IEEE JOURNAL OF SELECTED TOPICS IN QUANTUM ELECTRONICS, VOL. 25, NO. 1, JANUARY/FEBRUARY 2019

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Application of Scan-less Two-Dimensional Confocal
Microscopy Based on a Combination of Confocal Slit
With Wavelength/Space Conversion
Eiji Hase , Takeo Minamikawa , Shuji Miyamoto, Yasuhiro Mizutani, Tetsuo Iwata,
Hirotsugu Yamamoto, and Takeshi Yasui

Abstract—Confocal laser microscope (CLM) has been widelyused in the fields of non-contact surface topography, biomedical imaging, and other applications, as confocality gives twodimensional (2-D) optical-sectioning or three-dimensional (3-D)
imaging capability with depth selectivity. The combination of linefocused CLM with one-dimensional spectral encoding CLM enables us to obtain the 2-D confocal image without the need formechanical scanning. The so-called scan-less 2-D CLM is a unique
imaging modality; however, there have been no attempts to apply
this to practical applications. In this paper, we have constructeda scan-less 2-D CLM with an image acquisition time of 0.23 ms,lateral resolution of 1.2 µm, depth resolution of 2.4 µm, and haveapplied it to different kinds of applications to evaluate its practicalpotential.
Index Terms—Biomedical imaging, microscopy.

I. INTRODUCTIONCONFOCAL laser microscopy (CLM) [1] has been widelyused in the field of biomedical imaging, non-contact sur-

Manuscript received April 6, 2018; revised August 23, 2018 and September 4,
2018; accepted September 5, 2018. Date of publication September 10, 2018; dateof current version October 4, 2018. This work was supported in part by grants for
the Exploratory Research for Advanced Technology (ERATO) MINOSHIMAIntelligent Optical Synthesizer (IOS) Project from the Japanese Science andTechnology Agency under Grant JPMJER1304 and in part by a grant-in-aid
for Scientific Research (A) from the Ministry of Education, Culture, Sports,
Science, and Technology of Japan under Grant 15H02026. (Corresponding
author: Takeshi Yasui.)E. Hase and S. Miyamoto were with the Graduate School of Advanced Technology and Science, Tokushima University, Tokushima 770-8506,
Japan, and also with JST, ERATO, MINOSHIMA Intelligent Optical Synthesizer Project, 2-1, Tokushima 770-8506, Japan (e-mail:, hase@spring8.or.jp;
shuujimiyamoto@gmail.com).
T. Minamikawa, T. Iwata, and T. Yasui are with the Graduate School of Technology, Industrial and Social Sciences, Tokushima University, Tokushima 7708506, Japan, and also with JST, ERATO, MINOSHIMA Intelligent Optical Synthesizer Project, 2-1, Tokushima 770-8506, Japan (e-mail:, minamikawa.takeo@tokushima-u.ac.jp; iwata@tokushima-u.ac.jp; yasui.takeshi@tokushima-u.
ac.jp).
Y. Mizutani is with the Graduate School of Engineering, Osaka University,
Osaka 565-0871, Japan, and also with JST, ERATO, MINOSHIMA Intelligent
Optical Synthesizer Project, 2-1, Tokushima 770-8506, Japan (e-mail:, mizutani@mech.eng.osaka-u.ac.jp).
H. Yamamoto is with the Center for Optical Research and Education, Utsunomiya University, Tochigi 321-8585, Japan, and also with JST, ERATO,MINOSHIMA Intelligent Optical Synthesizer Project, 2-1, Tokushima 7708506, Japan (e-mail:,yamamoto@opt.utsunomiya-u.ac.jp).
Color versions of one or more of the figures in this paper are available online
at http://ieeexplore.ieee.org.
Digital Object Identifier 10.1109/JSTQE.2018.2869615

