Optical imaging through the intact mouse skull is challenging because of the skull-induced
aberration and scattering. We find that three-photon-excitation improves optical sectioning
compared to two-photon-excitation, even with the same excitation wavelength and imaging
system. Through the adult mouse skull, we demonstrate three-photon imaging of vasculature
at >500μm depth, and GCaMP6-calcium imaging over weeks in cortical layers 2/3 and
4 in awake mice, with 8.5 frames/s and hundreds of micrometers field-of-view.
Observing the mouse brain in its native environment is critical to the study of neural
network function and disease progression
1
. Cranial window implantation
2
and skull thinning
3
,
4
are the common minimally invasive procedures to obtain optical access to the mouse
brain; however, they can still cause perturbation to the physiological environment
in some cases. For example, mechanical stress during the surgeries induce unexpected
activation of microglia and astrocytes
3
,
5
; skull openings change intracranial pressure and affects fluid flow in paravascular
space, which may be important for waste disposal
6
. Therefore, impoving imaging performance through the intact skull will open new opportunities
to non-invasive brain research.
Non-fluorescence based technologies such as magnetic resonance imaging
7
, photoacoustic tomography
8
, and optical microaniography
9
can map brain structure and hemodynamics below an intact skull. However, these technologies
typically cannot achieve single-cell or sub-cellular resolution, and none of them
is established for direct cellular activity measurement with high temporal resolution
(e.g., > 1 Hz).
One-photon fluorescence can be used for imaging vasculature and neuronal activity
through skull, either with infrared dyes
10
, or skull clearing techniques
11
,
12
. However, such methods generally do not offer single-cell resolution, due to out-of-focus
fluorescence excitation.
Two-photon microscopy (2PM) is routinely used for in vivo deep brain imaging for its
optical sectioning capability in scattering media
13
; however, 2PM has poor resolution when imaging through an intact skull
14
. Although wavefront correction enabled 2PM to achieve submicron resolution, the corrected
field-of-view (FOV) was about 23×23 μm and the imaging depth was limited to approximately
50 μm in the cortex
14
. Recently, chemical treatment of the skull surface has been applied to improve the
contrast of structural 2PM in the shallow cortex
15
,
16
.
Three-photon microscopy (3PM) increases the imaging depth in the mouse brain because
of the weaker scattering at longer excitation wavelengths and the background suppression
by higher order nonlinear excitation, and 3PM imaging studies have revealed structure
and function in the mouse hippocampus through cranial windows in intact brains
17
,
18
. Recently, vasculature imaging through the intact mouse skull was demonstrated with
a synthesized dye of unusually large three-photon excitation (3PE) cross section at
1550 nm, reaching a depth of 300 μm
19
. In this study, we show that 3PM not only achieves more than 500 μm cortical depth
with conventional dyes but also is capable of calcium activity imaging at high spatial
and temporal resolution with hundreds of micrometers FOV.
The refractive index of the cranial bone (~1.55) is significantly higher
20
than that of water (1.32) or cerebrospinal fluid (~ 1.34)
21
. The high refractive index and the rough skull surface, especially after exposed
to air
11
, renders the skull opaque by scattering light like optical diffusers. We found that
the skull surface roughness can be reduced by using an index-matching glue, which
also seals the skull from the air and preserves transparency for chronic imaging (Fig.
1a, Supplementary Fig. 1). After the treatment, the effective attenuation length
17
(l
e
) of the skull was measured using the third harmonic generation (THG) signal from
the osteocytes, and we found l
e
~ 60 μm at 1320 nm (Fig. 1).
We compared 2PM at 920 nm and 3PM at 1320 nm by imaging fluorescein-labeled vasculature
of the same mouse, through the intact skull around the center of the parietal bone.
1320-nm 3PM resolved capillary vessels with high contrast (Fig. 1b), with signal-to-background
ratio (SBR) close to 100 at 120 μm cortical depth, and ~ 10 at 510 μm (Fig. 1c). In
comparison, 920-nm 2PM has substantially lower contrast, with tens of times higher
background in the unlabeled region of the brain even at shallow depths (Fig. 1c).
We verified that 920 nm generates negligible intrinsic autofluorescence in the emission
band of fluorescein by imaging the mouse before any dye injection. Therefore, the
background in 2PM is fluorescence from fluorescein.
