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Extended two-photon microscopy in live samples with Bessel beams: Steadier focus, faster volume scans, and simpler stereoscopic imaging

Authors:
  • Gentec-EO, Canada, Québec
  • CERVO Brain Research Centre

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Two-photon microscopy has revolutionized functional cellular imaging in tissue, but although the highly confined depth of field (DOF) of standard set-ups yields great optical sectioning, it also limits imaging speed in volume samples and ease of use. For this reason, we recently presented a simple and retrofittable modification to the two-photon laser-scanning microscope which extends the DOF through the use of an axicon (conical lens). Here we demonstrate three significant benefits of this technique using biological samples commonly employed in the field of neuroscience. First, we use a sample of neurons grown in culture and move it along the z-axis, showing that a more stable focus is achieved without compromise on transverse resolution. Second, we monitor 3D population dynamics in an acute slice of live mouse cortex, demonstrating that faster volumetric scans can be conducted. Third, we acquire a stereoscopic image of neurons and their dendrites in a fixed sample of mouse cortex, using only two scans instead of the complete stack and calculations required by standard systems. Taken together, these advantages, combined with the ease of integration into pre-existing systems, make the extended depth-of-field imaging based on Bessel beams a strong asset for the field of microscopy and life sciences in general.
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METHODS ARTICLE
published: 20 May 2014
doi: 10.3389/fncel.2014.00139
Extended two-photon microscopy in live samples with
Bessel beams: steadier focus, faster volume scans, and
simpler stereoscopic imaging
Gabrielle Thériault
1,2
,MartinCottet
2
, Annie Castonguay
2
, Nathalie McCarthy
1,2
and
Yves De Koninck
1,2,3
*
1
Département de Physique, de Génie Physique et d’Optique, Centre d’Optique, Photonique et Laser, Université Laval, Québec, QC, Canada
2
Centre de Recherche de l’Institut Universitaire en Santé Mentale de Québec, Québec, QC, Canada
3
Département de Psychiatrie et de Neurosciences, Université Laval, Québec, QC, Canada
Edited by:
Tycho M. Hoogland, Netherlands
Institute for Neuroscience,
Netherlands
Reviewed by:
Tim Murphy, The University of
British Columbia, Canada
Marc Guillon, Neurophotonics
laboratory, France
*Correspondence:
Yves De Koninck, Centre de
Recherche de l’Institut Universitaire
en Santé Mentale de Québec, 2601
de la Canardière, Québec,
QC G1J 2G3, Canada
e-mail: yves.dekoninck@
crulrg.ulaval.ca
Two-photon microscopy has revolutionized functional cellular imaging in tissue, but
although the highly confined depth of field (DOF) of standard set-ups yields great optical
sectioning, it also limits imaging speed in volume samples and ease of use. For this
reason, we recently presented a simple and retrofittable modification to the two-photon
laser-scanning microscope which extends the DOF through the use of an axicon (conical
lens). Here we demonstrate three significant benefits of this technique using biological
samples commonly employed in the field of neuroscience. First, we use a sample of
neurons grown in culture and move it along the z-axis, showing that a more stable
focus is achieved without compromise on transverse resolution. Second, we monitor 3D
population dynamics in an acute slice of live mouse cortex, demonstrating that faster
volumetric scans can be conducted. Third, we acquire a stereoscopic image of neurons
and their dendrites in a fixed sample of mouse cortex, using only two scans instead of
the complete stack and calculations required by standard systems. Taken together, these
advantages, combined with the ease of integration into pre-existing systems, make the
extended depth-of-field imaging based on Bessel beams a strong asset for the field of
microscopy and life sciences in general.
Keywords: nonlinear microscopy, depth of field, axicon, nondiffractive beam, temporal resolution, 3D imaging,
functional calcium imaging, cellular imaging
INTRODUCTION
Since its invention in 1990, two-photon microscopy (Denk et al.,
1990) has become an essential tool for biologists, especially
in the field of neuroscience (Zipfel et al., 2003). It can reveal
structures deep inside tissue (Helmchen and Denk, 2005), and
fluorescent markers can help track activity in networks of cells
(Stosiek et al., 2003; Lütcke and Helmchen, 2011). The intrinsic
optical sectioning of two-photon microscopy limits the focal vol-
ume to a very thin plane, which has been exploited to improve
axial resolution and limit photo damage around the focal vol-
ume (Zipfel et al., 2003). When the features of interest are
mainly located in the same plane or when a volume sample
is densely labeled, optical sectioning is a great advantage. But
ifthelabelingissparseandthecellsaredistributedatdif-
ferent depths in an extended volume, optical sectioning forces
the use of integrating multiple frames at different depths to
recover all the information. This limits the temporal resolu-
tion of the measurements. Optical sectioning therefore poses
challenges for scanning large volumes, in particular for func-
tional cellular imaging in live tissue or reconstructions of large
structures. Many research groups are attempting to address this
challenge (Göbel et al., 2007; Otsu et al., 2008; Reddy et al., 2008;
Grewe et al., 2010; Botcherby et al., 2012). Furthermore, a small
depth of field (DOF) can become problematic when the sample
moves vertically, as it often occurs during in vivo measurements
(Laffray et al., 2011).
In two-photon microscopy, it is possible to extend the DOF
of the system by generating a nondiffracting beam at the sam-
ple, while maintaining a good transverse resolution throughout
the sample. Different approaches have been recently proposed to
shape the distribution of light at the sample into a Bessel-Gauss
beam (Botcherby et al., 2006; Dufour et al., 2006; Thériault et al.,
2013), which is characterized by an intense central lobe and is
nondiffractive, i.e., the central lobe has a constant radius.
