Double-Clad Optical Fiber-Based Multi-Contrast Noncontact Photoacoustic and Fluorescence Imaging System

: A noncontact photoacoustic and ﬂuorescence dual-modality imaging system is proposed, which integrates a ﬁber-based ﬂuorescence imaging system with noncontact photoacoustic imaging using a specially fabricated double-cladding ﬁber (DCF) coupler and a DCF lens. The performance of the DCF coupler and lens was evaluated, and the feasibility of this new imaging system was demonstrated using simple tubing phantoms with black ink and ﬂuorophore. Our imaging results demonstrated that the multimodal imaging technique can simultaneously acquire photoacoustic and ﬂuorescence images without coming into contact with the sample. Consequently, the developed method is the ﬁrst noncontact scheme among multimodal imaging systems that is integrated with a photoacoustic imaging system, which can provide varied and complementary information about the sample. To characterize the ﬂuorescence excitation efﬁciency of the ﬁber probe as a function of distance, we conducted an experiment using a ﬂuorescent ﬁber. The ﬂuorescence emission signal was measured while varying the distance between the ﬂuorescent ﬁber and the probe tip using the linear stage. Figure 7 shows a photograph of the experimental setup and the results. As shown in Figure 7b, the detected ﬂuorescence signal decreases steeply with the increase in distance. The detectable distance of the FWHM for the ﬂuorescence signal is approximately 1000 µ m from the tip of the DCF lens, and the measurable distance of the 20% ﬂuorescence signal is approximately 2500 µ m, which implies that the FLI is dominated by the ﬂuorescence signal collected within this distance. excitation


Introduction
Multi-contrast images can be used to quantify or classify samples more effectively because they contain morphological, functional, and molecular information about the same biological specimens [1]. Because optical multimodal imaging techniques can provide multi-contrast information on different optical properties of a sample, various studies have focused on these techniques [2,3]. To acquire a multi-contrast image using a multimodal imaging system, various systems must be irradiated with light from different systems at a fixed location of a sample, and various techniques have been proposed to achieve such an image. Particularly, a method of coupling different lights in free space [2][3][4] or combining two lights using an optical fiber has been utilized to irradiate samples [5].
During the last decade, photoacoustic imaging (PAI) has been successfully demonstrated as a noninvasive, label-free, in vivo, volumetric imaging modality with microscale resolution and high optical contrast [4]. Because this technique is based on optical absorption relevant to physiological properties in biological samples, it can be used for monitoring tumor angiogenesis, blood oxygenation mapping, functional brain imaging, skin melanoma detection, etc. [5]. Because PAI integrates light and ultrasound, it can easily be used to construct a multimodal imaging technique by adding an optical system to a commercialized ultrasound device [6]. To provide a more comprehensive understanding of physiological processes in biological samples, the PAI system has been integrated with optical imaging modalities such as fluorescence imaging (FLI) [7,8], diffuse optical tomography, optical coherence tomography (OCT) [2,3,[9][10][11][12], and fluorescence confocal microscopy. Among them, FLI, which is capable of high-sensitivity molecular imaging, is cost effective and has the advantage of using nonionizing radiation; therefore, it is used for tumor detection, cell dynamics monitoring, and biological studies. Owing to the advantages of light absorption-based imaging techniques, FLI and PAI have been combined Electronics 2021, 10, 3008 2 of 13 to improve the understanding of pathophysiological processes [9,10,13,14]. For example, dual-modal imaging can distinguish between sentinel lymph nodes and blood vessels; fluorescence-based cancer targets and neovascularization; and inflammation, bacteria and immune angiogenesis [9,10,13,14].
Despite these advantages of the dual-modal FLI and PAI, there are fundamental limitations to the combination of PAI and FLI systems due to their fundamentally different detection regimes, i.e., ultrasound detection and optical detection, respectively. It is fundamentally difficult to integrate PAI into a common path with an optical detection regime that is based on an optical imaging modality (fluorescence imaging). This is because, to effectively measure ultrasonic signals, PAI requires an ultrasonic transducer to be in close contact with the sample surface, as well as an ultrasonic impedance matching process. To overcome these limitations while developing a multimodal PAI system, multimodal imaging techniques using noncontact PAI (ncPAI) have been studied [15,16], which can be easily combined with other optical imaging devices by measuring ultrasonic signals using a noncontact method [10][11][12]17]. ncPAI detects the vibrations of the sample surface that are induced by photoacoustic waves generated inside the sample by using optical interference, which does not require the use of a coupling agent (water or gel) for ultrasonic impedance matching. This noncontact optical ultrasound detection can be easily and simply configured with other optical imaging techniques and a common path of light irradiation, resulting in an easily configurable multimodal imaging system that can generate co-registered images without any image processing. Therefore, this can ease system development and minimize development costs. Moreover, the proposed method avoids the risk of sample contamination or secondary infection that may occur because of the use of water or an ultrasonic matching gel in image acquisition. It is expected that the proposed noncontact multimodal imaging technique can be used in fields where a noncontact method is absolutely necessary, such as ophthalmic imaging, brain imaging, and burnt tissue examination.
In this study, to achieve a noncontact photoacoustic and fluorescence dual-modality imaging system, we integrated a fiber-based FLI system into a ncPAI system using a specially fabricated double-cladding fiber (DCF) coupler. The unique fiber-based design of the DCF coupler improves the convenience of combining the two systems (PAI and FLI) while enabling the separation of fluorescence excitation and emission lights. We evaluated the feasibility of this new imaging system using a simple tubing phantom with black ink and fluorophore. To the best of our knowledge, the photoacoustic-fluorescence image obtained in this study using our dual-modality imaging system is the first one obtained using a noncontact scheme among all the multimodal imaging systems integrated with a PAI system. Consequently, the proposed method simultaneously acquires both photoacoustic and fluorescence images without coming into contact with the sample, which shows that varied and complementary information about the sample can be obtained.

