1
Line-field confocal optical coherence tomography for three-
dimensional skin imaging
Jonas Ogien
a
, Anthony Daures
a
, Maxime Cazalas
a
, Jean-Luc Perrot
b
, Arnaud Dubois
c
a
DAMAE Medical, 14 rue Sthrau, 75013 Paris, France.
b
Service dermatologie, CHU St-Etienne, 42055 Saint-Etienne, France.
c
Université Paris-Saclay, Institut d’Optique Graduate School, CNRS, Laboratoire Charles Fabry,
91127 Palaiseau, France.
ABSTRACT
This paper reports on the latest advances in line-field confocal optical coherence tomography (LC-OCT), a
recently invented imaging technology that now allows the generation of either horizontal (x×y) section
images at an adjustable depth or vertical (x×z) section images at an adjustable lateral position, as well as
three-dimensional images. For both two-dimensional imaging modes, images are acquired in real-time, with
real-time control of the depth and lateral positions. Three-dimensional (x×y×z) images are acquired from a
stack of horizontal section images. The device is in the form of a portable probe. The handle of the probe
has a button and a scroll wheel allowing the user to control the imaging modes. Using a supercontinuum
laser as a broadband light source and a high numerical microscope objective, an isotropic spatial resolution
of ~ 1 µm is achieved. The field of view of the three-dimensional images is 1.2×0.5×0.5 mm
3
(x×y×z).
Images of skin tissues are presented to demonstrate the potential of the technology in dermatology.
Keywords: Optical coherence tomography, microscopy, three-dimensional imaging, dermatology.
1. Introduction
Optical coherence tomography (OCT) is a technology based on low-coherence optical interferometry for
imaging biological tissues with micrometer-scale spatial resolution [1,2]. OCT is commonly used in several
medical fields [3], especially in ophthalmology to obtain images of the retina and the anterior segment of
the eye [4]. OCT has begun to be used in interventional cardiology [5], and in gastroenterology for the
detection and diagnosis of tumors [6,7]. OCT can be a useful tool for noninvasive imaging of brain tissues
[8,9]. OCT also shows promise in dermatology to improve the diagnosis process of skin lesions [10].
In the field of dermatology, high spatial resolution imaging is necessary to resolve subtle morphological
changes in skin tissues resulting from the early progression of lesions. Since the introduction of OCT about
30 years ago, significant progress has been achieved in the spatial resolution of OCT images. The axial
resolution in OCT is governed by the temporal coherence of the illumination light source [2]. Improvement
in the axial resolution of OCT images has been achieved through the emergence of efficient broadband light
sources. Axial resolutions down to ~ 1 µm have been achieved with mode-locked lasers [11] and more
recently with supercontinuum lasers [12-14]. The lateral resolution of the OCT images depends on how the
light beam is focused on the sample [2]. Depending on whether the signal is acquired as a function of the
optical frequency (in frequency/Fourier-domain OCT, referred to as FD-OCT) or as a function of time (in
time-domain OCT, referred to as TD-OCT), the beam focusing constraints differ, which has an impact on
the lateral resolution.
In TD-OCT, the reflectivity profile of the sample as a function of depth (an A-scan) is acquired by scanning
the sample depth [1,2]. In FD-OCT, the reflectivity profile of the sample as a function of depth is acquired
without scanning the depth, by measuring the spectrum of the interferometric signal [2]. In both TD-OCT
and FD-OCT, a B-scan (a vertical section image) is then obtained by lateral scanning of the light beam to
acquire several adjacent A-scans. En face images can also be obtained by scanning the beam in two lateral
directions. En face imaging has been implemented in both TD-OCT and FD-OCT [15].
2
FD-OCT is superior to TD-OCT in terms of acquisition rate and detection sensitivity
[16], but has
shortcomings including a limited lateral resolution
[17]. Since all points in the depth range in the sample
must be in focus simultaneously, a depth of field (DOF) at least equal to the depth range is needed, which
limits the beam focusing. Several approaches have been reported to try to circumvent this limitation, i.e.
improve the lateral resolution without sacrificing the imaging depth. One approach consists in increasing
the DOF using Bessel beams produced by axicon lenses [17-20] or coaxially focused multimode beams [20]
or apodized beams [21, 22]. Computational methods have also been reported, such as interferometric
synthetic aperture microscopy [23,24] or digital refocusing [25,26]. Another approach consists in
overlapping and fusing several B-scans acquired at different depths by using a phase plate [27] or multiple
light beams focused at different depths, in order to image over a depth range larger than the DOF [28,29].
This approach has also been combined with Bessel beam illumination [30], or using several beams with
different wavelengths associated to different depths [31,32]. The B-scans to combine can also be obtained
using a single beam refocused at different depths, a method referred to as C-mode scanning [33] or Gabor-
domain OCT [34,35]. Let us note that this approach has also been combined with digital refocusing to
improve its throughput [36].