Although highly promising, these previous reports of two-
photon microscopy with an extended DOF have only shown
results with powerful fluorescent samples, such as fluorescent
micro-beads or stained pollen grains. In order to demonstrate
to the neuroscience community that the Bessel extended DOF
microscope is suitable to this field, biologically relevant samples
must be used. To our knowledge, this paper is the first report of
such measurements.
In this paper, we demonstrate experimentally three advan-
tages of two-photon microscopy with an extended DOF using a
Bessel beam when compared to standard two-photon microscopy.
These benefits are: (1) a more robust focus when sample moves
in the z direction, (2) an increase in information throughput or
in scanning speed for volume samples, and (3) the possibility
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CELLULAR NEUROSCIENC
E
Thériault et al. Extended depth-of-field two-photon microscopy
of creating stereoscopic images with only two x-y scans, dra-
matically reducing the number of scans required to examine
the relationship between structures in the axial direction. We
illustrate each of these advantages with a set of measurements per-
formed on different biological samples that are commonly used in
neuroscience.
MATERIALS AND METHODS
CONFIGURATION OF A BESSEL EDOF TWO-PHOTON MICROSCOPE
Standard two-photon microscopes can easily be modified to
extend the D OF with a Bessel beam by placing an axicon and a
lens in the laser beam path (Figure 1). Axicons are refractive opti-
cal elements shaped as a cone (McLeod, 1954), which deviate lig ht
toward the optical axis by an angle β simply calculated from Snell’s
refraction law. The complete details of this method are presented
in a previous paper (Thériault et al., 2013).
Let us quickly recall the parameters of the focal line in the
extended DOF system. The transverse resolution ρ and the D OF
L of the two-photon excitation at full-width at half m aximum are
given by:
ρ = m
1.629λ
2π sin β
and L = m
2
0.577w
tan β
(1)
where λ is the wavelength of the laser beam and m = Ff
1
/f
2
f
α
is
the magnification applied to the Bessel beam while being relayed
to the sample, with focal lengths F of the microscope objective, f
1
and f
2
of relay lenses in the scanning system and f
α
of the lens after
the axicon. The advantage of this approach is that it allows adjust-
ing the DOF independently (without changing the resolution) by
using a simple telescope (Thériault et al., 2013).
CUSTOM-BUILT EXTENDED DOF MICROSCOPE
In this paper, experiments were carried out on two different sys-
tems. The first one is a custom-built laser-scanning microscope,
FIGURE 1 | Illustration of the set-up. A Ti:Sapphire laser generates an
ultra-short pulsed laser beam with a Gaussian profile. This beam is
expanded with a simple two-lens telescope. Once expanded, the beam
passes through an axicon followed by a lens. These two elements
transform the laser beam into an annulus of light. This annulus is imaged
into the back focal plane of the objective lens, which creates a tightly
focused Bessel-Gauss beam in the sample. The scanning system enables a
beam tilt in the back focal plane of the objective, leading to an x-y scan of
the beam in the sample. Fluorescence light is retro-collected with the
objective and directed to a photomultiplier tube with a dichroic mirror.
which includes a removable DOF extension module as illustrated
in Figure 1.
We used a Ti:Sapphire pulsed laser with central wavelength
λ = 900 nm (Chameleon, Coherent), relay lenses with a magnifi-
cation factor, f
1
/f
2
= 1.5 and an objective with a focal length F =
4.11 mm (Zeiss, W N-Achroplan 40×,0.75NA). To demonstrate
the flexibility of the Bessel extended DOF set-up, we used different
sets of parameters throughout this paper. These parameters are
detailed in Table 1 . Note that the transverse resolutions achieved
with this beam are significantly better than with a Gaussian
beam (0.5 μm). The ability to design an optical system with an
improved transverse resolution represents an added advantage of
this imaging configuration (April et al., 2012). The two-photon
signal intensity distributions (on-axis intensity and t ransverse
resolution) for each of these sets of parameters are presented in
Figure 2. Experimental measurements with micro-beads show an
excellent agreement with the theoretical curves.
Table 1 also includes the average laser power used for each
experiment, m easured at the sample plane. Laser power is an
important factor in extended D OF imaging because the flu-
orescence signal intensity is highly dependent on the length,
L (Thériault et al., 2013). One can also note that for a focal
line of approximately 50 μm, roughly 3 times more power was
required with the extended DOF set-up to obtain signal-to-noise
ratios similar to when using the standard two-photon set-up
with the same transverse resolution. Although more power is
sent to the sample during one scan, the volume in which this
power is focused is much larger than in the conventional set-up.
Therefore, the power per unit volume and the peak intensity of
the excitation beam are the same as with the standard two-photon
microscope to generate the same fluorescence signal. Therefore,
photobleaching and photodamage are not an issue.
MODIFIED COMMERCIAL MICROSCOPE
The second system was a Zeiss LSM510 coupled to a Ti:Sapphire
pulsed laser (Chameleon, Coherent) for two-photon imaging.
We modified this system by adding a simple double-convex lens
(f = 200 mm, Thorlabs) and an axicon (0.1
,UVFS,Altechna)
just before the laser injection porthole. With these parameters and
using an objective with a focal length F = 4.11 mm (Zeiss, W N-
Achroplan 40×,0.75NA), the system produces a focal line with
a transverse resolution of ρ = 1.1 μmandaDOFofL = 25 μm.