face topography, and other applications thanks to the benefitof the noninvasiveness, the optical sectioning capability, andthe stray light elimination. Usual CLMs are based on a pointscanning measurement. This is because a pinhole for a point
light-source, a focal point of the objective lens, and a pinhole
before a detector must have a conjugate relationship to eachother. Therefore, a two-dimensional (2D) image has to be acquired by a mechanical scanning of the focal point while maintaining the conjugate configuration. The mechanical scanningby a galvanometer mirror, polygon mirror, or MEMS (Micro
Electro Mechanical System) mirror leads to the image acquisition rate of several to a few tens frames per seconds (fps)[2]–[4]. The combination of a Nipkow disk with a micro-lensarray boosts the image acquisition rate up to 1000 fps [5], [6].
However, these mechanical scanning systems are susceptible
to environmental disturbances, such as mechanical or acoustic
vibration. Requirement for the stable experimental condition,such as an optical table, often limit the application of CLM inside the lab. To widely expand its application fields outside thelab, the mechanical scanning must be omitted from CLM.
Line-field CLM [7] is a one interesting approach to eliminate
the one-dimensional (1D) mechanical scanning. In this method,
the laser beam is focused on a sample as a line beam, and theline beam reflected from a sample passes through a confocal
slit before the detection. This slit gives the confocality for thewhole line image, and then the reflected light is captured as theconfocal 1D image in real time by a 1D imaging sensor (line sensor). However, this approach still needs additional mechanicalscanning of the line beam across the line illumination. Anotherinteresting approach is the use of 1D spectral encoding (1D-SE)
CLM [8], [9]. In 1D-SE CLM, a rainbow line beam is formed
by a combination of a broadband-spectral light and a diffraction
grating, and then are irradiated on the sample as the line beam of
the rainbow spectrum. This result in an encoding of 1D image
information onto the rainbow spectrum. Finally, the 1D image
is decoded from the spectrum acquired by a multi-channel spectrometer without the mechanical scanning. However, as well as
the line-field CLM, additional 1D mechanical scanning needs
to acquire the 2D image.
If the line-field CLM and the 1D-SE CLM are orthogonallycombined, 1D image information of each coordinates are individually and orthogonally developed in the 2D plane. By using
the multi-channel spectrometer equipped with the 2D imaging

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Fig. 1.

IEEE JOURNAL OF SELECTED TOPICS IN QUANTUM ELECTRONICS, VOL. 25, NO. 1, JANUARY/FEBRUARY 2019

Principle of operation of the proposed method.

sensor, the confocal 2D image can be decoded from a spectralline image, composed of 1D spatial dimension and 1D spectral
dimension, without the need for any mechanical scanning. The
image acquisition rate of this approach is limited by not the mechanical scanning but the frame rate of the 2D imaging sensor,
which is achieved up to Mfps in the state-of-art complementary
metal oxide semiconductor (CMOS) camera.
Such scan-less 2D CLM has a high potential as CLM due tothe fast image acquisition rate and the high robustness to theenvironmental disturbances [10]. However, its potential of confocal 3D or volume imaging has not been fully investigated fromthe viewpoint of practical applications. In this paper, we expand
the scan-less 2D CLM into confocal 3D imaging by help of apiezoelectric-transducer (PZT) scanner for objective lens, andthen apply it for three applications, namely biomedical imaging of tissue specimen, plant monitoring, and non-destructive
inspection of electronic board.
II. PRINCIPLE
Figure 1 shows a principle of operation for the scan-less 2D
CLM based on a combination of line-field CLM with 1D-SE
CLM. In this figure, while the optical axis is defined as a Z coordinate, the horizontal and vertical directions in the 2D plane normal to the optical axis are respectively corresponding to X and Y
coordinates. The X and Y coordinates are used for each 1D imaging by 1D-SE and line-field CLMs, respectively. The circularbeam shape of a broadband-spectral laser light is converted intoa line shape along the Y coordinate by the beam-shaping optics,
composed of a cylindrical lens and a lens. The wavelength disperser disperses each wavelength component at different angle
along the X coordinate depending on the wavelength. This results in a formation of “vertical rainbow" composed of the same
wavelength component along the Y coordinate and the different
wavelength components along the X coordinate. After passing
thorough an objective lens, the demagnified vertical rainbow isilluminated on a sample. This enables to encode the 2D information of the sample on the vertical rainbow at one-shot.
The back-reflected vertical rainbow from the sample inversely propagates the same optical components, and each wavelength component is spatially overlapped again. The resulting

Fig. 2. Experimental setup for scan-less 2D CLM. CL: cylindrical lens, BS:beam splitter, G: grating, L: lens, OL: objective lens, M: mirror.