To delineate the effects of longer wavelength and the higher order nonlinear excitation
in through-skull imaging, we injected Alexa 680 into the same mouse, and immediately
performed imaging by two-photon excitation (2PE) of Alexa 680 with 1320 nm, using
the same laser and microscope. Figs. 1c and 1d indicate that the 1320-nm 2PM has similar
SBR as 920-nm 2PM at all depths, with only limited improvement on contrast, presumably
by the reduced scattering and aberration at the longer excitation wavelength (Fig.
1b, the background was also verified to be fluorescence from Alexa 680). These results
show that longer excitation wavelength alone is not sufficient, and 3PE is necessary
for through-skull imaging.
With cranial windows, the SBR of 2PM remains high until the imaging depth is beyond
approximately 4 scattering lengths
13
. However, in our through-skull experiment, the SBR of 2PM, even with 1320 nm excitation,
is below 10 immediately beneath the skull (Figs. 1c and 1d). The skull accounts for
approximately 2 attenuation lengths, and has low labeling density due to the sparsity
of vasculature in the bone. Therefore, the background in 2PM must be caused by the
degradation of the point spread function (PSF), in both lateral (XY) and axial (Z)
dimensions. For example, the lateral broadening of PSF is indicated by the blurred
edges of capillaries in 2PM images (Fig. 1b); the axial elongation is indicated by
the observation that large, penetrating blood vessels are much brighter relative to
horizontal capillaries in 2PM than in 3PM (Fig. 1b). The higher order nonlinear excitation
of 3PM is more effective in accentuating the central peak (i.e., the signal) and suppressing
the unwanted fluorescence excitation from the side lobes (i.e., the background) of
the PSF. Therefore, 3PE helps to preserve spatial resolution and improve image contrast
with a degraded PSF. This explanation is further corroborated by the measured attenuation
length of the brain tissue for through-skull imaging, which is shorter in 3PM than
in 2PM (Fig. 1e, more discussion on the attenuation length is presented in the Supplementary
Note 1).
To provide an upper bound estimate of the spatial resolution of through-skull imaging,
we measured the fluorescence intensity profile of a dendrite (Figs 2a-d). The lateral
and axial FWHM is 0.96 μm and 4.6 μm, respectively, which provides the upper bound
of the spatial resolution due to the finite size of the neural processes. 2PM at 920
nm could not resolve any feature at high spatial resolution using the same GCaMP6
mice (Supplementary Fig. 2b).
We imaged spontaneous activities of GCaMP6s-labeled neurons in adult transgenic mice
(CamKII-tTA/tetO-GCaMP6s, 8–12 weeks, including both male and female, N=5). Fig. 2e
shows an imaging site in cortical layer 2/3 of an 8-week-old mouse with ~120-μm-thick
skull. The neuronal activity traces were recorded under awake condition (Fig. 2f).
The absolute signal-to-noise ratio (SNR) of the recording can be ascertained by the
raw photon counts (Supplementary Fig. 3), which is comparable to typical 2PM calcium
imaging through a cranial window. As imaging depth increases, the ΔF/F for through-skull
3PM appears to decrease due to increased background contribution (Fig. 1e). To maintain
the SNR and temporal resolution, the photon counts per neuron per second must be increased
by either delivering additional power to the focus or reducing the FOV. The deepest
activity imaging in this study was at 465 μm below the cortical surface (Supplementary
Fig. 4). While through-skull 3PM provides sufficient SNR for recording the activities
with GCaMP6s in cortical layer 2/3 neurons, it requires much higher average and peak
power than 2PM with cranial windows due to the combined effects of skull attenuation
and the inefficient higher order nonlinear excitation. These power requirements ultimately
limit the performance of through-skull 3PM (for a detailed discussion on the limits
of 3PM through-skull imaging, see Supplementary Note 2). Therefore, 2PM imaging through
cranial windows remains the preferred method if the experimental results are not expected
to be affected by the craniotomy.
We performed longitudinal study and recorded from the same neurons on 8 different
days over a period of 4 weeks after the initial skull preparation (Supplementary Figs.