This resolution is not optimal because of the mismatch between
the characteristics of the axicon that we had available and the
objective lens. An axicon of 0.2
would have yielded a resolution
of 0.44 μm. With this DOF, the laser power at the sample for these
experiments was 30 mW spread over the 25 μmoftheDOF.
This commercial microscopy system also supports multiple
modalities such as confocal imaging and a photon-counting unit
for fluorescence lifetime imaging (FLIM), which we used for the
increased information throughput demonstration. The confocal
modality was set at a wavelength of 488 nm and with a pinhole
opening of 1 Airy unit (0.61 λ/NA).
SAMPLE PREPARATION
As mentioned in Introduction, we present here three advantages
of the Bessel extended DOF microscope, using three different
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Thériault et al. Extended depth-of-field two-photon microscopy
Table 1 | Components and focal line parameters used for the experiments in this paper that were carried out on the custom-built set-up.
Type of DOF Figure w (mm) Axicon f
α
(mm) ρ (μm) L (μm) P/L (mW/μm)
Extended 4, 7, 8 1.0 5
, UVFS 45 0.43 54 71/54
62.31
, BK7 125 0.69 81 83/81
Standard 4, 6 2.3 N/A N/A 0.43 1.2 24/1.2
FIGURE 2 | Two-photon fluorescence distributions for the data
presented in this paper. The on-axis intensity (top) and the transverse
resolution (bottom) of a standard two-photon set-up (red lines) varies much
more rapidly along the optical axis t han those of the Bessel extended DOF
two-photon microscope (green and blue lines). Green dots are experimental
values measured with fluorescent microspheres (Molecular Probes,
Fluosphere 505/515, diameter 200 nm) mounted on a coverslip with
fluorescent mounting medium (Dako).
types of samples commonly used in neuroscience. To demonstrate
the improvement of focus stability, we use a thin sample, i.e., neu-
rons grown on a glass coverslip and transfected with a fluorescent
protein. To demonstrate the increased speed for volumetric scans,
we use thick acute slices of adult mouse cortex, stained with a
calcium indicator. Finally, to show the simplicity of stereoscopic
imaging, we use a fixed sample of mouse cortex, containing flu-
orescent protein-labeled neurons. In this section, we detail how
each type of sample was prepared.
Cultured cells
Primary dissociated neurons grown in culture were obtained as
described previously (Nault and De Koninck, 2010). Cells were
plated at 0–3 post-natal days at a density of approximately 1–
2 M cells per coverslip. From day 5, Ara-C (10 μM) was added
to the culture medium to kill cells in division and prevent pro-
liferation of glial cells. For DOF stability experiments, mEGFP
plasmid was t ransfected in hippocampal cells in culture at 12 days
in vitro using lipofectamin 2000 (Invitrogen) and 0.5 μgofDNA
per coverslip. Cells were allowed to express the fluorescent pro-
tein for 24 h before PFA fixation. Coverslips were fixed in a 4%
paraformaldehyde solution with PB 0.1 M at 37
C for 10 min.
Fixation was followed by 3 washes (10 min) in PB 0.05 M and
coverslips were mounted on glass slides using DAKO fluorescent
mounting medium.
Live acute brain slices
Acute slices were prepared using the following method. Adult
mice were deeply anesthetized with isoflurane and decapi-
tated. The brain was quickly removed and placed in ice-cold
solution ( 4
C) containing (in mM) 252 sucrose, 2.5 KCl,
1.5 CaCl
2
,6MgCl
2
, 10 glucose, 26 NaHCO
3
,1.25NaH
2
PO
4
,and
5 kynurenic acid (sACSF). Coronal slices of cortex were cut at
300 μm using a vibratome (VT 1200S, Leica) and kept in artifi-
cial cerebro-spinal fluid (ACSF) containing (in mM) 126 NaCl,
2.5 KCl, 2 MgCl
2
,2CaCl
2
,1.25NaH
2
PO
4
, 26 NaHCO
3
,and10
glucose, bubbled with 5% CO
2
/95% O
2
to adjust the pH to
7.4. Slices were incubated with a solution containing 1 μMFluo-
4 AM (Molecular Probes) for 30–60 min at room temperature
prior to imaging. To prevent mechanical damage, the slices were
placed on a mesh of nylon in a covered 12-well plate, which
was continuously bubbled. Slices were then transferred to a
recording chamber continuously perfused with ACSF or high
potassium solution for recordings to raise network activity. The
high K
+
solution contained: 80 NaCl; 50 KCl; 2 CaCl
2
;1MgCl
2
;
25 d-Glucose; 26 NaHCO
3
;1.25NaH
2
PO
4
, also gassed with 5%
CO
2
/95% O
2
to adjust the pH to 7.4.
Fixed tissue preparations
For comparison of images obtained from fixed tissue, 300 μm-
thick brain slices from Thy1::COP4-EYFP (Jackson Laboratories)
mice and whole dorsal root ganglions (DRGs) from C57 wildtype
mice 6 weeks after injection at P6 of a AAV9 viral vector encod-
ing for a GFP (U. Pennsylvania) were fixed by immersion in 4%
paraformaldehyde for 1 h.
RESULTS
STEADIER FOCUS
In standard two-photon microscopy, the DOF is approximately
L λ/NA
2
whenthebackapertureoftheobjectiveisproperly
filled (when the ratio of the objective back aperture to the beam
width is π/2). This means that at λ = 900 nm and with a 0.8
NA objective, then L = 1.4 μm. Such a thin optical section offers
a number of advantages, including the ability to resolve small
features in three dimensions, but it also comes with drawbacks,
such as not being able to resolve complete neurons in a single
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Thériault et al. Extended depth-of-field two-photon microscopy
frame and important fluorescence signal fluctuations due to small
perturbations along the vertical axis.