Fabrication of DCF Coupler
For a photoacoustic and fluorescence multi-contrast imaging configuration, a DCF coupler is employed to simultaneously guide lights of different wavelengths. A general single-clad optical fiber is composed of a single core and single cladding; conversely, the DCF is composed of one core and two claddings (inner and outer claddings). Unlike the single-clad fiber, which guides light only through the core, the DCF can guide light through the core as well as the inner cladding. Using these DCF features, the DCF coupler was fabricated to simultaneously irradiate the sample with two light sources of different wavelengths through the core and to separately detect the reflected and fluorescence lights through the core and inner cladding, respectively. The DCF coupler was fabricated by a fused biconical taper method, which couples only the inner cladding modes in both DCF pieces without affecting the core modes. First, the two DCFs, with the outer cladding Electronics 2021, 10, 3008 3 of 13 removed, were twisted around each other. Then, while heat was applied to the twisted part, the DCFs were pulled to opposite sides, causing the inner cladding of the DCFs to be fused together. Figure 1a shows the structure of the fabricated DCF coupler, and Figure 1b shows the guiding beam path in the proposed dual-modal imaging system. As shown in Figure 1b, when the inner claddings of the two DCFs are fused together, the beam of the through port can move to the cross port through the coupled region.
DCF pieces without affecting the core modes. First, the two DCFs, with the outer cladding removed, were twisted around each other. Then, while heat was applied to the twisted part, the DCFs were pulled to opposite sides, causing the inner cladding of the DCFs to be fused together. Figure 1a shows the structure of the fabricated DCF coupler, and Figure  1b shows the guiding beam path in the proposed dual-modal imaging system. As shown in Figure 1b, when the inner claddings of the two DCFs are fused together, the beam of the through port can move to the cross port through the coupled region.
The fluorescence excitation light of the proposed dual-modal system and the interrogation light of the PAI are transmitted through the core of the DCF coupler. The interrogation light that measures the photoacoustic signal without contact is backcoupled through the core of the DCF coupler and is detected by the PAI system. Conversely, the fluorescence excitation light generates fluorescence, which is backcoupled through the inner cladding of the DCF coupler. The fluorescence system detects the emitted fluorescent light passing through the coupled region of the DCF coupler.

Fabrication of Lensed Fiber Probe
A measurement probe for dual-modal imaging is manufactured using a DCF lens (lensed fiber) to focus the interrogation light and fluorescence excitation light on the sample and receive reflected light from the sample. Figure 2 shows the fabrication process of the DCF lens. The DCF lens is fabricated by splicing a short piece of coreless silica fiber (CSF) to the tip of the DCF coupler and then forming a lens curvature at the tip of the CSF using a fusion splicer. The fusion splicing of the DCF and CSF and formation of the lens curvature of the CSF are performed using an optical fiber fusion splicer. The fabricated DCF lens and a multimode fiber for transmission of the pulsed laser were attached sideby-side to create an optical probe for the dual-modal imaging system. The fluorescence excitation light of the proposed dual-modal system and the interrogation light of the PAI are transmitted through the core of the DCF coupler. The interrogation light that measures the photoacoustic signal without contact is backcoupled through the core of the DCF coupler and is detected by the PAI system. Conversely, the fluorescence excitation light generates fluorescence, which is backcoupled through the inner cladding of the DCF coupler. The fluorescence system detects the emitted fluorescent light passing through the coupled region of the DCF coupler.

