Rapid volumetric imaging with Bessel-Beam three-photon microscopy

Abstract

Owing to its tissue-penetration ability, multi-photon fluorescence microscopy allows for the high-resolution, non-invasive imaging of deep tissue in vivo; the recently developed three-photon microscopy (3PM) has extended the depth of high-resolution, non-invasive functional imaging of mouse brains to beyond 1.0 mm. However, the low repetition rate of femtosecond lasers that are normally used in 3PM limits the temporal resolution of point-scanning three-photon microscopy. To increase the volumetric imaging speed of 3PM, we propose a combination of an axially elongated needle-like Bessel-beam with three-photon excitation (3PE) to image biological samples with an extended depth of focus. We demonstrate the higher signal-to-background ratio (SBR) of the Bessel-beam 3PM compared to the two-photon version both theoretically and experimentally. Finally, we perform simultaneous calcium imaging of brain regions at different axial locations in live fruit flies and rapid volumetric imaging of neuronal structures in live mouse brains. These results highlight the unique advantage of conducting rapid volumetric imaging with a high SBR in the deep brain in vivo using scanning Bessel-3PM.

Full text

Rapid volumetric imaging with Bessel-Beam
three-photon microscopy
BINGYING CHEN,1,7 XIAOSHUAI HUANG,2,7 DONGZHOU GOU,2 JIANZHI
ZENG,3 GUOQING CHEN,2 MEIJUN PANG,2 YANHUI HU,4 ZHE ZHAO,5
YUNFENG ZHANG,4 ZHUAN ZHOU,2 HAITAO WU,5 HEPING CHENG,2 ZHIGANG
ZHANG,1 CHRIS XU,2,6 YULONG LI,3 LIANGYI CHEN,2,8 AND AIMIN WANG1,9
1State Key Laboratory of Advanced Optical Communication System and Networks, School of Electronics
Engineering and Computer Science, Peking University, Beijing 100871, China
2State Key Laboratory of Membrane Biology, Beijing Key Laboratory of Cardiometabolic Molecular
Medicine, Institute of Molecular Medicine, Peking University, Beijing 100871, China
3State Key Laboratory of Membrane Biology, School of Life Sciences, PKU-IDG/McGovern Institute for
Brain Research, Peking University, Beijing 100871, China
4School of Electronics Engineering and Computer Science, Peking University, Beijing 100871, China
5Department of Neurobiology, Institute of Basic Medical Sciences, Beijing, China
6School of Applied and Engineering Physics, Cornell University, Ithaca, NY 14853, USA
7These authors contributed equally to this work
8lychen@pku.edu.cn
9wangaimin@pku.edu.cn
Abstract: Owing to its tissue-penetration ability, multi-photon fluorescence microscopy
allows for the high-resolution, non-invasive imaging of deep tissue in vivo; the recently
developed three-photon microscopy (3PM) has extended the depth of high-resolution, non-
invasive functional imaging of mouse brains to beyond 1.0 mm. However, the low repetition
rate of femtosecond lasers that are normally used in 3PM limits the temporal resolution of
point-scanning three-photon microscopy. To increase the volumetric imaging speed of 3PM,
we propose a combination of an axially elongated needle-like Bessel-beam with three-photon
excitation (3PE) to image biological samples with an extended depth of focus. We
demonstrate the higher signal-to-background ratio (SBR) of the Bessel-beam 3PM compared
to the two-photon version both theoretically and experimentally. Finally, we perform
simultaneous calcium imaging of brain regions at different axial locations in live fruit flies
and rapid volumetric imaging of neuronal structures in live mouse brains. These results
highlight the unique advantage of conducting rapid volumetric imaging with a high SBR in
the deep brain in vivo using scanning Bessel-3PM.
© 2018 Optical Society of America under the terms of the OSA Open Access Publishing Agreement
OCIS codes: (110.0180) Microscopy; (180.2520) Fluorescence microscopy; (180.5810) Scanning microscopy;
(190.4180) Multiphoton processes.
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#322760
https://doi.org/10.1364/BOE.9.001992
Journal © 2018
Received 9 Feb 2018; revised 22 Mar 2018; accepted 23 Mar 2018; published 29 Mar 2018