UsinganextendedDOFcountersbothofthesedrawbacks.
First, the entire cells can be imaged in a single x-y scan. For exam-
ple, even in thin samples like neurons grown in cultures, fine
structures such as dendrites are generally located on the same
plane close to the substrate, but they also grow around and above
thicker cell bodies, often 10–15 μm thick, which means that the
total sample thickness is well over the standard DOF; the different
neurites end up not all located in the same plane and thus cannot
be captured in one scan (see Figure 3). With an extended DOF of
only 15–20 μm, complete cell bodies including the dendrites can
be imaged i n only one frame. Although this improvement does
not imply a very high gain in acquisition speed, it guarantees that
all the features of interest are imaged.
Second, small perturbations in the z direction and focus drift
do not affect the fluorescence signal when using an extended
DOF microscope. Focus drift is a major issue for live-cell imaging
(Waters, 2007) and it is often caused by changes in temperature,
an unstable stage or focusing mechanism, an uneven perfusion, or
movement of the specimen. Post-processing can be used for data
with a focus drift in the illumination plane, but when the drift is
vertical, the data are lost. This data loss is less an issue in systems
that include an autofocus control, which can compensate for slow
vertical drifts.
Finally, let us remark that it is also possible to slightly increase
a systems DOF by using an objective with a lower numerical aper-
ture (or, equivalently, under-filling the objective back-aperture).
For example, as mentioned above, at λ = 900 nm and NA = 0.8,
the DOF is L 1.4μm. But with a numerical aperture of 0.3, the
same system has a much larger DOF: L 10μm. However, the
major drawback of this approach is that the width of the focal
spot also increases: the transverse resolution in this examples goes
from 0.56 to 1.5 μm, when calculated with the Abbe criterion:
ρ = λ/2 NA. With a Bessel beam, in contrast, the transverse reso-
lution is not compromised and remains constant, even when the
DOF is extended.
Demonstration of focus stability
To demonstrate the enhanced focus stability provided by the
extended DOF modification, we used a thin sample of neuron
cultures grown on a glass coverslip that we moved in the z
FIGURE 3 | Extended DOF for imaging thin samples. With the standard
two-photon microscope (left), the focal plane is very thin and small
perturbations affect the fluorescence signal. With an extended DOF (right),
the focus is much more robust.
direction. Although two-photon microscopy is rarely needed to
image cultured samples, two-photon excitation was used here
because it offers several advantages: (1) it reduces the sidelobes in
the Bessel beam to a point where they are negligible; (2) it maxi-
mizes transverse resolution (in fact, the Bessel beam offers even a
better resolution than the Gaussian beam; April et al., 2012); (3)
using a pulsed laser is ideal for certain imaging modalities, such
as fluorescence lifetime imaging (FLIM) (Doyon et al., 2011). In
this section, we will compare the acquired images with a stan-
dard DOF system to the ones acquired with a Bessel-modified 2P
microscope.
The biological samples used in this section are neurons grown
on a glass coverslip and transfected with a fluorescent pro-
tein. Their preparation is detailed in Materials and methods.
Fluorescence images of the transfected cells were taken at a speed
of 1 s/frame (2 ms/line; 512 × 512 pixels) with the extended DOF
set-up described in Materials and methods. The excitation and
emission light were separated by a dichroic mirror at 665 nm
(Semrock). The emission light was truncated by a 633 nm short-
pass filter (Semrock). Between each image, the motorized stage
supporting the sample was translated 2 μminthezdirection.The
single frames shown in Figure 4 are the raw fluorescence images,
only brightness and contrast levels were adjusted. For the graph
in Figure 4A,thepixelvaluesoneachframewereaveraged,and
the data was normalized to the maximum of each curve.
At first glance, these results prove that the sig nal-to-noise ratio
of the extended DOF set-up is sufficient to discern single neurites,
even though the laser power is spread in a long focal line instead of
a tight spot. Furthermore, m easurements taken with the extended
DOF system can be considered more stable than with the standard
two-photon microscope, since external factors such as vibrations
of the stage or focus drift do not affect the fluorescence signal
intensity, within a range of a few tens of microns, whereas dis-
placements of only a few microns induce dramatic changes in the
standard two-photon set-up. This greater signal stability in turn
leads to less variability in the measured fluorescence levels, but a
possible drawback from this approach could be a higher chance
of recording from several dendrites belonging to different cells at
the same time if they are superimposed in the z axis.
Finally, in vivo measurements could also benefit from the
steadier focus that the extended DOF approach provides. For
example, when imaging spinal dorsal horn or brain stem fea-
tures in vivo in rats, breathing and cardiac movements induce
vertical displacements of tens of microns (Laffray et al., 2011).
These displacements make out-of-focus images unusable, which
dramatically reduces the temporal resolution. With an extended
DOF, all the acquired images can be used if the excitation line
is set long enough to keep the features of interest within the
excitation volume.
FASTER VOLUME SCANS
In many biological experiments, the features of interest are spread
out in a three-dimensional matrix. It is not always necessary
to know at which depths these features are, only their pres-
ence/absence, action/reaction or growth/retraction can provide
precious information. In such cases, a method to scan the entire
volume faster than raster-scanning each plane of interest could
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FIGURE 4 | Extended DOF microscopy provides a steadier focus. (A)
Average fluorescence signal from each frame in a z-stack of fixed neurons,
grown on a glass coverslip. The stage holder was translated 1 μminthez
direction between each frame. The signal in the extended DOF set-up (blue)
is stable over a larger distance than the signal in the standard set-up (red).