Fabrication of Lensed Fiber Probe
A measurement probe for dual-modal imaging is manufactured using a DCF lens (lensed fiber) to focus the interrogation light and fluorescence excitation light on the sample and receive reflected light from the sample. Figure 2 shows the fabrication process of the DCF lens. The DCF lens is fabricated by splicing a short piece of coreless silica fiber (CSF) to the tip of the DCF coupler and then forming a lens curvature at the tip of the CSF using a fusion splicer. The fusion splicing of the DCF and CSF and formation of the lens curvature of the CSF are performed using an optical fiber fusion splicer. The fabricated DCF lens and a multimode fiber for transmission of the pulsed laser were attached side-by-side to create an optical probe for the dual-modal imaging system.  Figure 3 shows the schematic of the proposed multimodal PAI and FLI system using the DCF coupler and DCF lens. The system consists of two major parts: a fiber-optic interferometry system for photoacoustic signal readout for noncontact PAI and a fiber-based FLI module.

Optical Interferometry for Noncontact Fiber-based PA imaging
The fiber-optic interferometry system for photoacoustic signal readout consists of a fiber-based Mach-Zehnder interferometer to detect the photoacoustic wave, owing to its high sensitivity and the advantages of the noncontact scheme. The interferometer uses a single-frequency laser (SFL1550S, Thorlabs, Newton, NJ, USA) at 1550 nm wavelength, single-mode fiber couplers, an acousto-optic modulator (AOM; Fiber-Q, Gooch & Housego, Ilminster, UK) with 80 MHz driving frequency, an AOM driver (Gooch & Housego), a polarization controller (FPC031, Thorlabs), an optical circulator, a 1550 nm optical fiber filter, a balanced photodetector (BPD; PDB430C, Thorlabs), and a self-built IQ demodulator. The laser source is split into reference and sample arms using the first coupler. The beam that is guided into the reference arm is frequency modulated by the AOM and enters the second coupler after passing through the polarization controller. The sample beam passes through the circulator and DCF lens and reaches the sample surface. The reflected beam from the surface enters the second coupler after backcoupling by the DCF lens. These two beams cause an optical interference, which contain the modulated frequency and phase information of the surface vibration; this is detected by the BPD and digitized by a digitizer (PCI-5142, National Instrument, Austin, TX, USA) after demodulation by the IQ demodulator. A detailed description of the configuration of the interferometer and measurement principle has been provided in our previous reports [10]. For the generation of the photoacoustic wave, a Q-switched neodymium-doped yttrium aluminum garnet (Nd:YAG) laser (Brilliant Ultra 50, Quantel, Newbury, UK) is employed, which is guided through the multimode fiber to the sample. The performance of the proposed fiber-based ncPAI system was discussed in our previous reports [10,11]. The lateral and axial resolutions of the PAI system are approximately 100 and 30 μm at a depth of 2.5 mm, respectively [10]. The displacement resolution of the optical fiber-based heterodyne interferometer is approximately 0.5 nm, corresponding to 18.7 kPa [10]. The detection sensitivity and measurement frequency bandwidth are 13.7 dB and 20 MHz, respectively [11].

Fiber-based Fluorescence Imaging System
The fiber-based FLI system is shown in the blue box in Figure 3. For guiding the fluorescence excitation and emission lights, a special fiber probe consisting of a self-built DCF coupler and DCF lens is used. A fiber pigtailed laser with a 405 nm center wavelength (OZ-1000-405-3, OZ Optics Ltd.) is used for generating the fluorescence excitation light, which is guided into the sample through the DCF core and lens. Moreover, a fluorescence  Figure 3 shows the schematic of the proposed multimodal PAI and FLI system using the DCF coupler and DCF lens. The system consists of two major parts: a fiber-optic interferometry system for photoacoustic signal readout for noncontact PAI and a fiberbased FLI module.

Fiber-Based Combined Photoacoustic (PA) and Fluorescence Imaging System
Electronics 2021, 10, x FOR PEER REVIEW 5 of 14 emission filter (MF460-60, Thorlabs) and an avalanche photodetector (APD; APD110A, Thorlabs) are used to detect the fluorescence emission that is backcoupled and separated by the DCF lens and coupler.

Noncontact Combined PA and Fluorescence Imaging System
The two light sources of both the imaging systems, i.e., the interrogating light and fluorescent excitation light, are combined by a wavelength division multiplexer (WDM) and guided to a sample through the core of the DCF. Then, the DCF lens focuses the two lights into the sample and collects the back-reflected beam of the interrogating light and the fluorescent emission beam, which are transmitted through the core and inner cladding of the DCF, respectively. The back-reflected beam is delivered back to Port 1 of the DCF coupler (in Figure 3), WDM, and optical circulator; then, it interferes with the reference beam that is modulated by the AOM. The interference signal is detected by the BPD and demodulated with the AOM driving signal using an IQ demodulator. Finally, the demodulated signals are digitized and estimated as photoacoustic signals. Conversely, the fluorescent emission beam is guided through the inner cladding of the DCF and delivered to Port 2 of the DCF coupler ( Figure 3) through the DCF lens and the coupled mode of the inner cladding. After passing through the emission filter, the fluorescence beam is detected by the APD. To acquire a multidimensional image, the fiber probe head is scanned using an XY motorized stage. All the devices in the system are controlled by a program based on the Laboratory Virtual Instrument Engineering Workbench.