line-shape beam passes through a confocal slit for the confocality of the 1D image along the Y coordinate. Then, the spectralline image, or the spatio-spectral image, is measured as an image with the wavelength dimension along the X coordinate andthe spatial dimension along the Y coordinate by a multi-channel
spectrometer equipped with a 2D imaging sensor. Since thisspatio-spectral image is corresponding to the demagnified vertical rainbow on the sample, the confocal 2D image of the sample can be decoded without the need for mechanical scanningsystem.
III. EXPERIMENTAL SETUP
Figure 2 shows a schematic diagram of the experimental setup. A broadband-spectral laser (Spectra-Physics, Inc.,
Santa Clara, CA, USA, InSight DeepSee, λC = 780 nm, ∆λ =10 nm, Pm ean = 900 mW) was used for the light source of
scan-less 2D CLM. The output light was line-focused ona diffraction grating (G, Thorlabs, Inc., Newton, NJ, USA,
GR25-1208, groove density = 1200 groove/mm, blaze wavelength = 750 nm) by a pair of a cylindrical lens (CL, f =200 mm) and a lens (L1, f = 200 mm) after passing through aspatial filter and a beam expander (BE). The direction of the focused line on the grating was orthogonal to the groove direction.
Each wavelength component of the light source was diffracted
at different angles depending on the wavelength and then was
focused on the sample by the combination of relay lenses (L2,f = 200 mm; L3, f = 200 mm) and the objective lens (OL,NA = 0.95, Magnification = 60). This results in the illumination of the vertical rainbow with a field of view (FOV) of 68 µm
× 156.2 µm on the sample plane. Although the vertical rainbow
is illustrated as a visible rainbow color for easy understanding, its actual color is considerably monochromatic in the nearinfrared region. The reflected or scattered light at the sample
inversely propagated the same optical path including the objective lens, the relay lenses, and the diffraction grating, leading
to the inverse process of 1D-SE. Such the inverse 1D-SE process recovers the horizontally line-focused light on the grating
again. After reflected by a beam splitter (BS), the horizontally

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Fig. 4. Depth profile of scan-less 2D CLM. Red and green plots show theexperimental depth profile with and without the confocal slit. Red and green
lines indicate the corresponding to curve fitting results with a Gaussian function.

Fig. 3. (a) Scan-less 2D confocal image of the test chart (image size =156.2 µm × 68.0 µm). Green and blue lines show the extracted portion forevaluation of the lateral resolution in X and Y directions. Differential curves ofthe extracted edge profile along (b) X direction and (c) Y direction. The plots
indicate the experimental value, whereas the line indicate the curve fitting resultswith a Gaussian function.

line-focused light was converted as the vertically line-focusedlight and then passes through the incident slit of a multichanneled spectrometer (Princeton instruments, Inc., Trenton,
NJ, USA, Acton SP2300) equipped with a CMOS camera (Thorlabs, Inc., Newton, NJ, USA, DCC1545M, 1280 × 1024 pixels,
pixel size = 5.2 µm square, bit depth = 10 bit, frame rate =22 fps). The incident slit of the multi-channel spectrometer canbe used for the confocal slit for the line-field CLM, too. Thus,
the width of the incident slit determines not only the spectral
resolution but also the confocality for the X and Z coordinates.
We here set the slit width to be 60 µm by taking into account of
the central wavelength of the light source, the NA of the objective lens, and the magnification of the lens before the incident
slit. This slit width is corresponding to the spectral resolution of
0.1 nm. In this way, the spectral line image was acquired by thespectrometer and the camera. Scanning the depth position of asample with the PZT-driven OL scanner (Physik Instrumente,GmbH, Karlsruhe, Germany, P-721, travel range = 100 µm)
enables us to acquire 3D confocal imaging of the sample.
IV. RESULTS
A. Imaging Performance
We first evaluated the lateral resolution for the X and Y coordinates in the constructed scan-less 2D CLM. A commercialized 1951 USAF resolution test chart with a negative pattern(Edmond Optics, Barrington, NJ, USA, #38-256) was placedat the focal position of the objective lens as a test sample.
Fig. 3(a) shows the confocal 2D image of the test chart (image size = 156.2 µm × 68.0 µm, exposure time of CMOS
camera = 0.23 ms) measured by the scan-less 2D CLM system.
In this image, X and Y coordinates were corresponding to theline images obtained by 1D-SE CLM and the line-field CLM,
respectively. The fringe pattern along the horizontal directionin Fig. 3(a) is due to the interference between the transmitted

Fig. 5. Reflection imaging of the two-layered scotch tape. (a) Schematic
drawing of the two-layered scotch tape. A blue rectangle is the measured portion.
(b) Reflection image at objective position = 0 µm. (c) Reflection image at
objective position = 45.1 µm (image size = 156.2 µm × 68.0 µm).