Single frames at 5 different positions in the z-stack are presented below, for
(B) the conventional set-up and (C) the Bessel extended DOF set-up. (D)
The DOF extension does not affect the transverse resolution of the system,
as shown by this comparison of average intensities from 10 frames for each
method.
be very useful. We demonstrate here that using the Bessel beam
extended DOF set-up to image thick live samples leads to a greater
throughput of information (more cells sampled) or faster volu-
metric scans than when using a standard two-photon microscope.
Various approaches can be envisaged when one needs to
increase the number of cells sampled within a specific time-
frame. Setting the sample’s geometry and staining density aside,
the approaches can be resumed by two categories: increasing the
scanning speed or the excitation volume.
Many techniques have been recently developed to increase the
scanning speed in two-photon microscopy (Lillis et al., 2008; Otsu
et al., 2008; Reddy et al., 2008; Grewe et al., 2010; Truong et al.,
2011; Botcherby et al., 2012; Katona et al., 2012). Nevertheless,
the most commonly used method to image thick samples still
remains the 3-D raster scan (Figure 5). This method consists in
rapidly tilting the laser beam with a set of mirrors. Particular
mirrors that are extremely fast can also be used to increase scan-
ning speeds [ex.: rotating polygon (Rajadhyaksha et al., 1999),
resonant mirrors (Göbel et al., 2007)]. The two set-ups that we
used in this paper feature the slower but more adaptable galvano-
metric mirrors. With these mirrors, line scans of up to 120 Hz
can be achieved (for a 4.3 Hz frame-rate with 512 × 512 pixels).
Nevertheless, all the results presented here could be reproduced
on systems with faster scanning mirrors.
Shaping the laser beam into a Bessel-type nondiffractive beam
as we implemented in our two set-ups is a technique that increases
the excitation volume. The excitation spot, now spread out into
a thin, long line, generates fluorescence signal at various depths
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Thériault et al. Extended depth-of-field two-photon microscopy
FIGURE 5 | Bessel beam 3-D raster scan vs. conventional DOF
volumetric imaging in a thick sample. (A) With the standard two-photon
microscope, the focal volume is very small and one must acquire a stack of
raster-scanned images to image the entire volume of interest. (B) With an
extended DOF, the entire volume of interest can be examined in a single x-y
scan, which leads to much faster volumetric scans without compromise on
transverse resolution.
simultaneously inside the sample. This way, many cells are sam-
pled in a single x-y scan, even when they are located at different
depths.
Demonstration: fluorescence lifetime imaging of dorsal root
ganglions with 2-photon extended DOF
One application of the extended 2-photon DOF in thick samples
would be for the acquisition of fluorescence lifetime data, using
the pulsed characteristics of the 2-photon laser for synchroniza-
tion. In the example provided, lifetime of a fluorescent protein
was acquired in DRG neurons (Figure 6).
Furthermore, the extended DOF set-up was implemented on
a commercial laser-scanning microscope, by simply adding and
aligning the lens and the axicon in the two-photon laser path
between the periscope and the microscope. This configuration
allowed for measurements of multiple cells located in a large vol-
ume. When comparing these measurements to confocal images,
where the DOF is very small, we can see that much fewer cells are
sampled in a single small-DOF image (Figure 6). As mentioned
above, this configuration also allows for fluorescence lifetime
imaging. In live tissue, time-lapse fluorescence lifetime imaging
could be achieved. Doing so would per mit to probe more cells
than with the conventional two-photon sectioning and ensure
that a maximum of cells would stay in the focal plane throughout
the recording.
The extended DOF modification to a two-photon setup also
offers a significant i ncrease in speed, especially for imaging cellu-
lar dynamics in live tissue. Indeed, a 40 μm thick volume can be
completely examined with a single scan. A confocal setup would
however require 20 x-y frames with a typical 2 μm-DOF, result-
ing in a 20-fold increase in acquisition time. This alone allows
imaging larger volume with a temporal resolution of a few Hz.
This enhancement in temporal resolution remains valid for high
speed rotating mirrors (polygons, resonant scanners, etc.) and
thus could potentially yield scan speeds on the order of a few kHz
(Rajadhyaksha et al., 1999).
On the other hand, the cell loading method must also be
optimized to eliminate contaminating background fluorescence.
Hence, the expression of genetically-encoded proteins of interest
FIGURE 6 | Extended DOF for fluorescence lifetime imaging of
dorsal root ganglion neurons (DRGs). (A) Confocal imaging of DRG
neurons expressing GFP. Images obtained at various focal depths, with
a DOF of 2.1 μm. The image on the far right is the resulting
z-projection of 7 images taken every 6 μm, between z =−18 μmand
z = 18 μm (example images on the left). (B) Extended depth of field
with 2-photon excitation and fluorescence lifetime imaging. The same
sample was imaged with 2-photon excitation. Left, single frame
obtained with a DOF of 20 μm. Center, average of 7 frames (same
acquisition time as for each confocal image). Right, color-coded lifetime
image of DRG neurons obtained from photons accumulated from 7
consecutive frames.
in mice and targeted viral infection are methods of choice to be
used with the extended DOF.
Application: time-lapse imaging of calcium fluctuations in thick
samples
To demonstrate that this technique is compatible with live tissue
imaging, we acquired a time-lapse sequence of calcium fluc-
tuations in a thick, acute slice of mouse cortex stained with
Fluo-4.
The biological sample used in this section is a thick acute
slice of adult mouse cortex, stained with a calcium indicator.
