore serves both as an excellent test of system performance, as
well as proof that wide-field three-photon excitation is possible for
chromophores other than QDs. To maximize the photocurrent, a
recently-developed opsin called CoChR17 was used. CoChR has a
higher photocurrent than other opsins perhaps in part due to slow
offkinetics that enables more charge to enter the cell for a given photon
dosage, as well as potentially superior membrane trafficking properties.
Use of this opsin maximizes the possibility of triggering an action
potential, given the low excitation efficiency of three-photon excitation
versus conventional one-photon excitation. Figure 3a illustrates that it
was possible to repeatedly trigger an action potential by three-photon
excitation at 1300 nm in cultured neurons. Tests were also performed
on non-transfected control cells, obtained from the same animal as the
transfected cells (Supplementary Fig. 9). Although the power levels
were occasionally high enough to trigger a damage-mediated action
potential (consistent with Hirase et al.36), any damage was evident by
an increase in the resting potential, and the results were not repeatable
?further excitation resulted in higher and higher resting potentials
and ultimately cell death. Power levels required to cause photodamage
were around 64 ? 10 mW (n = 22 cells) at the sample, as compared
with the power required to excite an action potential, which was
around 51 ? 11 mW (n = 14 cells); errors indicate one standard
deviation from the mean. This safety margin was found to be sufficient
in the majority of cases to excite a cell repeatedly without causing
significant damage, with damage defined as a pronounced rise in the
resting potential, such that more than 100 pA hold current was
required for a ? 60 mV voltage clamp. It is likely that this margin will
be more pronounced in vivo, as the neurons are likely to be healthier
compared with cell culture, and the presence of the vascular system
can help dissipate heat in the case of thermally mediated damage
Results indicate that three-photon excitation of action potentials is
consistently achievable (n = 14 cells). Further investigation of the
maximum excitation frequency (n = 9 cells) illustrates the low-pass
filtering response of the neurons under test; as the excitation frequency
rose, the fraction of successful excitation events dropped. This is
consistent with experiments performed using modulated one-photon
excitation at 470 nm (see Figure 3d and Supplementary Fig. 10),
as well as the mean minimum pulse duration required to excite an
action potential of 52 ms, ? 20 ms standard deviation (n = 7 cells).
It is therefore safe to conclude that the inability to excite action
Light from temporal
8 10 12 14 16 18 20 22
potentials at higher frequencies is not due to the efficiency of
threephoton excitation, since one-photon excitation suffers from the same
limitations. Rather, it is due to either the CoChR off-kinetics, the low
spontaneous spike rate of the patched neurons37, subtle effects due to
the cell culture preparation, or any combination thereof. Indeed, the
inability of our system to excite spike trains at a rate greater than ~ 10?
20 Hz in cell culture is not unique; the maximum excitation frequencies
in cell culture reported by several other authors are similar, including
for Channelrhodopsin-25,38, C1V1TT17 and ReaChR39.
The primary damage mechanism potentially places a strong
limitation on the penetration depth. If it is mediated by a
singlephoton absorption event, penetration will be limited as the tissue
surface will be damaged as the excitation power is increased to
compensate for tissue attenuation. If, however, it is mediated by
multiphoton absorption, damage will be confined to the temporal
focusing plane, and hence penetration depths will not be significantly
Literature results suggest that damage is primarily multiphoton
in nature40?44; penetration depths of well over a millimeter were
achieved without observable tissue damage3. Nevertheless, an
experiment was performed to establish the primary damage
mechanism. Cells were patched and illuminated as before, but
this time the microscope was defocused by ~ 180 ?m from the cell
surface, in order to maintain approximately the same photon flux
but reduce multiphoton excitation to negligible levels. Even with
average intensities at least double the previously determined
damage threshold, no cells suffered any observable damage for
either 4 ? 300 ms exposures, or a subsequent 1 ? 3 s exposure,
whereas by translating back to the focal plane afterwards and
illuminating the cell, all but two cells could be damaged (82%,
n = 11). Example plots of the membrane potential under current
clamp during these experiments can be seen in Supplementary Fig.
11. From this we conclude that photodamage is likely primarily
multiphoton in nature, but we should emphasize that, as with all
multiphoton experiments, there may also be a very small degree of
damage caused by single-photon heating.