light and the multiple reflection light in the beam splitter. We
extracted the amplitude profile across the pattern edge along
a green line and a blue line in Fig. 3(a), and then calculated
their differential curves [see green plots and blue plots in Figs.
3(b) and 3(c)]. These plots were considerably noisy due to thelow signal-to-noise ratio (SNR). To reduce the influence of suchnose in the profile, we first calculated the moving average of thedifferential curves in Figs. 3(b) and 3(c), and then performed
the curve fitting analysis using a Gaussian function [see a green
line and a blue line in Figs. 3(b) and 3(c)]. We here defined
the lateral resolution in X and Y coordinates (δX and δY ) as afull width of half maximum (FWHM) for the differential curvesof them. The resulting lateral resolution was determined to be
1.2 µm for δX and 1.2 µm for δY . From the diffraction limit oflight, theoretical δX and δY values were given by [7], [8], [11]
λ
(1)
NA
0.61λ
δY =
(2)
NA
where λ is a typical wavelength of light and NA is a numericalaperture of OL. Due to use of the confocal slit with the confocality along the X direction and without the confocality along
the Y direction, the theoretical δX value was smaller than thetheoretical δY value. From λ = 780 nm and NA = 0.95 in thepresent system, theoretical δX and δY values were estimated toδX = 0.48

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IEEE JOURNAL OF SELECTED TOPICS IN QUANTUM ELECTRONICS, VOL. 25, NO. 1, JANUARY/FEBRUARY 2019

0.4 µm and 0.5 µm, respectively. We have to further consider
the contribution of the spectral resolution in the spectrometerto the X lateral resolution because the X-line image is encodedon the optical spectrum in 1D-SE CLM. X dimension of FOV(= 156.2 µm) in Fig. 3(a) was corresponding to the spectral
range of 773 nm to 788 nm with a spectral resolution of 0.1 nm.
Therefore, the X lateral resolution determined by the spectral
resolution, δX λ , is estimated to be 0.5 µm. Since the theoretical
δX value was larger than the δX λ value, the former is a dominantfactor of the X lateral resolution in the present system. In thisway, the lateral resolution of the system is mainly determinedby the diffraction limit in the same manner as usual optical
microscopy. Difference of lateral resolution between the theoretical value and the experimental value is due to the imperfectly
filling NA in the objective lens and/or the residual aberration.
We next evaluated the depth resolution δZ along Z coordinate.
To this end, we measured the total signal intensity for 2 pixels
by 2 pixels around the center of image when the Z positionof the sample was moved at a step of 2 µm within the rangeof −18 µm to +18 µm. Red plots in Fig. 4 shows the depth
profile of the signal intensity with respect to Z position. For
comparison, the depth profile is indicated as green plots in thesame graph when the confocal slit was fully opened. Comparison
between them clearly indicated the sharp confocality along thedepth direction. From the curve fitting analysis with a Gaussianfunction, the experimental δZ value was 2.4 µm from FWHM ofthe red depth profile. Let here us consider theoretical δZ value
given by [7], [8], [11]λ√δz = 0.88
n − n2 − N A2

(3)

where n is a reflective index of the medium between OL and thesample. Substituting 780 nm for λ, 0.95 for NA, and 1 for n, we
obtained δZ = 1.0µm. Difference of depth resolution between
the theoretical value and the experimental value is due to theimperfectly filling NA in the objective lens and/or the residual
aberration similar to the lateral resolution.
We next performed depth-resolved confocal imaging, or volume imaging, of a standard sample with known dimension. toevaluate the imaging performance of the proposed system. We
here used a transparent scotch tape (thickness ! 46 µm) as astandard sample with known thickness. To make a two-layered
structure, two pieces of scotch tapes were attached orthogonallyto each other at a slide glass as shown in Fig. 5(a). Movie 1
shows a series of depth-resolved 2D confocal images when the
Z position of OL was scanned within the depth range of around
50 µm at 1.4 Hz. Fig. 5(b) and 5(c) show two typical images(camera exposure time = 21 ms, frame rate = 18 fps) at different
Z positions (Z = 0 µm and 45.1 µm), respectively. While thesurface of the first-layer scotch tape was focused at Z = 0 µm,
that of the second-layer scotch tape was focused at Z = 45.1 µm
Difference of Z position between them was 45.1 µm, which isin good agreement with the specification of this tape. In thisway, the depth-resolved confocal 2D image correctly reflects
the 3D surface profile of the sample. The image contrast of theFigs. 5(b) and (c) were sufficient to discriminate both layersof the transparent tape. Considering the high transparency inFig. 6. (a) Optical photograph of a tomato leaf. (b) Microscopic image ofthe tomato leaf (Magnification = 20, NA = 0.42). (c) Sketch of the leaf crosssection. (d) Depth-resolved 2D confocal images of the tomato leaf (image size
= 156.2 µm × 68.0 µm).