The sample preparation is detailed above. F luorescence images
of the calcium indicator Fluo-4 AM were taken at a frequency
of 0.5 Hz (one frame every 2 s; 2 ms/line; 512 × 512 pixels) with
the extended DOF set-up is described above. The excitation and
emission light were separated by a dichroic mirror at 665 nm
(Semrock). The emission light was truncated by a 633 nm short-
pass filter (Semrock). For measurements of calcium-dependent
changes, a sequence of fluorescence images w as acquired. To
stimulate cellular activity, the extra cellular solution was switched
from the standard solution to the high potassium solution
every 60 s.
In each sampling epoch, an average of the first 20 images
was calculated to set the baseline value, F
0
. Regions of inter-
est (ROI) were defined in the first image, and the normalized
fluorescence changes (F-F
0
)/F
0
were measured throughout the
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Thériault et al. Extended depth-of-field two-photon microscopy
image sequence. For the time-lapse video of the data presented
in Figure 7 (and Supplementary Video S1, available online), each
frame was smoothed with a gaussian filter with a 2-pixel radius to
reduce noise, brightness and contrast levels were also adjusted.
After the extended DOF time-lapse acquisition was completed,
we reverted to the standard two-photon set-up by removing the
axicon and lens and proceeded to acquire a stack of images with
enough laser power to resolve the recorded cells. With this stack
of images, it was possible to compare the features in each plane
to the ones observed during the extended DOF time-lapse acqui-
sition. We were therefore able to determine at which depth was
located the bulk of the features selected in each ROI used for
the extended DOF acquisition sequence. These depths are color-
coded in Figure 7 (top) and the ROIs are superimposed with the
summed stack from the standard DOF set-up.
We s e e in Figure 7 that several features located at different
depths in the sample have calcium levels that are partially syn-
chronized.Forexample,ROIs1,3,7,9,and11allshowthree
main peaks of calcium concentration at approximately the same
times, even though they span 60 μm in depth. We can therefore
infer that the cells in these five regions are part of a common 3-
dimensional network that receives synchronized inputs. It would
have been very difficult to observe this with a standard DOF since,
as discussed above, it would take at least 7 s to image a complete
60 μm-thick volume.
Comparison of high-speed microscopy approaches
Let us now compare our method’s speed to that of three of
the highest speed two-photon microscopy systems recently pub-
lished.
The first one (Cheng et al., 2011) uses a resonant mirror for
the fast-scanning axis to obtain frame rates of 250 Hz for single
images with 500 × 500 pixels. They split the beam into N = 4
separate delay lines to multiplex the excitation temporally and
add optical elements to these lines to separate the 4 beams axially,
causing the excitation beam to be focused at 4 different depths
during one pulse cycle. They therefore achieve a volume-scan
rate of 250 Hz/N = 62.5 Hz, but a large portion of the volume
is still not imaged since the DOF is approximately 0.8 μmand
wewishedtosample60μm depth. If this system were adapted to
multiplex the excitation beam at N = 20 different depths, span-
ning 60 μm, it would then be equivalent to our experiment w i th
the Bessel extended DOF microscope (although the complexity
of aligning a set-up with 20 delay lines is highly challenging).
The effective volume-scan rate of such a system would then be
250 Hz/N = 12.5 Hz, which is still three times faster than the
fastest volume-scan rate of our set-up. Nevertheless, both the
temporally multiplexed beam approach and the use of a resonant
scanning mirror are fully compatible with our proposed approach
and could be applied to further increase the volume scanning
speed.
FIGURE 7 | Volumetric imaging of calcium dynamics in mouse cortex.
(A) Positions of the ROIs in the specimen, acquired with a standard
two-photon stack at the end of the experiment (the summed stack of the raw
fluorescence images is shown here), and color-coded to indicate the depth of
each feature. (B) Single-cell calcium transients in an acute slice of mouse
cortex stained with Fluo-4 AM, imaged with the extended DOF set-up and
corresponding to the ROIs in (A). Dotted lines correspond to the extended
DOF single frames shown below. (C–G) (F-F
0
)/F
0
single frames from the
time-lapse acquisition of calcium dynamics (full video available online:
Supplementary Movie S1).
Frontiers in Cellular Neuroscience www.frontiersin.org May2014|Volume8|Article139
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Thériault et al. Extended depth-of-field two-photon microscopy
The second method with which we compare our systems per-
formance is the random-access scanning two-photon microscope
(Katona et al., 2012). This approach uses acousto-optic deflectors
to steer the laser beam instead of oscillating mirrors. In the 3D
line-scanning mode, this method can sample up to 500 points
per kHz. A volume containing 500 × 500 × 20 voxels would
then require an acquisition time of 10 s. In this case, our sys-
tem is at least 20 times faster. Although it is slow for complete
volume scans, the advantage of the random-access microscope is
that once the user knows where the regions of interest lie, only
a small subset of points must be sampled repetitively during the
remainder of the experiment. On the other hand, the problem of
focus drift or sample movement remains, so the random-access
approach might not be appropriate for all experiments, whereas
the Bessel-beam approach is more flexible.
The third fast two-photon method that we mention here uses
a spatial light modulator to shape the two-photon illumination
pattern in the focal plane (for example, see Nikolenko et al.,
2008). SLM microscopy is ongoing and its speed is theoretically
limited by the refreshing rate of the detecting module. The advan-
tages and disadvantages of this method are similar to those of the
random-access approach since it offers a very high throughput of
information on a fixed number of cells, and a previous knowledge
of the sample must be acquired and focus drift or sample move-
ment could affect the recorded sig nal if they are not monitored
and compensated in real-time.