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1. Introduction
Multi-photon microscopy (MPM) holds the promise of unique and impactful applications in
cell biology and neuroscience research, where it can enable the high-resolution imaging of the
neuronal structures buried deep inside the intact brain [1–3]. Benefiting from higher order
nonlinear excitation and longer excitation wavelengths, 3PM has shown better performance in
terms of the signal-to-background ratio (SBR) than has two-photon microscopy (2PM), and it
has extended the non-invasive functional imaging of the mouse brain to beyond 1.0 mm.
Using 3PM, the spontaneous activity recording in the hippocampal stratum pyramidale has
been realized [4–6].
MPM normally uses focused femtosecond laser pulses to illuminate the sample, which
limits the excitation volume to the focal point. Therefore, the sample must be imaged pixel by
pixel to obtain a 3D image stack. In particular, the mechanical movement of the objective to
perform the axial scanning decreases the imaging rate. This hampers its application in
recording the activity of the large neuronal circuits distributed in a 3D volume. Moreover, the
low repetition rate of femtosecond lasers (1-2 MHz) optimized for deep-tissue 3PE further
limits the temporal resolution of point-scanning 3PM [7].
Various alternative axial scanning methods have been proposed to address this problem,
such as fast z-scanning with remote focusing [8], wavefront shaping using spatial light
modulators (SLM) [9], and random access scanning with acousto-optic deflectors (AOD)
[10]. For sparsely labeled samples, using a Bessel beam to image a 3D volume is another
feasible approach. The Bessel beam has an axially elongated, needle-like optical intensity
Vol. 9, No. 4 | 1 Apr 2018 | BIOMEDICAL OPTICS EXPRESS 1993

