MEMS Actuators for Optical Microendoscopy

Growing demands for affordable, portable, and reliable optical microendoscopic imaging devices are attracting research institutes and industries to find new manufacturing methods. However, the integration of microscopic components into these subsystems is one of today’s challenges in manufacturing and packaging. Together with this kind of miniaturization more and more functional parts have to be accommodated in ever smaller spaces. Therefore, solving this challenge with the use of microelectromechanical systems (MEMS) fabrication technology has opened the promising opportunities in enabling a wide variety of novel optical microendoscopy to be miniaturized. MEMS fabrication technology enables abilities to apply batch fabrication methods with high-precision and to include a wide variety of optical functionalities to the optical components. As a result, MEMS technology has enabled greater accessibility to advance optical microendoscopy technology to provide high-resolution and high-performance imaging matching with traditional table-top microscopy. In this review the latest advancements of MEMS actuators for optical microendoscopy will be discussed in detail.


Introduction
Actuation and scanning mechanisms have played important roles in novel microendoscopic imaging systems. Common challenges in the development of these miniature instruments are in both design freedom and the integration of miniaturized opto-mechanical components. Microelectromechanical systems (MEMS) fabrication technologies play a valuable and instrumental role in solving the aforementioned issues in order to achieve similar performance as traditional microscopy counterparts. In optical microendoscopy, actuation and scanning mechanisms enable three-dimensional (3D) image formation in the tiny devices with ultra-compact form factors. The technical challenges in designing such kinds of components include the generation of a distortion-free scanning pattern with sufficient speed to mitigate in vivo motion artifacts with millimeter package dimensions. To realize in vivo tissue imaging on living subjects, 5 Hz or faster frame rates are usually required to accommodate movements induced by several factors, such as respiratory displacement, heart beating, and organ peristalsis.
The size of the MEMS actuators determines their mounted locations at either the proximal or distal end of the instrument. A much greater control of the focal volume, including axial scanning for imaging into the tissue, can be achieved with the MEMS actuators positioned distally. However, their typical sizes mounted at the distal end of the instrument should be less than 5.5 mm (for example, fitting the Olympus therapeutic endoscope) in order to be compatible with the tool channel of a standard medical endoscope [1]. used for in vivo imaging on small animal models or human patients [21][22][23], mass-producible MEMS actuator-enabled OCT microendoscopes will very likely become the future trend because MEMS-based micro-devices have many advantages, especially in terms of the miniaturization potential and repeatability. MEMS-based micro-devices may be based on various working principles and actuation mechanisms, such as electrostatic [24][25][26][27][28][29], electrothermal [18,[30][31][32][33], bulky PZT-based fiber scan tube [34][35][36][37][38][39], and electromagnetics [40].
Among the variety of micro-devices, the electrostatic comb-drive-actuated MEMS scanner is a popular one [24][25][26][27][28][29]. For example, one of the representative MEMS-based OCT microendoscopes, as shown in Figure 1 [28], has utilized an electrostatic MEMS scanner [25] driven by angled vertical comb (AVC) actuators for a large tilting angle. The effective mirror aperture's diameter is as large as 1 mm, which is sufficient to reflect the light beam for side-view high-resolution imaging with the MEMS-based endoscopic catheter. The single-mode fiber (SMF), fiber collimator, and the MEMS scanner are fully integrated in an aluminum-based packaging. The detailed fiber-based optical system design of the time-domain OCT imaging system and the real-time data acquisition system with high sampling speed are illustrated in Figure 2a. Three-dimensional OCT image volumes acquired in vitro from a hamster cheek pouch are shown in Figure 2b. Both horizontal (also called en-face) and vertical cross-sectional plane images extracted from the 3D OCT volume, in Figure 2c, have demonstrated high-resolution morphological changes inside the tissue specimen.