Overall, wide-field three-photon excitation overcomes many of the
limitations of three-photon point-scanning, all without the need
to significantly compromise axial resolution. We demonstrate
excitation in a wide array of samples, both in vivo and ex vivo, with
approximately double the penetration depth compared with two
photon, and we successfully excite action potentials in
opsinexpressing neurons. These results indicate that future moves to
excitation in live brain slices or even in vivo experiments are well
Construction of wide-field three-photon instrumentation will
become much easier with the development of commercially available
OPCPA designs offering several tens of Watts of power throughout the
visible and near-infrared spectrum45?47. Excitation can therefore be
performed faster, and over larger areas compared to the
Ti:Sapphirebased system used here.
Experiments were conceived by CJR, DP, OTB, ESB and PTCS.
Instrumentation and software was constructed by CJR, with help from DP. Quantum dots
from the laboratory of MGB were assembled into test samples by CJR and
OTB. The mouse bearing a cranial window was obtained from the laboratory of
DF and RKJ, and was prepared for imaging by OTB. For optogenetics
experiments, cells were prepared by DP and transfected by KP. Patch clamping
was performed by CJR and DP. The manuscript was written by CJR, with help
from all other authors.
We thank Professor Chris Xu for extremely helpful discussions during
preparation of the manuscript, Jessica Carr for measuring the QD excitation and
emission spectra, and Sylvie Roberge for preparing the mouse cranial window
model. The authors are also grateful to QD Vision for providing the green QD
sample. CJR and PTCS acknowledge support from NIH-5-P41-EB015871-27,
DP3-DK101024 01, 1-U01-NS090438-01, 1-R01-EY017656 -0, 6A1,
1-R01HL121386-01A1, the Biosym IRG of Singapore-MIT Alliance Research and
Technology Center, the Koch Institute for Integrative Cancer Research Bridge
Initiative, the Hamamatsu Inc., and the Samsung GRO program. CJR is grateful
for a fellowship to carry out this research; the fellowship was supported by the
Wellcome Trust 093831/Z/10/Z. ESB acknowledges funding from NIH
1R24MH106075, NIH 2R01DA029639, NIH 1R01MH103910, NIH
1R01GM104948, the MIT Media Lab, the New York Stem Cell
FoundationRobertson Award and NSF CBET 1053233. OTB is grateful for an EMBO
Longterm Fellowship to carry out this research. MGB and OTB acknowledge
support from NIH 5U54 CA151884-04 and 9-P41-EB015871-26A1. DF and
RKJ are grateful for NCI grants R35 CA197743 and P01 CA080124 to carry out
Supplementary Information for this article can be found on the Light: Science & Applications? website (http://www.nature.com/lsa).
1 Helmchen F , Denk W. Deep tissue two-photon microscopy . Nat Methods 2005 ; 2 : 932 - 940 .
2 Jacques SL . Optical properties of biological tissues: a review . Phys Med Biol 2013 ; 58 : R37 - R61 .
3 Horton NG , Wang K , Kobat D , Clark CG , Wise FW et al. In vivo three-photon microscopy of subcortical structures within an intact mouse brain . Nat Photon 2013 ; 7 : 205 - 209 .
4 Yu JH , Kwon SH , Petr??ek Z , Park OK , Jun SW et al. High-resolution three-photon biomedical imaging using doped ZnS nanocrystals . Nat Mater 2013 ; 12 : 359 - 366 .
5 Papagiakoumou E , Anselmi F , B?gue A , de Sars V , Gl?ckstad J et al. Scanless two-photon excitation of channelrhodopsin-2 . Nat Methods 2010 ; 7 : 848 - 854 .
6 Dodt HU , Leischner U , Schierloh A , J?hrling N , Mauch CP et al. Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain . Nat Methods 2007 ; 4 : 331 - 336 .
7 Suarez SS , Varosi SM , Dai X . Intracellular calcium increases with hyperactivation in intact, moving hamster sperm and oscillates with the flagellar beat cycle . Proc Natl Acad Sci USA 1993 ; 90 : 4660 - 4664 .
8 Rowlands CJ , Wu J , Uzel SGM , Klein O , Evans CL et al. 3D-resolved targeting of photodynamic therapy using temporal focusing . Laser Phys Lett 2014 ; 11 : 115605 .
9 Rowlands CJ , Bruns OT , Bawendi MG , So PTC . Objective, comparative assessment of the penetration depth of temporal-focusing microscopy for imaging various organs . J Biomed Opt 2015 ; 20 : 061107 .
10 Sela G , Dana H , Shoham SUltra-deep penetration of temporally-focused two-photon excitationIn :Periasamy A , K?nig K , So PTCeditors . Proceedings of SPIE 8588 , Multiphoton Microscopy in the Biomedical Sciences XIII 858824 . San Francisco, CA, USA: SPIE; 2013 .