distribution and maintains the same lateral resolution over an extended depth of field [11, 12].
Hence, a single scan can generate a 2D projection of the 3D volume image. Given that a
single neuron often has dendrites that are sparsely distributed in a 3D volume, Bessel beam
scanning is an excellent fast imaging method for obtaining volumetric information about the
neuronal structures and activities. Moreover, the image obtained with Bessel-3PM is a 2D
projection of a 3D object, which cannot offer the information in z-axis. Therefore, we can use
Bessel-3PM to obtain the temporal information at different depths and use point-scanning
3PM to obtain the imaging depth as assistance. Nevertheless, the sample must be sparsely
labeled; otherwise, this approach will cease to be effective if too much axial information is
stacked together.
The Bessel beam has been used to extend the depth of field in 2PM of neurons [13, 14].
However, the intensity distribution of the zeroth-order Bessel beam in the transverse plane
has strong side lobes under two-photon excitation (2PE) that generate an undesirable
background and decrease the SBR. Because 3PE and 2PE fluorescence obey the cubic and
quadratic dependence on the excitation power, respectively, a Bessel beam with 3PE has
much smaller side lobes than does that with 2PE. In this paper, we compared the performance
of 2PE and 3PE Bessel-beam using femtosecond lasers at 920 nm and 1300 nm, respectively,
and demonstrate the superior SBR of volumetric Bessel 3PM in imaging zebrafish larva, fruit
flies and mice in vivo.
2. Method and materials
2.1 Three- and two-photon imaging systems with Bessel-beam or Gaussian-beam
excitation
A schematic diagram of the optical setup is shown in Fig. 1.
Fig. 1. Schematic diagram of the apparatus. Flipper mirrors: F1 was used to switch between
920 nm and 1300 nm lasers; F2 and F3 were used to switch between the Gaussian and Bessel
beam excitation. Axicon, α = 2 degrees. Lenses: L1 and L2, 50 mm (or 75, 100 mm) focal
length, determines the effective excitation NAs and lengths of the Bessel beam. L3 (100 mm),
L4 (100 mm), SL (54 mm) and TL (200 mm) were used to create multiple conjugate planes of
the back focal plane (BFP) of the objective in the light path; L5, collection lens (30 mm).
Galvo mirrors: GMX/GMY, for the x/y direction of beam scanning. DM, 735-nm long-pass
dichroic mirror; a 770-nm short pass filter and a 525/40 nm bandpass filter were used here
(Filters) for emission collection.
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The light source for 3PM was a non-collinear optical parametric amplifier (NOPA,
Spectra Physics) pumped by a 1040 nm high-power femtosecond laser (Spirit, Spectra
Physics). The NOPA produced 50-fs pulses at 1300 nm with an average power over 500 mW
and a repetition rate of 400 kHz. The femtosecond laser at 1300 nm could also be switched to
a home-built femtosecond Nd:fiber laser, which delivered up to 800 mW, 150-fs pulses at 930
nm with a repetition rate of 50 MHz [15, 16]. This configuration enabled a comparison of the
performance of 2PM and 3PM using the same specimen. After using a pair of scan mirrors
(6215H, Cambridge Technology), the laser pulses were directed into a 54-mm scan lens and a
200-mm tube lens. A water immersion objective lens (XLPLN25XWMP2, 25X, NA 1.05,
Olympus) was used to focus the light into the sample. The fluorescence emitted from the
sample was epi-collected and directed to a GaAsP PMT detector (H7422p-40, Hamamatsu)
by a long-pass dichroic mirror (FF735-Di02-25 × 36, Semrock). A collection lens, a short-
pass filter (ET770sp-2p 1500IR, Chroma) and a 525/40 nm bandpass filter (FF02-525/40-25,
Semrock) were used to collect the fluorescence signals. The microscope was compatible with
two scanning modes: the Bessel-beam scanning mode and the point-scanning of Gaussian
focal spot mode. For the Bessel-beam mode, a plano-convex axicon (1-APX-2-VIS-G254-P,
Altechna) was used to generate the Bessel beam to excite the sample. The axial FWHM of
Bessel beams depends on the different combinations of the angle α of the axicon (angle
between the sloped and plane faces) and the focal length of Lens 2. For the point-scanning
mode, lens L1 was used to collimate the beam before directing to the scan mirror. The
transmission efficiencies through the system under both modes were approximately 25%.
The length of the Bessel-beam depends on optical components; however, for a certain
biological experiment, the maximum extension is limited by the sample’s property and the
single pulse energy of the laser source. For tissues with high transparency, a long Bessel
beam could be used to illuminate the entire imaging region (e.g. zebrafish). However, the
length of the Bessel-beam should not be much longer than the thickness of the sample;
otherwise, the excessive background fluorescence would be produced. Moreover, because of
the limited excitation power, the beam length should be adjusted to even shorter to increase
the imaging depth (e.g. mouse brain in our case). Hence, the appropriate length of depends on
the sample’s thickness and transparency, the depth of the imaging region, the single pulse
energy of the laser source and other factors.
2.2 Sample preparation
In this work, three representative tissues (zebrafish, Drosophila and mouse brain) were used
to characterize the properties of the imaging system.
The zebrafish was used to compare the SBR between 2PE and 3PE. The vascular
epithelial cells were labeled with EGFP in the Tg (kdrl:EGFP) transgenic fish line. For
imaging, the fish was raised in an embryo medium containing 0.002% phenylthiourea (PTU,
Sigma) to suppress pigmentation synthesis. Prior to live imaging, the fish was anaesthetized
with 0.01% tricaine (Sigma) to minimize the movement artifacts during imaging. Then, the
anaesthetized zebrafish was embedded in a 1% ultra-pure agarose (Invitrogen) and immersed
in E3 medium containing 0.01% tricaine.
For functional imaging, flies expressing GCaMP were reared at 25 °C on standard
cornmeal for 8-12 days after eclosion before experiments. The procedures used for mounting
and dissecting the flies for imaging were the same as previously described [17]. Flies were
mounted to a small dish by tape, and the antennae were exposed to the air. The cuticle, fat
bodies and air sacs between the eyes were removed to expose the brain. The exposed brains
were perfused with adult-like hemolymph (ALH). Isoamyl acetate (IA, Sigma-Aldrich; Cat#
306967) was initially diluted by 100-fold in mineral oil (Sigma-Aldrich; Cat# 69794) (100 μl
IA in 900 μl mineral oil) and then placed in a glass bottle. During olfactory stimulation, the
airflow carrying IA (200 μl/min) was mixed with purified air (1000 ml/min) and delivered to
the antennae of the flies. The identification of glomeruli was based on the map of the antenna
Vol. 9, No. 4 | 1 Apr 2018 | BIOMEDICAL OPTICS EXPRESS 1995