biological samples such as tissues and cells, this result implies
that the proposed microscopy has a potential for 3D imaging ofpractical biomedical applications.
B. Observation of Plant Leaf
Non-invasive observation of plant leaf is required for healthmonitoring of crops because dynamics of the plant leaf isclosely related with the activity of living crops [12]. For example, stoma plays an important role to maintain internal
environment by continuously exchanging gases with the atmosphere and diffusing water vapor through stoma for transpiration [13]. Therefore, opening and closing of stoma is agood indicator for the plant growth and health. One potential
application of the scan-less 2D CLM is in an observation ofplant leaf in situ. As a demonstration, we here used a tomato
leaf as a plant sample. Fig. 6(a) and 6(b) shows the opticalphotograph of the sample and its microscopic image (image
size = 156.2 µm × 68.0 µm). Although the microscopic image
was obtained by a normal optical microscope (Magnification= 20, NA = 0.42), it is difficult to observe the distribution of
stomata in the tomato leaf due to the non-confocality. Figure6(c) shows a sketch of leaf cross section. We here tried to acquire depth-resolved 2D confocal images from the side of upper
epidermis. Movie 2 shows a series of depth-resolved 2D confocal images when the Z position of OL was scanned within thedepth range of 40 µm at 0.5 Hz. Fig. 6(d) shows three typicalimages at different Z positions (Z = 0 µm, 10 µm, and 30 µm).
At Z = 0 µm, as the leaf surface was out of focus, no structureappeared in the image. At Z = 10 µm, while guard cells werefocused as bright regions, the stoma appeared as a black regionof vertical oval inside guard cells, indicating the stoma opening.
At Z = 30 µm, upper epidermis and/or palisade mesophyll werevisualized as compartments. In this way, scan-less 2D CLM

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Fig. 7. (a) HE stained image of a rat’s skin specimen. (b) Magnified image(image size = 0.84 mm × 0.28 mm) of a blue rectangle region in Fig. 7(a).
MC; muscle cell, FCT; fibrous connective tissue. Scan-less 2D confocal images(image size = 1.18 mm × 0.28 mm) of the rat’s skin (c) without and (d) withthe confocal slit.

enables us to visualize 3D structure of leaf plant with high contrast images in real time.
C. Imaging of Biological Tissue
Staining method is a powerful tool in histopathology [14].
For the correct diagnosis with histopathology, a thin layer of lesion (typical thickness ! 5 µm) has to be appropriately extracted
from a thick specimen of biopsy tissue before the staining. However, if a position of the sliced specimen is inappropriate, it might
be the wrong diagnosis. Optical-sectioning imaging capability
with the micrometer depth selectivity in the scan-less 2D CLM
has a potential to find an application as a sample pre-processing
tool in the histopathology.
To investigate a potential for such application, we demonstrated imaging of biological tissues. In this demonstration, toenlarge FOV, a lower-NA OL (NA = 0.25) was used for thescan-less 2D CLM. Using this system, δx of 13.9 µm, δY of
2.4 µm, and δZ of 29.4 µm were achieved in the image size of
1.18 mm × 0.28 mm. Fig. 7(a) shows the Hematoxylin-Eosin(HE) stained image of a sliced rat’s skin sample (thickness= 5 µm). The enlarged view of the blue rectangle region in
Fig. 7(a) is show in Fig. 7(b). The HE staining method enables
the selective visualization of muscle tissue and extracellularmatrix as pink color. In Fig. 7(b), muscle cells enveloped by
fibrous connective tissue were clearly observed in the whole ofimage. We next measured the same sample by using the proposed method. Figures 7(c) and 7(d) show a comparison of thescan-less 2D image without and with confocal slit. While thetissue was visualized with some blur in Fig. 7(c) due to nonconfocallty, Fig. 7(d) the enhances the image contrast due to thestray light elimination rather than the depth selectivity in theconfocality. More importantly, the image quality in Fig. 7(b) issimilar to that in Fig. 7(d); each unit of the muscle fiber bundles
were clearly distinguished in the image.
D. Non-Destructive Inspection of Electric Board
Finally, we measured an electric circuit board by the same
lower-NA OL. as a demonstration for the non-destructive
inspection in industrial filed. Figure 8(a) shows an opticalphotograph of the measured sample. The measured region wasFig. 8. (a) Optical photograph of an electric circuit board. The blue rectangle
shows the measured region. (b) Depth-resolved scan-less 2D confocal images
at z = −150, 450, and 600 µm. (c) 3D confocal image reconstructed by a seriesof depth-resolved scan-less 2D confocal images.