Finally, let us remark on the choice of DOF. Although it could
appear interesting to increase the DOF extensively in order to
obtain a greater g ain in speed, one should be aware that as the
DOF increases, the probability of two or more labeled features
being super-imposed also increases. The DOF should therefore
be adjusted to the labeling density. Larger depths of field should
only be used with sparsely labeled samples to avoid measurement
errors from super-imposed cells.
SIMPLER STEREOSCOPIC IMAGING
In the previous section, we mentioned that the information about
depth cannot be retrieved from a single extended DOF image.
A simple way to circumvent this disadvantage is to compose a
stereoscopic pair by inducing a tilt in the focal line at the sam-
ple (Botcherby e t al., 2006). With an extended DOF set-up, it is
possible to acquire a stereoscopic image with only two x-y scans,
one for each viewpoint. To illustrate this, we present an example
of stereoscopic imaging with a sample of protein-labeled neurons
from a fixed slice of mouse cortex.
The approach we used to induce a tilt in the focal line is illus-
trated in Figure 8A. When a lateral shift x is applied to either
theaxiconoritsassociatedlens,theringoflightincidentonthe
back aperture of the objective is shifted, and the focal line is tilted
with respect to the optical axis with the parallax angle θ,dened
below (adapted from Botcherby et al., 2006):
θ =
f
2
f
1
F
x
cos
(
mβ
)
(2)
To verify that displacing the lens does induce a tilt in the focal line
throughout the field of view, we acquired a stack of images from
FIGURE 8 | Simple method to produce a stereoscopic pair of images.
(A) A small displacement of either the lens after the axicon induces a tilt θ
of the focal line at the sample, with respect to the optical axis. We scanned
a random distribution of small fluorescent beads (Molecular Probes,
Fluosphere 505/515, diameter 500 nm) at different depths, spanning 60 μm,
to produce a stack of images. The z-projection of these stack show that
when (B) no shift is applied, the point of view is perfectly vertical, and
when (C) the lens is slightly shifted, the point of view is tilted with respect
to the vertical. (D) Two superimposed cells can be distinguished with this
method if they are separated by a distance z = d/sinθ.
fluorescent microspheres (Molecular Probes, Fluosphere 505/515,
diameter 500 nm) mounted on a 150 μm thick coverslip with flu-
orescent mounting medium (Dako). A z-projection of this stack
shows whether or not the point of view is shifted. When there is
no shift, the point of view is perfectly vertical and all the beads
appear as regular circles (Figure 8B). When the lens is displaced,
the focal line is shifted and the beads appear stretched out along
the tilted axis (Figure 8C). We can also see from this projected
image that the transverse resolution of the system has not been
degraded by the displacement of the lens.
The biological sample used in this section is a fixed slice of
cortex from a transgenic mouse in which genetically encoded flu-
orescent markers are expressed in a subset of cells. The sample
preparation is detailed in S ection Sample Preparation.
A set of two images was acquired wi th the extended DOF sys-
tem in order to get a stereoscopic image. To displace the lens,
we mounted it on a computer-controlled motorized translation
stage. For the first image (Figure 9A), the lens was displaced
by x =−100 μm, inducing a 2.5
tilt in the focal line. For
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Thériault et al. Extended depth-of-field two-photon microscopy
FIGURE 9 | Stereographic imaging of neurons in only two frames. With
the extended DOF microscope, two images were acquired, with different
values of x: (A) x =−10 0 μmand(B) x =−100 μm. The composite
image (C) can be viewed with 3-D perception using red-cyan glasses.
thesecondimage(Figure 9B), the lens was displaced by x =
100 μm, inducing a 2.5
tilt in the focal line. To improve the
signal-to-noise ratio, each line was averaged 10 times in both
images.
Although a stereoscopic pair would better be viewed using a
3D display and the matching goggles, we have chosen to present
our results as a red-cyan anaglyph. To form a stereogram that
can be viewed with red-cyan glasses, a black-to-red colormap
was assigned to the first image and a black-to-cyan colormap was
assigned to the second image. Adding the two images formed the
composite image shown in Figure 9C. When viewing this image
with the appropriate glasses, one can see that the cell bodies are
located at different depths (for example, the cell at the lower right
of the image appears much higher than the one at the upper left).
Even small details such as the fine dendr ites are resolved with
depth perception.
With this method, it is possible to recover information about
relative depths. As illustr ated in Figure 8D,whentwocellsare
superimposed in the z-axis, it is possible to distinguish them if
they are separated by a distance z = d/sinθ , where d is the cell
diameter. For example, w h en the total tilt between the two images
is θ = 10
and if we approximate a cell body to a 10-micron
sphere, then the minimal distance at which two superimposed
cells can be distinguished is z = 60 μm. With these parameters,
the cell bodies would appear as a single circle in one image and as
two touching circles in the second image.
To produce the same image with a standard two-photon
microscope, it is necessary to acquire a stack of at least 30 images
(one image every 2 μm, spanning 60 μm) and to recompose the
left and right image by calculating projections of the stack accord-
ing to two different angles. All of these steps and calculations are
necessary to generate a similar composite image comparable to
the one obtained in Figure 9C with only two scanned frames.
The method we presented here, using an axicon, is therefore dra-
matically simpler and faster, and could be implemented into a
conventional two-photon microscopy system with a 3D screen to
view samples stereoscopically in real time.
DISCUSSION
In this paper, we have presented a simple modification to the
standard two-photon microscope, which consists in extending the
DOF of the system without compromising on transverse resolu-
tion, by adding two optical elements in the laser beam path: an
axicon (conical lens) and a regular lens.