lobe published previously [18]. The Fiji software was used to process the images of
functional imaging experiments, including subtracting the background, smoothing with a
Gaussian blur filter and measuring the fluorescence within the ROIs. The Origin software was
used to process the raw data with binning and plot the traces. The pseudo-color snapshots
(color-encoded by ΔF/F0) generated by Matlab with custom scripts were the averaged results
of 2-4 repeats.
We used male C57BL/6J mice (20 g, 8 postnatal weeks, Thy-1-YFP) for in vivo brain
imaging. Animals were prepared using the methods described in Ref [4]. The craniotomies
were performed centered at 2.2 mm posterior and 3 mm lateral to the Bregma point. All
procedures were approved by the Peking University Animal Use and Care Committee and
complied with the standards of the Association for Assessment and Accreditation of
Laboratory Animal Care, including the animal breeding and experimental manipulation.
3. Results and discussions
Following the analysis of Xu et al. [19], we can obtain the n-photon fluorescence:
()
n0
C() ()
nn
n
Ft I t dS=rr (1)
where n
C is a constant that depends on the dye concentration, the n-photon fluorescence
cross-section, and the collection efficiency of the system. 0()
n
I
t and ()
n
Sr are, respectively,
the temporal and spatial intensity distributions of the excitation beam. Because the Bessel
beam was transformed from a Gaussian beam by an axicon, ()Sr is given by [13]
()
12
,()()Srz S zS r=(2)
()
22 22
12
0
4πNA 2NA
()
zz
S z exp w
λ
−
=(3)
()
2
20
2NA
()
r
Sr J
π
λ
=(4)
where NA is the effective numerical aperture, λ is the wavelength of the laser, 0
w is the
initial Gaussian beam width, and z, r and θ are the cylindrical coordinates. Using the
equations above, the calculated point-spread functions (PSFs) in the x-z plane are shown in
Fig. 2(a) (3PM) and Fig. 2(b) (2PM). The x-y intensity distributions after integrating the z-
axis are shown in Fig. 2(e) (3PM) and Fig. 2(g) (2PM).
The total fluorescence of the m-th lobe for n-photon excitation can be calculated as
()
0
0
1
22NA
n0 1 2
02NA
C()() ()
m
m
x
nn n
nm x
Ft I t S dz d rzSrdr
λ
π
λ
θ
−
π
π
= (5)
where 0
m
x
is the m-th node (m > 0) of the zeroth order Bessel function, and 0
0
x
is 0. From
Eqs. (4) and (5), and with '2NA
r r
π
λ
=, we obtain the ratio of fluorescence in the m-th lobe
to fluorescence in the main lobe:
()
()
0
0
1
0
1
'2n
0
'2n
10
0
(') '
.
(') '
m
m
x
x
nm
x
n
rJ r dr
Ft
Ft rJ r dr
−
=

(6)
Vol. 9, No. 4 | 1 Apr 2018 | BIOMEDICAL OPTICS EXPRESS 1996

Equation (2) shows that the transverse distribution of the fluorescence generated by the
Bessel beam is invariant along the z-axis, and Eq. (6) shows the relative fluorescence in the
side lobes does not depend on the excitation NA, the length of the Bessel beam, or the pulse
width. We define the fluorescence in the main lobe as the signal, and we define the rest,
including the fluorescence in the side lobes and in regions far away from the focal area, as the
background.
As shown in Fig. 2(e) and Fig. 2(g), the three-photon fluorescence in the 2nd lobe (i.e.,
m = 2) is approximately 5.3% that in the main lobe, while the two-photon fluorescence in the
2nd, 3rd and 4th lobes is approximately 27.2%, 15.2% and 10.6% that in the main lobe,
respectively. The fluorescence generated by the side lobes of two-photon Bessel-beam
significantly degrades the SBR of the image.
Fig. 2. Theoretically simulated and experimentally determined PSFs. (a)(b), simulated PSFs of
3PM and 2PM along the x-z planes. (c)(d), the experimental results. Scale bars, 100 μm (z), 2
μm (x). 0.5-μm fluorescent beads were used to measure xyz profiles of Bessel-3PM and
Bessel-2PM. (e)(g), x-y cross section after integration of the z-axis of the Bessel beam to
numerically simulate the fluorescence distribution of the side lobes (e, 3PM; g, 2PM), (f)(h),
the experimental results. Scale bar, 2 µm. (i-l), comparison of blood vessels in live zebrafish
imaged with Bessel-3PM and 2PM. (i), a representative example of the fish under Bessel 2PM
and 3PM, in which the boxed region was magnified in j and k. (l), fluorescence intensities
along the line across the same blood vessels.
We measured the lateral intensity distribution of fluorescent beads throughout the length
of the Bessel beam for 2PE and 3PE. A stack of x-y images of 0.5-µm yellow/green
(505/515) beads (Invitrogen) were recorded with a 2-µm depth increment using a constant
excitation power. Figure 2(c), 2(d) show the axial PSFs of the Bessel beam of 3PM and 2PM.
For focal length of L2 of 50 mm, the axial FWHMs of the Bessel beam were ~250 µm for
3PM and ~300 µm for 2PM. These values are in good agreement with theoretical predictions.
Vol. 9, No. 4 | 1 Apr 2018 | BIOMEDICAL OPTICS EXPRESS 1997