Micromachines 2018, 9, x FOR PEER REVIEW 4 of 19 MEMS actuator-enabled OCT microendoscopes will very likely become the future trend because MEMS-based micro-devices have many advantages, especially in terms of the miniaturization potential and repeatability. MEMS-based micro-devices may be based on various working principles and actuation mechanisms, such as electrostatic [24][25][26][27][28][29], electrothermal [18,[30][31][32][33], bulky PZT-based fiber scan tube [34][35][36][37][38][39], and electromagnetics [40]. Among the variety of micro-devices, the electrostatic comb-drive-actuated MEMS scanner is a popular one [24][25][26][27][28][29]. For example, one of the representative MEMS-based OCT microendoscopes, as shown in Figure 1 [28], has utilized an electrostatic MEMS scanner [25] driven by angled vertical comb (AVC) actuators for a large tilting angle. The effective mirror aperture's diameter is as large as 1 mm, which is sufficient to reflect the light beam for side-view high-resolution imaging with the MEMS-based endoscopic catheter. The single-mode fiber (SMF), fiber collimator, and the MEMS scanner are fully integrated in an aluminum-based packaging. The detailed fiber-based optical system design of the time-domain OCT imaging system and the real-time data acquisition system with high sampling speed are illustrated in Figure 2a. Three-dimensional OCT image volumes acquired in vitro from a hamster cheek pouch are shown in Figure 2b. Both horizontal (also called enface) and vertical cross-sectional plane images extracted from the 3D OCT volume, in Figure 2c, have demonstrated high-resolution morphological changes inside the tissue specimen.  Most recently, a novel MEMS-based OCT microendoscope with circumferential-scanning has been developed by the engineering team led by Xie from the University of Florida [41] through a unique optical design utilizing multiple electrothermal MEMS scanners. An array of ultra-compact electro-thermally actuated MEMS scanners (Figure 3a) are integrated at the distal end of the catheter to reflect collimated beams, as shown in Figure 3b,c. Flexible printed circuit boards (FPCB) provide MEMS actuator-enabled OCT microendoscopes will very likely become the future trend because MEMS-based micro-devices have many advantages, especially in terms of the miniaturization potential and repeatability. MEMS-based micro-devices may be based on various working principles and actuation mechanisms, such as electrostatic [24][25][26][27][28][29], electrothermal [18,[30][31][32][33], bulky PZT-based fiber scan tube [34][35][36][37][38][39], and electromagnetics [40]. Among the variety of micro-devices, the electrostatic comb-drive-actuated MEMS scanner is a popular one [24][25][26][27][28][29]. For example, one of the representative MEMS-based OCT microendoscopes, as shown in Figure 1 [28], has utilized an electrostatic MEMS scanner [25] driven by angled vertical comb (AVC) actuators for a large tilting angle. The effective mirror aperture's diameter is as large as 1 mm, which is sufficient to reflect the light beam for side-view high-resolution imaging with the MEMS-based endoscopic catheter. The single-mode fiber (SMF), fiber collimator, and the MEMS scanner are fully integrated in an aluminum-based packaging. The detailed fiber-based optical system design of the time-domain OCT imaging system and the real-time data acquisition system with high sampling speed are illustrated in Figure 2a. Three-dimensional OCT image volumes acquired in vitro from a hamster cheek pouch are shown in Figure 2b. Both horizontal (also called enface) and vertical cross-sectional plane images extracted from the 3D OCT volume, in Figure 2c, have demonstrated high-resolution morphological changes inside the tissue specimen.  Most recently, a novel MEMS-based OCT microendoscope with circumferential-scanning has been developed by the engineering team led by Xie from the University of Florida [41] through a unique optical design utilizing multiple electrothermal MEMS scanners. An array of ultra-compact electro-thermally actuated MEMS scanners (Figure 3a) are integrated at the distal end of the catheter to reflect collimated beams, as shown in Figure 3b,c. Flexible printed circuit boards (FPCB) provide Most recently, a novel MEMS-based OCT microendoscope with circumferential-scanning has been developed by the engineering team led by Xie from the University of Florida [41] through a unique optical design utilizing multiple electrothermal MEMS scanners. An array of ultra-compact electro-thermally actuated MEMS scanners (Figure 3a) are integrated at the distal end of the catheter to reflect collimated beams, as shown in Figure 3b,c. Flexible printed circuit boards (FPCB) provide driving current for electrothermal scanners. All of the micro-optical components and MEMS-based circumferential scanning systems have been fully integrated and assembled in a compact form factor (Figure 3e) for potential in vivo imaging application in the human gastrointestinal (GI) tract. The fiber-based collimating system is used for laser excitation, as shown in Figure 3f. driving current for electrothermal scanners. All of the micro-optical components and MEMS-based circumferential scanning systems have been fully integrated and assembled in a compact form factor (Figure 3e) for potential in vivo imaging application in the human gastrointestinal (GI) tract. The fiberbased collimating system is used for laser excitation, as shown in Figure 3f.