the electrode of the chip capacitor. We acquired a series of 2D
confocal images (image size = 1.18 mm by 0.28 mm, exposure
time = 9.15 ms, frame rate = 22 fps) when the sample position
was axially scanned at a step of 50 µm from Z = −150 µm to
Z = 900 µm. Figure 8(b) shows typical 2D confocal images at
two different Z positions. At Z = −150 µm, no signal appeared
due to out of focus for the sample. The image signal started
to appear from Z = 0 µm, indicating the sample surface (notshown). The surface structure of the electrode of the chip capacitor was clearly observed with high image contrast at Z =450 µm, whereas that of the electric board was observed at Z =600 µm. We then reconstructed the volume image of this sample
from a series of the depth-resolved 2D image. Figure 8(c) andMovie 3 show the reconstructed volume image, indicating thedetailed 3D structure of the electric board. The total acquisition
time of this volume image was 525 ms.
V. DISCUSSION
We achieve the scan-less 2D CLM with the 0.23-ms acquisition time, the 1.2-µm lateral resolution, and the 2.4-µm depth
resolution. We here compare this performance with that of theprevious researches. Table I summarizes the imagining performance of the proposed method and other CLMs. There is no
significant difference of lateral resolution and depth resolution
among them because they depend on the diffraction limit of
OL rather than the scanning method or the scan-less method.
The two former methods have the confocality for X, Y, and Z
axes while the three later methods have the confocality for X
and Z axes; however, the lateral resolution for X and Y axes
were almost similar to each other. Advantage or disadvantageof those methods are highlighted in the frame rate. Although the7101407

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TABLE IIMAGING FEATURE OF SEVERAL CLM APPROACHES

between the space and the wavelength. Conversion of the excitation light into the fluoresce by a sample cancels the spectral information and hence lose the one-to-one correspondence.
However, use of wavelength sweeping light [16] or an intensitymodulated broadband-spectrum light [17] enables the scan-lessconfocal fluorescent imaging.
VI. CONCLUSIONS

four former methods can achieve higher frame rate than the proposed method, the requirement for the mechanical scanning may
hamper the practical application of CLM mentioned in the introduction. The frame rate of 2D image in the proposed method
is limited by the camera frame rate, and the present frame rate
remained at 22 fps by use of usual CMOS camera. However,
one can use mega-fps CMOS camera now [15]. In other words,
the proposed method has a potential to achieve mega-fps CLM.
Such high frame rate cannot be achieved by other CLMs in principle. This comparison implies a high potential of the proposedsystem for practical CLM applications.
We next discuss the acquisition rate of volume imaging. In
usual CLM, the acquisition rate is limited by the lateral scanning,the depth scanning, and/or the camera frame rate. Thanks to
2D scan-less capability based on a combination of the linefield CLM and the 1D-SE CLM, the present system does notneed the lateral scanning. The acquisition rate of the present
system was limited by the PZT-driven OL scanner (scan rate
∼ a few Hz) rather than the camera frame rate (=22 fps). On
the other hand, the moderate image contrast was obtained evenwith the exposure time of 0.23 ms [see Fig. 3(a)]. If a faster
depth scanner and a higher-frame-rate camera are used for thescan-less 2D CLM, the acquisition rate of volume imaging will
be further increased. Fortunately, there is still space to increase
the scan rate of depth scanner and the frame rate of camera.
For example, usual PZT-driven OL scanners have a resonant
frequency of a few hundred Hz; use of a lightweight OL withthe limited stroke will further increase its depth scan rate. Also,
combination of OL with a focus-tunable lens will be useful forthe fast depth scanning. Regarding the camera frame rate, thestate-of-art CMOS camera can boost the frame rate up to 106 fps[15]. Use of these approaches enables us to achieve the videorate volume imaging of moving objects with micrometer spatial
resolution; this will find new applications not only in biomedical
application but also industrial inspection.
Finally, we discuss expansibility of the proposed system
for scan-less confocal fluorescent microscopy because thefluorescent microscopy is an important imaging modality inbiomedical application. Unfortunately, the present system cannot be directly applied for the scan-less confocal fluorescentimaging because it depends on the one-to-one correspondence