Sample Preparation for the Experiment using the PA and Fluorescence Dual Imaging System
To evaluate the fluorescence detection sensitivity along with the different concentrations of fluorescent solution and detection efficiency corresponding to the distance, a water-soluble fluorescent solution (WB-402UV, Flamak Chemical Industry Co., Ltd., Germany) and fluorescent plastic fiber (ECCBFH-10, Eagle Claw) with a diameter of 0.29 mm

Optical Interferometry for Noncontact Fiber-Based PA Imaging
The fiber-optic interferometry system for photoacoustic signal readout consists of a fiber-based Mach-Zehnder interferometer to detect the photoacoustic wave, owing to its high sensitivity and the advantages of the noncontact scheme. The interferometer uses a single-frequency laser (SFL1550S, Thorlabs, Newton, NJ, USA) at 1550 nm wavelength, single-mode fiber couplers, an acousto-optic modulator (AOM; Fiber-Q, Gooch & Housego, Ilminster, UK) with 80 MHz driving frequency, an AOM driver (Gooch & Housego), a polarization controller (FPC031, Thorlabs), an optical circulator, a 1550 nm optical fiber Electronics 2021, 10, 3008 5 of 13 filter, a balanced photodetector (BPD; PDB430C, Thorlabs), and a self-built IQ demodulator. The laser source is split into reference and sample arms using the first coupler. The beam that is guided into the reference arm is frequency modulated by the AOM and enters the second coupler after passing through the polarization controller. The sample beam passes through the circulator and DCF lens and reaches the sample surface. The reflected beam from the surface enters the second coupler after backcoupling by the DCF lens. These two beams cause an optical interference, which contain the modulated frequency and phase information of the surface vibration; this is detected by the BPD and digitized by a digitizer (PCI-5142, National Instrument, Austin, TX, USA) after demodulation by the IQ demodulator. A detailed description of the configuration of the interferometer and measurement principle has been provided in our previous reports [10]. For the generation of the photoacoustic wave, a Q-switched neodymium-doped yttrium aluminum garnet (Nd:YAG) laser (Brilliant Ultra 50, Quantel, Newbury, UK) is employed, which is guided through the multimode fiber to the sample. The performance of the proposed fiber-based ncPAI system was discussed in our previous reports [10,11]. The lateral and axial resolutions of the PAI system are approximately 100 and 30 µm at a depth of 2.5 mm, respectively [10]. The displacement resolution of the optical fiber-based heterodyne interferometer is approximately 0.5 nm, corresponding to 18.7 kPa [10]. The detection sensitivity and measurement frequency bandwidth are 13.7 dB and 20 MHz, respectively [11].

Fiber-Based Fluorescence Imaging System
The fiber-based FLI system is shown in the blue box in Figure 3. For guiding the fluorescence excitation and emission lights, a special fiber probe consisting of a self-built DCF coupler and DCF lens is used. A fiber pigtailed laser with a 405 nm center wavelength (OZ-1000-405-3, OZ Optics Ltd.) is used for generating the fluorescence excitation light, which is guided into the sample through the DCF core and lens. Moreover, a fluorescence emission filter (MF460-60, Thorlabs) and an avalanche photodetector (APD; APD110A, Thorlabs) are used to detect the fluorescence emission that is backcoupled and separated by the DCF lens and coupler.

Noncontact Combined PA and Fluorescence Imaging System
The two light sources of both the imaging systems, i.e., the interrogating light and fluorescent excitation light, are combined by a wavelength division multiplexer (WDM) and guided to a sample through the core of the DCF. Then, the DCF lens focuses the two lights into the sample and collects the back-reflected beam of the interrogating light and the fluorescent emission beam, which are transmitted through the core and inner cladding of the DCF, respectively. The back-reflected beam is delivered back to Port 1 of the DCF coupler (in Figure 3), WDM, and optical circulator; then, it interferes with the reference beam that is modulated by the AOM. The interference signal is detected by the BPD and demodulated with the AOM driving signal using an IQ demodulator. Finally, the demodulated signals are digitized and estimated as photoacoustic signals. Conversely, the fluorescent emission beam is guided through the inner cladding of the DCF and delivered to Port 2 of the DCF coupler (Figure 3) through the DCF lens and the coupled mode of the inner cladding. After passing through the emission filter, the fluorescence beam is detected by the APD. To acquire a multidimensional image, the fiber probe head is scanned using an XY motorized stage. All the devices in the system are controlled by a program based on the Laboratory Virtual Instrument Engineering Workbench.