MEMS-Based Photoacoustic Microendoscopy
A MEMS scanner-based photoacoustic microscope (PAM) system's conceptual design has been demonstrated by Chen [42] by taking advantages of both an optical micro-ring resonator and electrostatic comb-drive-actuated MEMS scanner. The ultrasensitive micro-ring resonator with broad bandwidth, developed by Ling [43], is one type of micro-/nano-photonic device which sense an ultrasonic signal using optical approaches. As shown in Figure 4a, a fiber-based optical system setup with pulse laser excitation (wavelength 532 nm), MEMS mirror driving system, real-time data acquisition system has been described in the schematic drawing. The electrostatic MEMS scanner within the package, in Figure 4b, provides the lateral laser beam point-scanning in raster scanning mode at a slow rate. This new PAM imaging system can provide ex vivo optical resolution photoacoustic images of the tissue. To detect the weak photoacoustic signal, the micro-ring resonator is located right under the tissue specimen with acoustic signal coupling media, such as water or ultrasonic gel. To realize the photoacoustic microscopic system in more portable or endoscope-friendly form factor, researchers have been making tremendous efforts on the miniaturization of the imaging

MEMS-Based Photoacoustic Microendoscopy
A MEMS scanner-based photoacoustic microscope (PAM) system's conceptual design has been demonstrated by Chen [42] by taking advantages of both an optical micro-ring resonator and electrostatic comb-drive-actuated MEMS scanner. The ultrasensitive micro-ring resonator with broad bandwidth, developed by Ling [43], is one type of micro-/nano-photonic device which sense an ultrasonic signal using optical approaches. As shown in Figure 4a, a fiber-based optical system setup with pulse laser excitation (wavelength 532 nm), MEMS mirror driving system, real-time data acquisition system has been described in the schematic drawing. The electrostatic MEMS scanner within the package, in Figure 4b, provides the lateral laser beam point-scanning in raster scanning mode at a slow rate. This new PAM imaging system can provide ex vivo optical resolution photoacoustic images of the tissue. To detect the weak photoacoustic signal, the micro-ring resonator is located right under the tissue specimen with acoustic signal coupling media, such as water or ultrasonic gel. driving current for electrothermal scanners. All of the micro-optical components and MEMS-based circumferential scanning systems have been fully integrated and assembled in a compact form factor ( Figure 3e) for potential in vivo imaging application in the human gastrointestinal (GI) tract. The fiberbased collimating system is used for laser excitation, as shown in Figure 3f.