11 Papagiakoumou E , B?gue A , Leshem B , Schwartz O , Stell BM et al. Functional patterned multiphoton excitation deep inside scattering tissue . Nat Photon 2013 ; 7 : 274 - 278 .
12 Oron D , Papagiakoumou E , Anselmi F , Emiliani V . Two-photon optogenetics . Prog Brain Res 2012 ; 196 : 119 - 143 .
13 Yuan F , Salehi HA , Boucher Y , Vasthare US , Tuma RF et al. Vascular permeability and microcirculation of gliomas and mammary carcinomas transplanted in rat and mouse cranial windows . Cancer Res 1994 ; 54 : 4564 - 4568 .
14 Jain RK , Munn LL , Fukumura D. Dissecting tumour pathophysiology using intravital microscopy . Nat Rev Cancer 2002 ; 2 : 266 - 276 .
15 Dubertret B , Skourides P , Norris DJ , Noireaux V , Brivanlou AH et al. In vivo imaging of quantum dots encapsulated in phospholipid micelles . Science 2002 ; 298 : 1759 - 1762 .
16 Stroh M , Zimmer JP , Duda DG , Levchenko TS , Cohen KS et al. Quantum dots spectrally distinguish multiple species within the tumor milieu in vivo . Nat Med 2005 ; 11 : 678 - 682 .
17 Klapoetke NC , Murata Y , Kim SS , Pulver SR , Birdsey-Benson A et al. Independent optical excitation of distinct neural populations . Nat Methods 2014 ; 11 : 338 - 346 .
18 Chow BY, Han X , Dobry AS , Qian XF , Chuong AS et al. High-performance genetically targetable optical neural silencing by light-driven proton pumps . Nature 2010 ; 463 : 98 - 102 .
19 Oron D , Tal E , Silberberg Y. Scanningless depth-resolved microscopy . Opt Express 2005 ; 13 : 1468 - 1476 .
20 Zhu GH, van Howe J , Durst M , Zipfel W , Xu C . Simultaneous spatial and temporal focusing of femtosecond pulses . Opt Express 2005 ; 13 : 2153 - 2159 .
21 Cianci GC , Wu JR , Berland KM . Saturation modified point spread functions in twophoton microscopy . Microsc Res Tech 2004 ; 64 : 135 - 141 .
22 Choi H , Tzeranis DS , Cha JW , Cl?menceau P , de Jong SJ et al. 3D-resolved fluorescence and phosphorescence lifetime imaging using temporal focusing wide-field two-photon excitation . Opt Express 2012 ; 20 : 26219 - 26235 .
23 Moreels I , Lambert K , Smeets D , De Muynck D , Nollet T et al. Size-dependent optical properties of colloidal PbS quantum dots . ACS Nano 2009 ; 3 : 3023 - 3030 .
24 Zhang F , Vierock J , Yizhar O , Fenno LE , Tsunoda S et al. The microbial opsin family of optogenetic tools . Cell 2011 ; 147 : 1446 - 1457 .
25 Zipfel WR , Williams RM , Webb WW . Nonlinear magic: multiphoton microscopy in the biosciences . Nat Biotechnol 2003 ; 21 : 1369 - 1377 .
26 Pitzschke A , Lovisa B , Seydoux O , Haenggi M , Oertel MF et al. Optical properties of rabbit brain in the red and near-infrared: changes observed under in vivo, postmortem, frozen, and formalin-fixated conditions . J Biomed Opt 2015 ; 20 : 025006 .
27 B?gue A , Papagiakoumou E , Leshem B , Conti R , Enke L et al. Two-photon excitation in scattering media by spatiotemporally shaped beams and their application in optogenetic stimulation . Biomed Opt Express 2013 ; 4 : 2869 - 2879 .
28 Mohanty SK , Reinscheid RK , Liu XB , Okamura N , Krasieva TB et al. In-depth activation of channelrhodopsin 2-sensitized excitable cells with high spatial resolution using twophoton excitation with a near-infrared laser microbeam . Biophys J 2008 ; 95 : 3916 - 3926 .
29 Prakash R , Yizhar O , Grewe B , Ramakrishnan C , Wang N et al. Two-photon optogenetic toolbox for fast inhibition, excitation and bistable modulation . Nat Methods 2012 ; 9 : 1171 - 1179 .