Sample Preparation for the Experiment Using the PA and Fluorescence Dual Imaging System
To evaluate the fluorescence detection sensitivity along with the different concentrations of fluorescent solution and detection efficiency corresponding to the distance, a water-soluble fluorescent solution (WB-402UV, Flamak Chemical Industry Co., Ltd., Germany) and fluorescent plastic fiber (ECCBFH-10, Eagle Claw) with a diameter of 0.29 mm were used. The water-soluble fluorescent solution was filled in silicon tubes (inner diame-ter: 250 µm, and outer diameter: 750 µm) and the emitted fluorescence light was imaged using the dual imaging system.
To obtain the photoacoustic and fluorescence images, a 5% mixed solution of milk and water containing silicon tubings was used. The 5% mixed solution was prepared by diluting 5 mL milk in 95 mL water. The solution was poured into a Petri dish containing four silicon tubings (inner diameter: 500 µm and outer diameter: 1500 µm). Two tubes were filled with black ink and the other two tubes were filled with fluorescent particles (SPHERO TM Fluorescent Light Yellow), which were placed in the Petri dish. The fluorescent particles have major excitation and emission wavelengths of 405 and 450 nm, respectively.

Characterization of DCF Coupler and DCF Lens
After fabricating the DCF coupler, the transmission rate of the inner cladding from the through port to the cross port of the DCF coupler was measured using a broadband source (600-900 nm) and an optical spectrum analyzer. Figure 4a shows that the transmission rate of the inner cladding of the DCF coupler is approximately 32% higher than the illumination spectrum. Figure 4b shows the mode field images when the light source is launched into the core and inner cladding of the DCF coupler. The image on the left in Figure 4b represents the inner cladding mode of the DCF coupler when the light source is launched from the through port to the cross port. The image on the right of Figure 4b shows the mode field of the core mode in the DCF coupler when the light source is launched from the core of the DCF coupler to the through port. were used. The water-soluble fluorescent solution was filled in silicon tubes (inner diameter: 250 μm, and outer diameter: 750 μm) and the emitted fluorescence light was imaged using the dual imaging system. To obtain the photoacoustic and fluorescence images, a 5% mixed solution of milk and water containing silicon tubings was used. The 5% mixed solution was prepared by diluting 5 mL milk in 95 mL water. The solution was poured into a Petri dish containing four silicon tubings (inner diameter: 500 μm and outer diameter: 1500 μm). Two tubes were filled with black ink and the other two tubes were filled with fluorescent particles (SPHERO TM Fluorescent Light Yellow), which were placed in the Petri dish. The fluorescent particles have major excitation and emission wavelengths of 405 and 450 nm, respectively.

Characterization of DCF Coupler and DCF Lens
After fabricating the DCF coupler, the transmission rate of the inner cladding from the through port to the cross port of the DCF coupler was measured using a broadband source (600-900 nm) and an optical spectrum analyzer. Figure 4a shows that the transmission rate of the inner cladding of the DCF coupler is approximately 32% higher than the illumination spectrum. Figure 4b shows the mode field images when the light source is launched into the core and inner cladding of the DCF coupler. The image on the left in Figure 4b represents the inner cladding mode of the DCF coupler when the light source is launched from the through port to the cross port. The image on the right of Figure 4b shows the mode field of the core mode in the DCF coupler when the light source is launched from the core of the DCF coupler to the through port.   Figure 1; right: the through port of the DCF coupler shown in Figure 1).
We evaluated the characteristics of the DCF lens. Figure 5 shows the optical coupling power of the back-reflected light for the measurement of the working distance of the DCF lens and the beam size. The optical coupling power of the back-reflected light on a mirror was monitored using a 1550 nm light source and power meter, as the mirror which is fixed on a translation stage is moved away. From Figure 5a, it can be observed that the normalized power of the back-reflected light on the mirror increases and decreases again when the mirror moves away from the DCF lens tip. Therefore, the working distance (or focal length) of the DCF lens, which indicates the distance of the peak value in the graph ( Figure  5a), is approximately 485 μm. The beam width of the lensed fiber was evaluated by the  Figure 1; right: the through port of the DCF coupler shown in Figure 1).
We evaluated the characteristics of the DCF lens. Figure 5 shows the optical coupling power of the back-reflected light for the measurement of the working distance of the DCF lens and the beam size. The optical coupling power of the back-reflected light on a mirror was monitored using a 1550 nm light source and power meter, as the mirror which is fixed on a translation stage is moved away. From Figure 5a, it can be observed that the normalized power of the back-reflected light on the mirror increases and decreases again when the mirror moves away from the DCF lens tip. Therefore, the working distance (or focal length) of the DCF lens, which indicates the distance of the peak value in the graph (Figure 5a), is approximately 485 µm. The beam width of the lensed fiber was evaluated by the line spread function (LSF), which is obtained by measuring the spatial derivative of the edge spread function (ESF) [17]. The optical coupling power of the back-reflected light was measured at the working distance of the DCF lens by moving a knife-edge mirror in the lateral direction; the results are shown in Figure 5b with black squares. The ESF was Electronics 2021, 10, 3008 7 of 13 obtained by fitting the original data (red solid line in Figure 5b), and the LSF was estimated by measuring the first derivative of the ESF, which is represented by the blue solid line in Figure 5b. The full width at half maximum (FWHM) of the LSF indicates that the beam size of the DCF lens was approximately 5.1 µm.
Electronics 2021, 10, x FOR PEER REVIEW 7 of 14 line spread function (LSF), which is obtained by measuring the spatial derivative of the edge spread function (ESF) [17]. The optical coupling power of the back-reflected light was measured at the working distance of the DCF lens by moving a knife-edge mirror in the lateral direction; the results are shown in Figure 5b with black squares. The ESF was obtained by fitting the original data (red solid line in Figure 5b), and the LSF was estimated by measuring the first derivative of the ESF, which is represented by the blue solid line in Figure 5b. The full width at half maximum (FWHM) of the LSF indicates that the beam size of the DCF lens was approximately 5.1 μm.