With this modification, we performed measurements on three
different types of biological samples, all of which are commonly
used in the field of neurosciences: a thin sample of cells grown in
culture on a glass coverslip; a thick, live sample of acute brain slice
from a mouse; a thick, fixed sample of transgenic mouse cortex.
Thefirstsamplewaslabeledbytransfectionofagreenfluores-
cent protein, the second sample was stained with a calcium-ion
indicator to track cell activity ex vivo with variations in fluo-
rescence intensity, and the third sample contained genetically
encoded fluorescent markers expressed in a subset of cells. All
of these marking techniques are common tools in neuroscience,
which shows that the extended DOF system is compatible with
the current biological techniques.
With each sample, we highlighted the benefits of using an
extended DOF system based on a Bessel beam, when compared to
the standard two-photon microscope. For thin samples, or spec-
imens in which most features of interest are generally located
in the same plane, we have shown that the extended DOF pro-
vides a more stable focus, which can protect against vibrations or
focus drift. The same benefit could be exploited for in vivo mea-
surements, to avoid measurement biases due to small movements
(e.g., due to breathing). For thick samples where the features of
interest are dispersed into a 3-dimensional matrix, an extended
DOF improves the speed of volumetric scans (up to 30 times
faster), which allows resolving dynamics on a shorter time-scale.
Despite the fact that the information about depth is lost, we
showed that it is possible to recover this information at the end of
the experiment by removing the DOF extension add-on, reduc-
ing the DOF of the set-up to a standard two-photon microscope.
Finally, we presented stereoscopic imaging in fixed tissue, a far
simpler and faster way of obtaining depth information than with
the standard image stack method. We introduced an efficient
approach to achieve the stereoscopic image with minimal degra-
dation of the focal line, in which only two images needed to be
Frontiers in Cellular Neuroscience www.frontiersin.org May2014|Volume8|Article139
| 9
Thériault et al. Extended depth-of-field two-photon microscopy
acquired, instead of a complete stack (e.g., 60 images) and a 3-D
reconstruction algorithm required with a standard two-photon
system.
Further extension of the DOF could be envisaged, however this
comes with power limitations as the power is distributed along the
focal line. Furthermore, given that this is a two-photon effect, the
power loss is squared with the increase in focal line. The maxi-
mum focal length extension possible is thus limited by the power
of the laser source available, by the density of the labeled fea-
tures and by the fluorescence collection capabilities of the system
(Sergeeva et al., 2010). Yet, phototoxicity or photobleaching are
not necessarily increased if the power is tuned so that the fluo-
rescence signal remains the same at each p oint along the focal
line. The same peak excitation intensities at each point within
the sample can thus be achieved with both the Bessel beam and
a conventional Gaussian beam.
A critical advantage of the proposed approach is that it allows
integration of the axicon into a standard laser-scanning micro-
scope. The system is thus fully retrofittable into existing com-
mercial systems and can be designed to offer both Bessel beam
and Gaussian beam illumination in the same system, allowing
both types of imaging to be performed sequentially on the same
sample to exploit the advantage of both techniques at the same
time. This key feature will likely result in a broad acceptance of
the technology by the community, further amplifying and accel-
erating its impact. We believe that due to the flexibility, simplicity
and accessibility of the extended DOF method, combined with all
the benefits it provides (steadier focus, faster volume scans and
simpler stereography), this technology w ill have a transforming
impact for life sciences in general.
ACKNOWLEDGMENTS
This work was supported by the National Sciences and
Engineering Research Council of Canada (NSERC), the Canadian
Institutes of Health Research (CIHR), the Canadian Institute for
Photonic Innovations (CIPI) and the Fonds de recherche du
Québec—Nature et technologies (FRQNT). Gabrielle Thériault
was supported by a studentship from the CIHR Neurophysics
training program. The authors would also like to thank Feng
Wang for providing the fixed DRG samples and Louis Thibon for
his support with the integration and alignment of the extended
DOF in the Zeiss microscope.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online
at: http://www.frontiersin.org/journal/10.3389/fncel.2014.
00139/abstract
Movie S1 | Extended DOF two-photon imaging of calcium transients in an
acute slice of adult mouse cortex.
(F-F
0
)/F
0
images from the x-y-t
time-lapse sequence of a 190 × 190 μm scan field depict single-cell
calcium transients in an acute slice of mouse cortex stained with Fluo-4
AM, imaged with the extended DOF set-up.
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Conflict of Interest Statement: The authors declare that the research was con-
ducted in the absence of any commercial or financial relationships that could be
construed as a potential conflict of interest.
Received: 27 January 2014; accepted: 30 April 2014; published online: 20 May 2014.
Citation: Thériault G, Cottet M, Castonguay A, McCarthy N, and De Koninck Y
(2014) Extended two-photon microscopy in live samples with Bessel beams: steadier
focus, faster volume scans, and simpler stereoscopic imaging. Front. Cell. Neurosci.
8:139. doi: 10.3389/fncel.2014.00139
This article was submitted to the journal Frontiers in Cellular Neuroscience.
Copyright © 2014 Thériault, Cottet, Castonguay, McCarthy, and De Koninck. This
is an open-access article distributed under the terms of the Creative Commons
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is per mitted, provided the original author(s) or licensor are c redited and that the orig-
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No use, distribution or reproduction is per mitted which does not comply with these
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Frontiers in Cellular Neuroscience www.frontiersin.org May 2014 | Volume 8 | Article 139
| 11
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