30 Packer AM , Russell LE , Dalgleish HWP , H?usser M. Simultaneous all-optical manipulation and recording of neural circuit activity with cellular resolution in vivo . Nat Methods 2014 ; 12 : 140 - 146 .
31 Zhu P , Narita Y , Bundschuh ST , Fajardo O , Sch?rer YP et al. Optogenetic dissection of neuronal circuits in zebrafish using viral gene transfer and the tet system . Front Neural Circuits 2009 ; 3 : 21 .
32 Andrasfalvy BK , Zemelman BV , Tang JY , Vaziri A . Two-photon single-cell optogenetic control of neuronal activity by sculpted light . Proc Natl Acad Sci USA 2010 ; 107 : 11981 - 11986 .
33 Packer AM , Peterka DS , Hirtz JJ , Prakash R , Deisseroth K et al. Two-photon optogenetics of dendritic spines and neural circuits . Nat Methods 2012 ; 9 : 1202 - 1205 .
34 Paluch-Siegler S , Mayblum T , Dana H , Brosh I , Gefen I et al. All-optical bidirectional neural interfacing using hybrid multiphoton holographic optogenetic stimulation . Neurophotonics 2015 ; 2 : 031208 .
35 Hsiao PY , Tsai CL , Chen MC , Lin YY , Yang SD et al. Non-invasive manipulation of Drosophila behavior by two-photon excited red-activatable channelrhodopsin . Biomed Opt Express 2015 ; 6 : 4344 - 4352 .
36 Hirase H , Nikolenko V , Goldberg JH , Yuste R . Multiphoton stimulation of neurons . J Neurobiol 2002 ; 51 : 237 - 247 .
37 Mizuseki K , Diba K , Pastalkova E , Buzs?ki G . Hippocampal CA1 pyramidal cells form functionally distinct sublayers . Nat Neurosci 2011 ; 14 : 1174 - 1181 .
38 Campagnola L , Wang H , Zylka MJ . Fiber-coupled light-emitting diode for localized photostimulation of neurons expressing channelrhodopsin-2 . J Neurosci Methods 2008 ; 169 : 27 - 33 .
39 Lin JY , Knutsen PM , Muller A , Kleinfeld D , Tsien RY . ReaChR: a red-shifted variant of channelrhodopsin enables deep transcranial optogenetic excitation . Nat Neurosci 2013 ; 16 : 1499 - 1508 .
40 D?barre D , Olivier N , Supatto W , Beaurepaire E. Mitigating phototoxicity during multiphoton microscopy of live drosophila embryos in the 1.0-1.2 ?m wavelength range . PLoS One 2014 ; 9 : e104250 .
41 Daddysman MK , Tycon MA , Fecko CJ . Photoinduced damage resulting from fluorescence imaging of live cells . Methods Mol Biol 2014 ; 1148 : 1 - 17 .
42 Koester HJ , Baur D , Uhl R , Hell SW . Ca2+ fluorescence imaging with pico- and femtosecond two-photon excitation: signal and photodamage . Biophys J 1999 ; 77 : 2226 - 2236 .
43 Hopt A , Neher E . Highly nonlinear photodamage in two-photon fluorescence microscopy . Biophys J 2001 ; 80 : 2029 - 2036 .
44 Chu SW , Tai SP , Ho CL , Lin CH , Sun CK . High-resolution simultaneous three-photon fluorescence and third-harmonic-generation microscopy . Microsc Res Tech 2005 ; 66 : 193 - 197 .
45 Riedel R , Stephanides A , Prandolini MJ , Gronloh B , Jungbluth B et al. Power scaling of supercontinuum seeded megahertz-repetition rate optical parametric chirped pulse amplifiers . Opt Lett 2014 ; 39 : 1422 - 1424 .
46 Prandolini MJ , H?ppner H , Hage A , Schulz M , Tavella F et al. First experimental results towards a 100 W wavelength tunable femtosecond OPCPA . In: Clarkson WA , Shori R Keditors. SPIE LASE 9342 , Solid State Lasers XXIV : Technology and Devices, 93421E . San Francisco, CA, USA: SPIE; 2015 .
47 Kraemer D , Cowan ML , Hua RZ , Franjic K , Dwayne Miller RJ . High-power femtosecond infrared laser source based on noncollinear optical parametric chirped pulse amplification . J Opt Soc Am B 2007 ; 24 : 813 - 818 . This work is licensed under a Creative Commons Attribution 4.0 International License . The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material . To view a copy of this license , visit http://creativecommons.org/licenses/by/4.0/ r The Author(s) 2017