4a and 5 show longitudinal recordings from 3 sites at different depths). THG signal
from the blood vessels was used to locate approximately the same imaging FOV for each
recording session. Despite the long exposure time (cumulatively over 6 hours per recording
site), no visible adverse effects had been observed on neuronal structure or activities,
indicating the average power and the peak intensity were safe for imaging. We also
performed immunostaining in the brains after imaging (Methods). The results confirmed
that there is no measurable tissue damage under the imaging conditions for activity
recording in this study (Supplementary Fig. 6). We observed some degradation of skull
transparency over time, as indicated by the power needed to image the same sites (e.g.
35–50% more power after 4 weeks, Supplementary Fig. 5). Nonetheless, successful recordings
of neuronal activity were performed for all imaging sessions.
In addition to 1320-nm 3PM, we tested 1700-nm 3PM for through-skull structural imaging
using Texas Red labeled vasculature and red-fluorescent-protein labeled neurons, both
reaching more than 500 μm cortical depth (Fig. 2g, Supplementary Fig. 7).
We have demonstrated through-skull 3PM of mouse brain structure and function, with
high spatial and temporal resolution, large FOV, and at significant depth. Furthermore,
by comparing 2PM and 3PM at the same excitation wavelength, we show unequivocally
that 3PE is necessary for imaging through the intact skull, regardless of the imaging
depth and the labeling density. This work demonstrates the advantage of higher order
nonlinear excitation for imaging through a highly scattering layer, which is in addition
to the previously reported advantage of 3PM in deep imaging of densely labeled samples.
The demonstrated technique will open new opportunities for non-invasive studies of
living biological systems.
Online Method Section
Experimental set up:
The laser and microscope setup is similar to that in our previous work
18
. Any difference in system parameters is stated in this method section.
Excitation source:
The excitation source for 1320-nm 3PM is a noncollinear optical parametric amplifier
(NOPA, Spectra Physics) pumped by a regenerative amplifier (Spirit, Spectra Physics).
A two-prism (SF11 glass) compressor is used to compensate for the normal dispersion
of the optics of the light source and the microscope, including the objective. The
NOPA operates at wavelength centered at 1320 nm, and provides an average power of
~ 700 mW (1750 nJ per pulse at 400 kHz repetition rate). The pulse duration (measured
by second-order interferometric autocorrelation) under the objective is ~ 37 fs after
optimizing the prism compressor. An optical delayed line is used to double the laser
repetition rate to 800 kHz, by splitting the excitation beam into two of equal powers
and introducing ~ 10 ns delay between their pulse trains.
The excitation source for 2PM is a mode-locked Ti:Sapphire laser (Tsunami, Spectra
Physics) centered at 920 nm. The 920-nm beam and 1300-nm beam are spatially overlapped
and directed to the same microscope, and then combined by a dichroic mirror.
The excitation source for 1700-nm 3PM was solitons centered at 1700 nm generated by
soliton self-frequency shift in a photonic crystal fiber pumped by a fiber laser at
1550nm. The excitation source was described in more detail in ref.
22
.
Imaging setup:
The images were taken with a custom-built multiphoton microscope with a high-numerical
aperture objective (Olympus XLPLN25XWMP2, 25X, NA 1.05). The objective was under-filled
to reduce unnecessary loss of power in the marginal rays, and the 1/e2 beam diameter
of the excitation beam was ~11 mm, which is ~70% of the objective back aperture diameter.
The signal is epi-collected through the objective and then reflected by a dichroic
beam splitter (FF705-Di01–25×36, Semrock) to the detection system. There are two detection
channels: one for fluorescence signal and the other for third harmonic generation
(THG). For GCaMP6s imaging, we used a photomultiplier tube (PMT) with GaAsP photocathode
(H7422–40, Hamamatsu) for the fluorescent signal and an ultra bialkali PMT (R7600–200,
Hamamatsu) for the THG signal. A 488-nm dichroic beam splitter (Di02-R488–25×36, Semrock)
was inserted at 45 degrees to the signal beam path to separate and direct fluorescence
and THG to their respective PMTs. Fluorescence generated by fluorescein and GCaMP6s
was selected by a 520±30 nm band-pass filter (Semrock), and THG around 440 nm was
selected by a 435±20 nm filter (Semrock). For Alexa 680 imaging, we switched to a
GaAs PMT (H7422–50, Hamamatsu) for the fluorescence channel, which has higher sensitivity
at longer wavelength. The fluorescence filter was also changed to 716±20 nm (Semrock)
filter to pass Alexa 680 fluorescence while blocking the second harmonic of 1320 nm.