In this paper, we developed a scan-less 2D CLM by the combination of line-field CLM with the 1D-SE CLM, and further
expanded it to 3D confocal imaging, or volume imaging, by helpof a PZT-driven OL scanner. After the evaluation of the imaging
performance, we provided a proof-of-principle demonstration
for three potential applications. These demonstrations imply ahigh potential of the proposed system for these practical applications. If the scan-less 2D CLM can be integrated as a hand-heldor endoscope CLM by help of a compact light source (e.g., LED
or SLD) and fiber optics, its robust and simple characteristics
will find the clinical applications including the intraoperative
diagnosis.
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HASE et al.: APPLICATION OF SCAN-LESS TWO-DIMENSIONAL CONFOCAL MICROSCOPY

Eiji Hase was born in Tokushima, Japan, in 1989.
He received the B.S., M.S., and Ph.D. degrees inengineering from Tokushima University, Tokushima,
Japan, in 2012, 2014, and 2017, respectively.
From 2014 to 2017, he was a Research Associate with the JST, ERATO, MINOSHIMA Intelligent Optical Synthesizer Project. Since 2017, he has
been a Research Scientist with the Research and Utilization Division, Japan Synchrotron Radiation Research Institute, Sayo, Japan, and a Visiting Associate
Professor with the Graduate School of Technology,
Industrial and Social Sciences, Tokushima University. He is also with the JST,
ERATO, MINOSHIMA Intelligent Optical Synthesizer Project, 2-1, Tokushima,
Japan. His research interests include biomedical imaging and biomechanicalanalysis using femtosecond laser and synchrotron X-ray.
Dr. Hase was the recipient of The Optical Society of Japan Best Presentation
Award in 2016, The Japanese Society for Medical and Biological Engineering
Symposium Award in 2016, The International Society for Optical Engineering(SPIE) BiOS2016 in Photonics West 2016 Student Poster Awards in 2016, andSPIE Structured light 2018 Best Paper Award. He is a member of SPIE.

Takeo Minamikawa was born in Ibaraki, Japan, in
1983. He received the B.S., M.S., and Ph.D. degrees
in engineering from Osaka University, Osaka, Japan,
in 2006, 2008, and 2010, respectively.
From 2010 to 2013, he was with the Research Fellowship for Young Scientists, Japan Society for the
Promotion of Science. From 2013 to 2015, he was an
Assistant Professor with the Department of Pathology and Cell Regulation, Graduate School of Medical
Science, Kyoto Prefectural University of Medicine.
Since 2015, he has been an Associate Professor with
the Department of Mechanical Science, Division of Science and Technology,
Graduate School of Technology, Industrial and Social Sciences, Tokushima
University, Tokushima, Japan. He is also with the JST, ERATO, MINOSHIMAIntelligent Optical Synthesizer Project, 2-1, Tokushima, Japan. He is the authorof two book chapters, over 30 articles, is credited with more than 15 inventions,as well as the holder of three patents. His research interests include Raman
microspectroscopy and optical frequency comb.
Dr. Minamikawa’s was the recipient of The Japan Society for Precision
Engineering Best Presentation Award in 2016, The Japanese Society of Pathology Poster Presentation Award in 2015, and Funai Foundation for Information
Technology Research Award in 2011. He is a member of the Optical Society ofAmerica (OSA), the Japan Society of Applied Physics, the Optical Society ofJapan, the Laser Society of Japan, the Japan Society of Mechanical Engineers,
as well as the Japan Society for Precision Engineering.