The intensity modulation along the z-axis was caused by the round tip of the axicon [20].
Figures 2(f) and 2(h) show the x-y cross sections after integration of the z-axis of the Bessel
beam using 3PE and 2PE. The lateral resolution (defined as the intensity FWHM) for 3PE and
2PE are approximately 0.7 µm and 0.6 µm, respectively, with an effective NA of 0.4. The
lateral resolution can be improved with higher NA (e.g., 0.6 to 0.8). After the subtraction of
the background, defined as the average pixel value from regions without fluorescent beads,
the total fluorescence from all planes was integrated to estimate the side lobes, as shown in
Fig. 2(f) and 2(h). The three-photon fluorescence of the 2nd lobe is 5.1% of the main lobe,
and the two-photon fluorescence of the 2nd, 3th and 4th lobes are 27.8%, 14.6% and 10.1%
of the main lobe, respectively, in good agreement with the calculated values. By integrating
the fluorescence intensity from the first six side lobes, the combined fluorescence intensity of
Bessel-3PM (8.7%) is less than one-eighth that of the Bessel-2PM (73.1%), indicating a
much-improved SBR for Bessel-3PM.
We compared the performance of Bessel-3PM and Bessel-2PM in imaging blood vessels
from a large volume (500 μm × 500 μm × 300 μm) in the same zebrafish (3 days post-
fertilization) in vivo. We used the Tg (kdrl:EGFP) transgenic fish, in which EGFP was
selectively expressed in vascular epithelial cells. The average power after the objective was
90 mW at 1300 nm for 3PM and 127 mW at 920 nm for 2PM. In agreement with the
theoretical predictions and the experimental results with fluorescent beads, images obtained
with Bessel-3PM were sharper than those obtained with Bessel-2PM [Fig. 2(i-l)].
Fig. 3. Projection of volumetric imaging of GCaMP-labeled neuronal Ca2+ transients in
olfactory centers of Drosophila in vivo. We stimulated the fly with isoamyl acetate (IA) for 5 s
to trigger neuronal activities at the LH and the AL (DM2 glomerulus) of ePNs. (a), 3D
neuronal structures under the point-scanning 3PM with color-coded by depth. (b), projection of
3D volume of neuronal structure under the Bessel-3PM. (c)(d), pseudo-color snapshots of the
Ca2+ signals upon mineral oil and IA. (e), time-dependent Ca2+ responses from regions at the
LH and the DM2 were recorded at a scanning rate of 5 Hz in experiments (c) and (d). Blue bar,
stimulation duration. Individual traces are averaged results of 2-4 repeats and are processed
with 2 × binning. Error bars, ± s.e.m. (f-j), similar experiments as (a)-(e), except that Kenyon
cells at the MB were imaged. Axial FWHM of the Bessel beam, 110 μm. NA, 0.6.
Given the superior temporal resolution and comparative spatial resolution with point-
scanning 3PM, we used the Bessel-3PM to monitor the fast dynamics of different olfactory
centers of Drosophila along the axial direction in vivo. We genetically expressed GCaMP5
(GH146>GCaMP5 strain) [21] in the excitatory projection neurons (ePNs) at the antenna lobe
(AL), where they relay crucial olfactory information from peripheral olfactory receptor
neurons to higher brain centers, the mushroom body (MB) and the lateral horn (LH). The
Vol. 9, No. 4 | 1 Apr 2018 | BIOMEDICAL OPTICS EXPRESS 1998