Detection Sensitivity of Fiber-based Fluorescence Imaging
Before the evaluation of fluorescence detection sensitivity along with the different concentrations of fluorescent solution and detection efficiency corresponding to the distance, the fluorescence spectra of the water-soluble fluorescent solution and fluorescent plastic fiber were measured using an excitation laser (405 nm) and a spectrometer (QE65000, Ocean Optics). Figure 6a,b show the fluorescence emission spectra of the watersoluble fluorescent solution and fluorescent fiber, respectively, where the first small peak indicates the wavelength of the fluorescence excitation laser source, and the subsequent peak(s) presents the emission spectra of the fluorescent solution/fiber.

Detection Sensitivity of Fiber-Based Fluorescence Imaging
Before the evaluation of fluorescence detection sensitivity along with the different concentrations of fluorescent solution and detection efficiency corresponding to the distance, the fluorescence spectra of the water-soluble fluorescent solution and fluorescent plastic fiber were measured using an excitation laser (405 nm) and a spectrometer (QE65000, Ocean Optics). Figure 6a,b show the fluorescence emission spectra of the water-soluble fluorescent solution and fluorescent fiber, respectively, where the first small peak indicates the wavelength of the fluorescence excitation laser source, and the subsequent peak(s) presents the emission spectra of the fluorescent solution/fiber. To characterize the fluorescence excitation efficiency of the fiber probe as a function of distance, we conducted an experiment using a fluorescent fiber. The fluorescence emission signal was measured while varying the distance between the fluorescent fiber and  To characterize the fluorescence excitation efficiency of the fiber probe as a function of distance, we conducted an experiment using a fluorescent fiber. The fluorescence emission signal was measured while varying the distance between the fluorescent fiber and the probe tip using the linear stage. Figure 7 shows a photograph of the experimental setup and the results. As shown in Figure 7b, the detected fluorescence signal decreases steeply with the increase in distance. The detectable distance of the FWHM for the fluorescence signal is approximately 1000 µm from the tip of the DCF lens, and the measurable distance of the 20% fluorescence signal is approximately 2500 µm, which implies that the FLI is dominated by the fluorescence signal collected within this distance. the fluorescence excitation laser source (405 nm), and the second peak presents the emission spectrum. (b) mission spectrum of the fluorescent fiber; the first small peak embedded in the main peak indicates the the fluorescence excitation laser source (405 nm), and the other peaks present the emission spectrum of the r.
To characterize the fluorescence excitation efficiency of the fiber probe as a function of distance, we conducted an experiment using a fluorescent fiber. The fluorescence emission signal was measured while varying the distance between the fluorescent fiber and the probe tip using the linear stage. Figure 7 shows a photograph of the experimental setup and the results. As shown in Figure 7b, the detected fluorescence signal decreases steeply with the increase in distance. The detectable distance of the FWHM for the fluorescence signal is approximately 1000 μm from the tip of the DCF lens, and the measurable distance of the 20% fluorescence signal is approximately 2500 μm, which implies that the FLI is dominated by the fluorescence signal collected within this distance.  The fluorescence detection sensitivity of the DCF probe was measured by scanning silicon tubes filled with water-soluble fluorescent solutions of varying concentrations. The scanning area was 1500 µm × 100 µm, and the scanning step size was 10 µm. Figures 8  and 9 show the fluorescence images of the silicon tubes and line profile along with the different concentrations of fluorescent solution. As shown in Figure 8, the fluorescence image of the 1% concentration of the fluorescence solution was hardly discernible. The line profile of the obtained fluorescence images from 0.1 to 100% concentration, as shown in Figure 9, indicates that the detection limit of the fiber-based FLI is at 0.1% concentration of the fluorescent solution.
Electronics 2021, 10, 3008 9 of 13 silicon tubes filled with water-soluble fluorescent solutions of varying concentrations. The scanning area was 1500 μm × 100 μm, and the scanning step size was 10 μm. Figure 8 and Figure 9 show the fluorescence images of the silicon tubes and line profile along with the different concentrations of fluorescent solution. As shown in Figure 8, the fluorescence image of the 1% concentration of the fluorescence solution was hardly discernible. The line profile of the obtained fluorescence images from 0.1 to 100% concentration, as shown in Figure 9, indicates that the detection limit of the fiber-based FLI is at 0.1% concentration of the fluorescent solution.