Unlike FD-OCT, TD-OCT offers the possibility to continuously adjust the focus as a function of depth,
making TD-OCT more suitable for producing images with high lateral resolution. Dynamic focus tracking
in TD-OCT with free-space optics has been reported, but the tracking rate was slow [37,38]. A
microelectromechanical systems (MEMS) mirror was designed for high-speed dynamic focus tracking, but
without demonstration of the imaging capability in vivo [39]. Another approach consists of acquiring a
sequence of images by gradually shifting the focus onto the sample and then fusing together the in-focus
imaging zones [40]. This process results in a trade-off between lateral resolution and image acquisition
speed. Another method is to collect multiple foci simultaneously with a multifocus fiber tip array [41].
Despite these advances, however, high lateral resolution TD-OCT imaging using dynamic focusing remains
difficult since a high tracking speed is required.
Line-field confocal optical coherence tomography (LC-OCT) is a recently invented imaging technology that
can produce images without the lateral resolution limitation of FD-OCT and without the speed limitation of
conventional dynamically-focused TD-OCT. This paper reports on the latest LC-OCT prototype. Compared
to the previous prototype capable of acquiring either horizontal (en face, x×y) section images at an adjustable
depth or vertical section images (B-scan, x×z) at an adjustable lateral position [42], three-dimensional
images (x×y×z) can now be acquired. The technical characteristics of the different imaging modes are
described. The device is now in the form of a portable probe. The handle of the probe has a button and a
scroll wheel allowing the user to control the imaging modes. Three-dimensional images of skin tissues are
presented to demonstrate the potential of LC-OCT in dermatology.
2. The LC-OCT technique
2.1 General principle and interest
Line-field confocal optical coherence tomography (LC-OCT) is an imaging technique based on TD-OCT
with illumination of the sample with a line of light and detection using a line camera [42-47]. This differs
from conventional TD-OCT where the sample is illuminated point by point and a vertical section image is
obtained from several A-scans acquired sequentially. In conventional TD-OCT, the scan of the sample depth
is repeated after each A-scan acquisition, which requires depth scanning at high speed to obtain an image in
real-time. In LC-OCT, all the A-scans of a vertical section image are acquired in parallel. The speed of the
depth scan in LC-OCT can therefore be reduced compared to point-scanning TD-OCT, without increasing
the image acquisition time. Producing a vertical section image in real time with LC-OCT requires scanning
the sample depth at a frequency of a few Hertz only. Obtaining a vertical section image at the same frame
rate in point-scanning TD-OCT requires depth scanning at a frequency increased by a factor equal to the
number of A-scans (factor of 2048 compared to LC-OCT using a line camera with 2048 pixels). The
significantly slower depth scan in LC-OCT makes it possible to dynamically focus a microscope objective
at a speed suitable for the real-time acquisition of high-resolution vertical section images. In LC-OCT,
vertical section images can thus be obtained without the lateral resolution limitation of FD-OCT and without
the speed limitation of conventional dynamically-focused TD-OCT. In addition, with the recent LC-OCT
prototypes, it is also possible to obtain high-resolution horizontal section images by scanning the
illumination line laterally on the sample [47].
3
Figure 1. Schematic diagram of the latest LC-OCT prototype.
2.2 Latest prototype
The experimental setup of the latest LC-OCT prototype is shown schematically in Figure 1. It is based on a
Linnik-type interferometer with a piezoelectric (PZT) stage for depth (z) scanning and a mirror
galvanometer for lateral (y) scanning. The reference surface of the interferometer is mounted on a
piezoelectric (PZT) chip that can oscillate to generate a phase modulation. A supercontinuum laser is used
as a broadband light source at a detected central wavelength of about 750 nm. A cylindrical lens is employed
to generate a line of light that is focused on the sample. The image of this line is projected on a line-scan
camera. All the elements used for illumination and imaging (laser, optics, camera) are the same as those of
the previously reported LC-OCT system [47]. The microscope objectives are also the same (20x, numerical
aperture of 0.5). Silicone oil is used as an immersion medium with a refractive index of 1.4, close to the
mean refractive index of human skin. Glass plates are placed in both arms of the interferometer under the
microscope objectives for subject stabilization and for providing a low reflectivity (3.5%) reference surface.
The whole reference arm of the interferometer and the microscope objective in the sample arm (elements in
the dashed frame in Figure 1) are mounted on the piezoelectric stage for vertical section imaging. The
general operation of the device and the image display are controlled by a new C++ software. The device is
now in the form of a portable probe (see Figure 2). The dimensions of the probe are 215 mm (length) × 120
mm (width) × 208 mm (height) and the weight is 1.2 kg. A button on the probe handle allows the user to
record an image in both real-time imaging modes (vertical or horizontal). A double-click on the button
enables to switch from one real-time imaging mode to another. The probe handle is also equipped with a
scroll wheel, which allows the user to adjust the lateral/depth positions in the horizontal/vertical real-time
imaging modes. When clicked, the scroll wheel also allows the user to choose between video recording and
three-dimensional acquisition.
4
Figure 2. LC-OCT probe and cart used by a dermatologist in a clinical setting.
3. The three imaging modes
Compared to the previous LC-OCT prototype capable of acquiring either horizontal (en face, x×y) section
images at an adjustable depth or vertical section images (B-scan, x×z) at an adjustable lateral position [42],
three-dimensional images (x×y×z) can now be acquired with the new device. The operating principle and
the technical characteristics of the three imaging modes are described below.