application of odor isoamyl acetate (IA) could evoke calcium responses specifically in the
DM2 glomerulus of the AL, as reported by point-scanning 2PM [22]. Figure 3(a) shows the
3D neuronal structure with depth coded by color under point-scanning 3PM. We acquired a
120-μm-deep stack taken at 2-μm depth increments, and each stack was averaged from 3
frames. Figure 3(b) shows the projective neuronal structure under Bessel-3PM (averaged
from 10 frames). When imaging at the same frame rate (without averaging), point-scanning
3PM takes 60 times longer than Bessel-3PM. The small structures discernible in Fig. 3(a) and
3(b) (labeled by arrow) indicate the comparable spatial resolution for Bessel-3PM. Using
Bessel-3PM, we simultaneously detected IA-evoked responses from discrete regions of the
DM2 glomerulus and the LH at a frame rate of 5 Hz (256 × 256 pixels/frame). The
application of the control mineral oil evoked no response [Fig. 3(c)], indicating the specificity
of responses [Fig. 3(d)]. We also probed Ca2+ responses in Kenyon cells (KCs) at the MB (in
OK107>GCaMP6s strain) [23], which are the third-order olfactory neurons thought to be
critical for olfactory associative learning [24]. As shown in Fig. 3(f)-3(j), the amplitudes of
IA-evoked Ca2+ responses from the calyx and the vertical lobe of the MB, which are 40 μm
apart in axial distance, could be recorded simultaneously.
Fig. 4. In vivo three-photon images of neural cortex in a Thy1-YFP transgenic mouse. (a),
projection of 3D volume neurons and neurites in cortex of the awake mouse taken with Bessel-
beam at a frame rate of 1 Hz (image averaged from 10 frames afterwards). (b), mean intensity
projection of a 65-μm-thick image stack collected at 1-μm z steps in point-scanning mode,
from the same region as in (a). The stack covers from 620 μm to 685 μm below the dura, with
structures color-coded by depth. The image of each layer was averaged from 3 frames, with a
post objective power of 10 mW for Gaussian beam scanning and 110 mW for Bessel beam
scanning.
Furthermore, we benchmarked the performance of Bessel-3PM to point-scanning 3PM in
imaging 3D neuronal structures deeply buried in mice. We altered the focal length of L2 to
100 mm, which resulted in a 60 μm focal-depth Bessel beam for 3PM and an NA of 0.8. We
used Bessel-beam scanning 3PM to image the brain of an awake mouse from 620 μm to 685
μm below the dura. The imaging speed was 1 Hz (512 × 512 pixels/frame) for a volume of
300 μm × 300 μm × 65 μm. Imaging the same volume with point-scanning 3PM required 1
min, which might contribute to the skewed structures due to motion artifacts (Fig. 4). These
data highlights the importance and unique advantage of conducting rapid volumetric imaging
in deep brain in vivo with scanning Bessel-3PM.
4. Summary
In this work, we presented the results of using a rapid volumetric 3PM method based on the
axially elongated Bessel beam in imaging zebrafish larva, fruit flies and mice in vivo. The
proposed method enables volume scanning as large as 500 µm × 500 µm × 300 µm at 5 Hz
rate. Bessel-3PM has superior performance in terms of the penetration depth, SBR, and
Vol. 9, No. 4 | 1 Apr 2018 | BIOMEDICAL OPTICS EXPRESS 1999

imaging speed and potentially provides a new multi-photon imaging tool to study the
information processing within three-dimensional neural circuits in vivo.
Funding
The authors gratefully acknowledge funding from the National Natural Science Foundation of
China (31327901, 61475008, 31428004, 31570839, and 31521062) and the National Science
and Technology Major Project Program (2016YFA0500400).
Acknowledgment
The authors thank Hao Rong and Wan Yang for their help with the mechanical design. The
Tg (kdrl:EGFP) fish was provided by Jingwei Xiong at Peking University.
Disclosures
The authors declare that there are no conflicts of interest related to this article.
Vol. 9, No. 4 | 1 Apr 2018 | BIOMEDICAL OPTICS EXPRESS 2000