PA Imaging and Fluorescence Imaging Performance
Phantom imaging was performed using the proposed dual-modal PAI and FLI system. The pulsed laser illuminates the phantom tube through the multimode fiber to excite

PA Imaging and Fluorescence Imaging Performance
Phantom imaging was performed using the proposed dual-modal PAI and FLI system. The pulsed laser illuminates the phantom tube through the multimode fiber to excite the photoacoustic signal of the black ink tube in the phantom. The radiant exposure of the pulsed Nd:YAG laser in free space was approximately 18 mJ/cm 2 . The DCF probe was positioned perpendicular to the phantom surface, and the photoacoustic and fluorescence signals were measured simultaneously. Because the two systems are combined as one imaging probe by the DCF coupler and lens, we can acquire the photoacoustic and fluorescent signals from the same position and register the dual images, simultaneously. Five photoacoustic signals were obtained per detection point and averaged. Figure 10a shows an imaging sample with four tubes filled with black ink and fluorescent particles. The excitation laser was illuminated at a wide angle to recognize the black ink and fluorescent tubes. The red arrow indicates the scanning direction. To acquire 3D photoacoustic images and 2D projected fluorescence images, the dual-modal imaging system scans over an area of 10 mm × 10 mm in steps of 50 µm using the XY linear stage. Figure 10b shows the 3D photoacoustic image and 2D projected fluorescence images, which are the maximumintensity projection images of the 3D rendered image on the XZ, XY, and YZ planes. The 3D rendered image size was 10 mm × 10 mm × 7 mm. The two tubes with black ink can be clearly observed in the XZ, XY, and YZ plane-view images, which represent the photoacoustic images, and the two tubes filled with fluorescence particles for FLI, are also clearly recognized in the projected images on the XY plane view.

Discussion
Studies on multimodal imaging systems that simultaneously apply various imaging technologies are being studied in multiple ways because of the advantage of providing information about samples with spatiotemporal consistency [12]. The optoacoustic imaging technique, which uses short pulses of light to generate ultrasonic waves inside the sample for acquiring sample information, not only combines the advantages of optical and ultrasound imaging techniques but can also easily combine additional optical imaging techniques. Therefore, research on multimodal imaging systems based on the PAI is being actively conducted. The combination of OCT, which measures the structural changes in the refractive index of a sample, and PAI, can simultaneously provide physical and optical information according to the change in the refractive index and optical absorption of a sample. Consequently, this imaging technique is often used in physiological