3.1 Vertical section imaging
A vertical section image is acquired by activating the PZT stage to scan the sample depth. The displacement
of the PZT stage is analogically controlled by a custom electronic board that sends a periodic driving current
(see Figure 3). The PZT oscillates at a frequency of
f
!"#
= 8$Hz
according to asymetrical sawthooths. The
amplitude of the oscillation is 500 µm, but due to the non-linearity of the PZT stage displacement at the
edges of the sawtooths, the images are acquired over only 80% of the total amplitude of the oscillation. The
vertical section images are thus acquired over an effective depth range of Z = 400 µm. The camera is
synchronized with the oscillation of the PZT. The camera frame rate is set at 70 kHz, so that the step between
two consecutive lines corresponds to a displacement of the PZT of
δ
= 71 nm, i.e. to a phase shift of p/2.
A stack of
Z/δ = 5,600
lines is acquired during each positive slope of the depth scan. A vertical section
image is obtained by processing the acquired stack using a five-frame fringe envelope detection algorithm
[48]. Acquisition and processing of stacks is repeated continuously during the round-trips of the PZT stage.
The images are displayed in real-time at 8 frames/s in logarithmic scale with auto-adjusted contrast after
being appropriately rescaled. The size of each vertical section image is 2048 ´ 680 pixels (x´z),
corresponding to a field of view of 1.2 mm ´ 0.4 mm (x´z).
0.8$T$
Current$(a.u.)$
Times$(s)$
0.1$ 0.2$
T$
0.3$0.0$
5
Figure 3. Periodic current driving the oscillation of the PZT stage. The asymmetric triangle signal has a frequency of
f
!"#
= 1/T = 8'Hz
and a duty cycle of 80%. Images are acquired only during the slow positive ramps.
In the vertical section imaging mode, the scroll wheel on the probe handle allows the user to modify the
value of a direct current sent to the mirror galvanometer by the electronic board. This enables to laterally
navigate within the sample being imaged, in real-time. The accuracy of the lateral position is determined by
the noise in the rotation of the mirror galvanometer. In practice, the position of the laser line focused on the
sample does not fluctuate more than the lateral resolution of the imaging system. Therefore, the positioning
accuracy can be considered to be better than 1 µm. This is of course a much higher positioning resolution
than can be achieved by moving the probe manually.
3.2 Horizontal section imaging
In the horizontal section imaging mode, the mirror galvanometer oscillates according to asymmetrical
sawtooths, at a frequency
f
$%&'(
= 8$Hz
(see Figure 4) to scan the illumination line on the sample over a
field of 500 µm. Let us note that the scanning field of 500 µm represents 95% of the total scanning amplitude
of the mirror galvanometer, to avoid non-linearities at the edges of the sawtooths. The mirror galvanometer
is analogically controlled by the same custom electronic board as the PZT stage. The camera frame rate is
set to
f
)%*
= 100$kHz
. Two consecutive lines acquired by the camera are thus separated by a lateral
distance of 47 nm. A stack of
10,600
lines is acquired during each positive slope of the lateral scan. The
reference surface of the interferometer oscillates sinusoidally to generate a phase modulation. The current
driving the oscillation is generated by the same custom electronic board that controls the PZT stage and
mirror galvanometer. It is amplified before being sent to the PZT chip. A sinusoidal phase-shifting algorithm
is used to calculate each line of the horizontal section image from an algebraic combination of five
consecutive lines acquired by the camera [48]. The frequency of the phase modulation and its amplitude are
set empirically at 8 kHz and 1 µm respectively to optimize the image quality. Acquisition and processing is
repeated continuously during the round-trips of the mirror galvanometer. The horizontal section images are
displayed in real-time at 8 frames/s in logarithmic scale with auto-adjusted contrast after being appropriately
rescaled. The size of each horizontal section image is 2048 ´ 850 pixels (x´y), corresponding to a field of
view of 1.2 mm ´ 0.5 mm (x´y).
Figure 4. Periodic current driving the oscillation of the mirror galvanometer. The asymmetric triangle signal has a
frequency of
f
$%&'(
= 1/T = 8'Hz
and a duty cycle of 90%. Images are acquired only during the slow positive
ramps.
In the horizontal section imaging mode, the scroll wheel on the probe handle allows the user to control the
value of a direct current sent to the PZT stage by the electronic board. This makes it possible to control at
what depth the sample is imaged, between 0 and 500 µm, in real time and with an accuracy determined by
the resolution of the PZT stage which is much better than the axial resolution of the imaging system of ~
1 µm.
3.3 Three-dimensional imaging
Three-dimensional images can be produced from a stack of horizontal section images acquired at successive
depths. In the three-dimensional imaging mode, horizontal section images are continuously acquired while
a ramp signal is sent by the electronic board to the PZT stage to displace it from 0 to 500 µm. The ramp
0.9$T$
Current$(a.u.)$
Times$(s)$
0.1$ 0.2$
T$
0.3$0.0$