Shuji Miyamoto was born in Tokushima, Japan,
in 1992. He received the B.S. and M.S. degrees inmechanical engineering from Tokushima University,
Tokushima, Japan, in 2015 and 2017, respectively.
From 2014 to 2017, he studied nonlinear optics under Prof. Takeshi Yasui, Tokushima University. Since
2017, he has been an Engineer with SCREEN Semiconductor Solutions Company Limited, Shiga, Japan.
He is also with the JST, ERATO, MINOSHIMA Intelligent Optical Synthesizer Project, 2-1, Tokushima,
Japan. His research interests include dual-opticalcomb spectroscopy and scan-less confocal microscope using broadband lightsource.
Mr. Miyamoto was a recipient of The Japanese Society for Medical andBiological Engineering Branch Conference Young Researcher Award in 2016.

7101407

Yasuhiro Mizutani received his Ph.D. degree in mechanical engineering from Tokyo university of Agriculture & Technology, Tokyo, Japan, in 2008. He
also received B.E. and M.E. degrees in nuclear engineering from Osaka University, in 1997 and 1999,
respectively.
From 1999 to 2003, he was a Researcher at Panasonic Corporation. Then, from 2003 to 2009, heworked as a Research Associate with the Departmentof Mechanical Systems Engineering, Tokyo University of Agriculture & Technology, Japan. In 2009, hejoined the University of Tokushima, Japan, in 2009 as an Associate Professor.
Since 2015, he has been working as an Associate Professor at Osaka University.
He is also with the JST, ERATO, MINOSHIMA Intelligent Optical Synthesizer Project, 2-1, Tokushima, Japan. He has authored or coauthored more than
100 papers in refereed international journals and conferences in these areas.
His research interests include interferometry, polarimetry, 3-D surface measurement, optical trapping, and 3-D lithography.
Dr. Mizutani is a member of SPIE and OSA.

Tetsuo Iwata received the Ph.D. degree in engineering from Osaka University,
Osaka, Japan, in 1984.
From 1984 to 1998, he worked at JASCO Corporation, Japan. He is currently aProfessor at the Graduate School of Technology, Industrial and Social Sciences,
Tokushima University, Tokushima, Japan. He is also with the JST, ERATO,MINOSHIMA Intelligent Optical Synthesizer Project, 2-1, Tokushima, Japan.
His research interests include fluorescent measurement and spectroscopy.

Hirotsugu Yamamoto received the Ph.D. degree in information science andtechnology from the University of Tokyo, Tokyo, Japan, in 2008.
From 1996 to 2014, he worked at Tokushima University, Japan. He iscurrently an Associate Professor at the Graduate School of Engineering, Utsunomiya University, Utsunomiya, Japan. He is also with the JST, ERATO,MINOSHIMA Intelligent Optical Synthesizer Project, 2-1, Tokushima, Japan.
His research interests include Information optics.

Takeshi Yasui received the first Ph.D. degree inengineering from the University of Tokushima,
Tokushima, Japan, in 1997, and the second Ph.D.
degree in medical science from the Nara Medical
University, Yagi, Japan, in 2013.
From 1997 to 1999, he worked as a Postdoctoral
Research Fellow in the National Research Laboratory of Metrology, Japan. He was with the Graduate School of Engineering Science, Osaka University
from 1999 to 2010, and was briefly with the University of Bordeaux I in 2007 and 2012, and with the
University of the Littoral Opal Coast in 2010 as an Invited Professor. He iscurrently a Professor at the Graduate School of Technology, Industrial and Social Sciences, Tokushima University, Tokushima, Japan and Vice Director of
Research Support in the same university since 2016, and an Invited Professor
at the Graduate School of Engineering Science, Osaka University, Toyonaka,
Japan. He is also with the JST, ERATO, MINOSHIMA Intelligent Optical Synthesizer Project, 2-1, Tokushima, Japan. His research interests include THz
instrumentation and metrology, second-harmonic-generation microscopy, andoptical frequency comb.
Prof. Yasui is a member of The Optical Society (OSA), The International
Society for Optical Engineering (SPIE), the Japan Society of Applied Physics,
the Optical Society of Japan, the Laser Society of Japan, the Japanese Society
for Medical and Biological Engineering, and the Japan Society of MechanicalEngineers. He was the recipient of the Award for the Most Promising Young
Scientist from the Optical Society of Japan in 1998, the Sakamoto Award fromthe Japan Society of Medical Electronics and Biological Engineering in 2006,
the Optics Paper Award from the Japan Society of Applied Physics, and theFunai Award from the Japan Society of Mechanical Engineers in 2009, and theOriginal Paper Award from the Laser Society of Japan in 2013.