Discussion
Studies on multimodal imaging systems that simultaneously apply various imaging technologies are being studied in multiple ways because of the advantage of providing information about samples with spatiotemporal consistency [12]. The optoacoustic imaging technique, which uses short pulses of light to generate ultrasonic waves inside the sample for acquiring sample information, not only combines the advantages of optical and ultrasound imaging techniques but can also easily combine additional optical imaging techniques. Therefore, research on multimodal imaging systems based on the PAI is being actively conducted. The combination of OCT, which measures the structural changes in the refractive index of a sample, and PAI, can simultaneously provide physical and optical information according to the change in the refractive index and optical absorption of a sample. Consequently, this imaging technique is often used in physiological studies related to the structure of blood vessels and oxygen saturation at the same time [3,15]. A combination of ultrasound and optoacoustic imaging can be used when a greater imaging depth is required than that provided by OCT [16]. In this study, a system combining PAI and FLI was constructed to simultaneously acquire the absorption and fluorescence characteristics of a sample.
The PAI technology, which expresses an image by measuring the ultrasound generated from a sample, requires a process of matching the ultrasound impedance between the sample and the ultrasound transducer. Because of this inherent limitation, it is necessary to simultaneously place the sample and the ultrasonic transducer in liquids such as oil and water or use an ultrasonic impedance matching medium in the imaging process. Furthermore, as this entails complicated pre-preparation of the sample and leads to the possibility of contamination and infection of the sample, there has been a demand for a technology that can obtain photoacoustic images using a noncontact method [17]. The ncPAI system was developed to measure the surface vibration or change in refractive index of the sample using an optical detection method [17]. In our previous work, we measured surface vibrations using an optical fiber-based heterodyne interferometer [10,11]. Thus, the ultrasonic signal is detected by measuring the optical interference signal generated on the sample surface and the phase modulation caused by the surface displacement. Therefore, it should be noted that, in the noncontact method of detecting the displacement of the sample surface, the ultrasonic sensitivity and frequency characteristics depend on the properties (elasticity, acoustic impedance, etc.) of the sample.
This noncontact detection method has enabled research on the development of multimodal imaging systems that combine PAI and other optical imaging techniques, by overcoming the limitations of the existing PAI systems.
In this study, photoacoustic and fluorescence signals were acquired using a noncontact method, and short-pulsed light and fluorescent excitation light for dual-modal imaging were delivered to the sample through an optical fiber. The ultrasonic signal generated from the sample was acquired using the fiber-based optical interference technology, and the fluorescence signal was also acquired using the optical fiber technique. Therefore, the imaging probe that generates and detects signals is only composed of optical fibers without the use of electrical components. The compact size of the probe configuration suggests that the results of this study can be applied to an ultrasmall probe configuration and the endoscopic configuration of PAI and FLI system.
The combined PAI and FLI system can combine OCT, which can provide highresolution structural information of samples as well as molecular images based on the absorption and fluorescence properties of the sample. This multimodal imaging system can be used as an imaging tool for diagnosis that overlays the physiological, pathological, and immunovascular information, such as information about cancer tissue, inflammation, organelles, lymph nodes, and lymphatic vessels, which are targeted by fluorescent contrast agents, along with the structural information of the sample [14,[17][18][19][20][21]. The system proposed in this paper consists of a fiber-optic system for FLI and a fiber interferometer for photoacoustic signal detection. If an optical interferometer for OCT is added [22], the configuration of three noncontact multimodal imaging systems can be easily achieved. Moreover, research on ultrasmall probes for lensed-fiber-based OCT systems has been conducted [23], which shows the possibility of acquiring OCT signals using the DCF lensed fiber. However, the wavelengths of the light sources used in the proposed system are 405 and 1550 nm, which correspond to the ultraviolet and infrared wavelengths, respectively. To achieve a multi-image system with a high signal-to-noise ratio, an optical system that is optimized for each light source must be designed and applied.
The proposed noncontact multimodal imaging technique is used to determine the dynamic changes in physiology, such as blood speed, hemoglobin concentration, oxygenation, and blood volume. To apply this system in the medical field, real-time functioning of the entire system is required, including real-time data acquisition, processing, and visualization. Recently, the speed of the entire system has increased owing to the high speed of the light source and detection system, making it possible to achieve real-time systems. Moreover, effective visualization techniques using various image processing techniques can be studied to effectively apply the proposed multimodal imaging technique in clinical practice. If real-time system implementation and image processing techniques for multi-contrast images are developed, it can be applied in real-time image-guided diagnosis and surgery.
When considering the advantages of the noncontact optoacoustic fluorescence fusion imaging technology proposed by this research team (direct coupling, simple system configuration, noncontact, optical fiber-based configuration), we expect the development of a new type of multifunctional integrated imaging technology. Furthermore, if the multi-contrast imaging through photoacoustic/fluorescence techniques is integrated with minimally invasive endoscopes or surgical imaging systems that require continuous technological developments, it is expected to demonstrate a more accurate diagnostic potential for diseases.

Conclusions
For a noncontact photoacoustic and fluorescence dual-modality imaging system, we integrated a fiber-based FLI system with ncPAI using a specially fabricated DCF coupler. The unique fiber-based design of the DCF coupler could combine and separate the lights of two imaging systems (photoacoustic and fluorescence), and we proved the feasibility of this new noncontact multimodal imaging system by simultaneously acquiring the photoacoustic and fluorescence images of the simple phantom, which shows that varied and complementary information regarding a sample can be obtained.
With this multimodal imaging capability, we expect that the proposed method can simultaneously provide functional and structural information of biological tissues and can be applied as a minimally invasive and improved diagnostic tool in medical diagnosis as well.