For signal sampling, the PMT current is converted to voltage by a transimpedance amplifier
with 10MHz bandwidth (C9999, Hamamatsu), providing adequate temporal resolution for
photon counting. For analog signal acquisition, an additional 1.9 MHz low pass filter
(Minicircuts, BLP-1.9+) is used before digital sampling. Analog-to-digital conversion
is performed by a data acquisition card (NI PCI-6115, National Instruments). The signal
acquisition system displayed shot-noise limited performance, and light shielding was
carefully done to achieve dark counts of 20–40 photons per second under actual imaging
conditions without laser scanning. ScanImage 3.8 running on MATLAB (MathWorks) was
used to acquire images and control a 3D translation stage to move the sample (M-285,
Sutter Instrument Company). All imaging depths and thickness are reported in raw axial
movement of the motorized stage, unless otherwise stated. The refractive indices of
skull and brain tissue (1.55 for the bone and 1.35 to 1.37 for the cortex
21
) are higher than that of water (1.32), which result in slight under-estimate of the
actual depth in the experiments (~ 12% for the skull thickness and ~3% for the depth
in the brain tissue).
High resolution structural images were typically taken with 512×512 pixels/frame,
0.5 Hz frame rate, and multiple frame averages at each depth. Neuronal activities
were recorded using 256×256 pixels/frame at 8.49 Hz frame rate. Each site was recorded
for 30 to 50 minutes in each imaging session. The conversion from pixel values to
photon counts was performed according to the method described in the previous publication
18
.
For in vivo imaging, the mouse was placed on a tip-tilt stage under the objective
lens, and it is important to ensure the skull surface and the coverslip is parallel
to the imaging plane of the objective. In most cases, the skull is curved, and it
is preferable to adjust the tilt angle so that the apex of the curvature is directly
above the imaging site.
Resolution Measurement on Dendrites:
To eliminate the fluctuation in brightness caused by changing calcium concentration,
a neuron was intentionally damaged by continuous laser scanning for 4 minutes at high
intensity (~9 nJ at the focus) and small FOV (20 μm x 20 μm). The damaged neuron has
a constant brightness for the GCaMP6s fluorescence. A z-stack was then taken around
the soma and its apical dendrites at 0.2 μm step. Intensity profiles of the dendrite
were plotted either laterally or axially to estimate the resolution.
Image Processing for Activity Recording:
Mechanical drift in the horizontal plane, if any, was corrected by TurboReg plug-in
in ImageJ. Regions of interest (ROIs) were generated by manual segmentation of neuron
bodies. In MATLAB, fluorescence intensity traces were low-pass filtered with a hamming
window of a time constant of 1.06 s. Spikes were inferred according to ref. 24 assuming
shot-noise limited detection. Baselines of the traces (F0) were then determined by
excluding the spikes as well as their rising and falling edges. Traces (F) were normalized
according to the formula (F- F0)/F0. Examples of the raw activity recording videos
played at the recording frame rate of 8.49 Hz with 2 frames rolling average are presented
in Supplementary Video 1. For visual representation of calcium activities in Supplementary
Video 2, raw image sequence was first rolling averaged with 17 frames, and then the
local background was subtracted to improve contrast (ImageJ). The activity movie was
further processed by Kalman filter with a gain from 0.7–0.9 for noise reduction
23
(ImageJ).
Image Processing for Structural Imaging:
Structural images were processed with a median filter of 1-pixel radius, and then
normalized by linear transform of pixel intensities to saturate the brightest 0.2–0.5%
pixels of each frame. Three-dimensional reconstruction of the stacks was rendered
in Volocity (version 6.3). For the purpose of visualization, some of the high-resolution
images in Fig. 2e, Supplementary Fig. 4 and 5 were displayed with a gamma correction
value of 0.8–0.95 to reduce brightness contrast between different neurons.
Statistics and Data Analysis:
All data analysis was performed in MATLAB 2016. The effective attenuation length in
Fig. 1e was derived by least-square linear regression of fluorescent signal data.
For all representative results, the number of successful independent experiments on
different animals is indicated in the corresponding figure legend, and more details
on the ages of the animals are included in “Life Sciences Reporting Summary”.
Animal Procedures
All animal experimentation and housing procedures were conducted in accordance with
Cornell
University Institutional Animal Care and Use Committee guidance.
Skull Preparation for Chronic Imaging:
Before the surgery, animals were anesthetized with isoflurane (3% in oxygen for induction,
and 1.5–2% for surgery to maintain a breathing frequency around 1 Hz). Body temperature
was kept at 37.5 °C with a feed-back controlled blanket (Harvard Apparatus), and eyes
were covered with eye ointment. Glycopyrrolate (0.01mg/kg body weight), dexamethasone
(0.2mg/kg body weight), and ketoprofen (5mg/kg body weight) were administrated intramuscularly.
Dexamethasone and ketoprofen were also administrated in two consecutive days following
the surgery. The anesthetized animal was fixed on stereotaxic, and hair was removed
from scalp with scissors and Nair. The scalp was further sterilized by alcohol wipes,
and then cut open and removed to expose both parietal plates as well as the bregma
and lambda. Sterile saline was applied to the skull immediately after the exposure,
and it is critical to keep the entire bone surface covered by saline to insulate from
air. Fascia and connective tissue on the skull were gently removed with forceps and
sterile wet cotton tips to avoid any internal bleeding inside the brain. At this point,
the whole skull was transparent, with blood vessels underneath visible with sharp
edges. The saline covering the skull was then wiped completely dry with cotton tips,
and the following actions were taken quickly before the bone turns opaque. Ultra-violet
curable glue (Loctite 4305) was applied to the skull surface within 2 seconds afterwards.
A sterile and dry round coverslip of 5-mm diameter (#1 thickness, Electron Microscopy
Sciences) was placed on the skull, centered at 2.5 mm lateral, and 2 mm caudal from
the bregma point. The coverslip was pressed closely against the skull surface by forceps
to minimize the amount of glue between the coverslip and the skull. The glue was left
to cure by itself for about 5 minutes without any ultra-violet light, during which
time the skull transparency tends to increase visually. Afterwards, an ultra-violet
light source (385–515nm, Bluephase Style 20i, Ivoclar vivadent) was used to completely
cure the glue, with roughly 1s on and 1s off for 3s. The coverslip is necessary to
keep the glue layer as thin as possible (down to ~10 μm at the thinnest part on the
skull), and to form a flat interface to reduce aberration. The exposed part of the
skull surrounding the coverslip was further covered with dental cement. Supplementary
Fig. 1 shows an example of successful preparation. For awake imaging, a head-bar for
head fixation during imaging was glued to the exposed parts of the skull surrounding
the coverslip by metabond glue.
Imaging Procedures:
For imaging of anesthetized animal, the mice were anesthetized using isoflurane (1–1.5
% in oxygen, maintaining a breathing frequency at 2 Hz) and placed on a heat blanket
to maintain body temperature at 37.5 °C. Eye ointment was applied and the animal was
place on a 3D motorized stage for navigation under the microscope. For structural
imaging, vasculature of wild-type mice (N=3, 10–12 weeks, male, C57BL/6J, The Jackson
Laboratories) was labeled through retro-orbital injection with fluorescein, Alexa
680, and Texas Red (25mg dextran conjugate dissolved in 200 μl sterile saline, 10kDa
molecular weight, Invitrogen).
For awake imaging, the animal was fixed on a custom-made stereotaxic plate by attaching
its head-bar to the metal holders. The body of the animal was further secured in a
tube of slippery inner walls to reduce motion.
Immunohistochemistry for tissue damage assessment:
Anesthetized mice were continuously imaged with 1320-nm 3PM with the laser operating
at 400 kHz repetition rate. The scanned region is located at ~2.5-mm lateral and 1-mm
caudal to the bregma point. A 200×200 μm FOV at ~200-μm cortical depth was continuously
scanned for 30 minutes, at 60 mW average power under the objective lens for one mouse
and at 120 mW for the other. After 16 hours, mice were transcardially perfused, and
the brains were postfixed. The brain fixation and antibody staining procedures followed
the protocol as described in a previous work
24
(detailed information on the antibodies used in this study is included in “Life Sciences
Reporting Summary”). Coronal sections were cut in 50-μm thickness step through the
center of the imaged region, and alternating slices were labeled for heat shock protein
(HSP) and glial fibrillary acidic protein (GFAP). The brain slices were imaged with
a Zeiss 780 confocal microscope.
Supplementary Material
1
2
3
4
video 1
video 2