MEMS-Based Photoacoustic Microendoscopy
A MEMS scanner-based photoacoustic microscope (PAM) system's conceptual design has been demonstrated by Chen [42] by taking advantages of both an optical micro-ring resonator and electrostatic comb-drive-actuated MEMS scanner. The ultrasensitive micro-ring resonator with broad bandwidth, developed by Ling [43], is one type of micro-/nano-photonic device which sense an ultrasonic signal using optical approaches. As shown in Figure 4a, a fiber-based optical system setup with pulse laser excitation (wavelength 532 nm), MEMS mirror driving system, real-time data acquisition system has been described in the schematic drawing. The electrostatic MEMS scanner within the package, in Figure 4b, provides the lateral laser beam point-scanning in raster scanning mode at a slow rate. This new PAM imaging system can provide ex vivo optical resolution photoacoustic images of the tissue. To detect the weak photoacoustic signal, the micro-ring resonator is located right under the tissue specimen with acoustic signal coupling media, such as water or ultrasonic gel. To realize the photoacoustic microscopic system in more portable or endoscope-friendly form factor, researchers have been making tremendous efforts on the miniaturization of the imaging To realize the photoacoustic microscopic system in more portable or endoscope-friendly form factor, researchers have been making tremendous efforts on the miniaturization of the imaging system design and the distal scanhead with MEMS technologies. A new handheld photoacoustic microscope (PAM) probe [40], as shown in Figure 5, has been developed recently for potential clinical application. The distal scanhead of the handheld PAM system, 17 mm in diameter and a weight of 162 g, mainly consists of the fiber-based collimator, ultrasound detector, acoustic and photonic beam coupler, and beam scanning system. The handheld PAM system has integrated a newly custom-developed electromagnetic MEMS 2D scanner, shown in Figure 5a. The schematic drawing of the full imaging system is illustrated in Figure 5b, including the high-speed data acquisition system, ultrasonic transducer, and fiber based optics. High-resolution imaging quality with a large FOV using this handheld PAM system has been demonstrated by imaging the blood vessel of a mouse ear, shown in Figure 6. The PAM imaging system has also been used to delineate a human mole to demonstrate its clinical application in delineating melanoma which has the highest death rate among skin cancers and may cause about 9730 deaths the United States.
Micromachines 2018, 9, x FOR PEER REVIEW 6 of 19 system design and the distal scanhead with MEMS technologies. A new handheld photoacoustic microscope (PAM) probe [40], as shown in Figure 5, has been developed recently for potential clinical application. The distal scanhead of the handheld PAM system, 17 mm in diameter and a weight of 162 g, mainly consists of the fiber-based collimator, ultrasound detector, acoustic and photonic beam coupler, and beam scanning system. The handheld PAM system has integrated a newly customdeveloped electromagnetic MEMS 2D scanner, shown in Figure 5a. The schematic drawing of the full imaging system is illustrated in Figure 5b, including the high-speed data acquisition system, ultrasonic transducer, and fiber based optics. High-resolution imaging quality with a large FOV using this handheld PAM system has been demonstrated by imaging the blood vessel of a mouse ear, shown in Figure 6. The PAM imaging system has also been used to delineate a human mole to demonstrate its clinical application in delineating melanoma which has the highest death rate among skin cancers and may cause about 9730 deaths the United States.  Based on an advanced ultra-compact electrothermal MEMS 2D scanner, a novel miniaturized MEMS-based photoacoustic (PA) microendoscope has been recently developed through collaborative team work led by Xi and Xie [44]. This work has been the most advanced PA microendoscopic imaging system and is close to clinical application. The cross-sectional view photograph of the endoscopic packaging is shown in Figure 7a, including a GRIN lens-based fiberbased collimator, electrothermal MEMS scanner, and optical and acoustic coupler. The new MEMSbased PA microendoscope can acquire high-resolution photoacoustic images of tissue specimens. The image performance of this new photoacoustic microendoscope has been demonstrated on a mouse ear, as shown in Figure 8c. system design and the distal scanhead with MEMS technologies. A new handheld photoacoustic microscope (PAM) probe [40], as shown in Figure 5, has been developed recently for potential clinical application. The distal scanhead of the handheld PAM system, 17 mm in diameter and a weight of 162 g, mainly consists of the fiber-based collimator, ultrasound detector, acoustic and photonic beam coupler, and beam scanning system. The handheld PAM system has integrated a newly customdeveloped electromagnetic MEMS 2D scanner, shown in Figure 5a. The schematic drawing of the full imaging system is illustrated in Figure 5b, including the high-speed data acquisition system, ultrasonic transducer, and fiber based optics. High-resolution imaging quality with a large FOV using this handheld PAM system has been demonstrated by imaging the blood vessel of a mouse ear, shown in Figure 6. The PAM imaging system has also been used to delineate a human mole to demonstrate its clinical application in delineating melanoma which has the highest death rate among skin cancers and may cause about 9730 deaths the United States.  Based on an advanced ultra-compact electrothermal MEMS 2D scanner, a novel miniaturized MEMS-based photoacoustic (PA) microendoscope has been recently developed through collaborative team work led by Xi and Xie [44]. This work has been the most advanced PA microendoscopic imaging system and is close to clinical application. The cross-sectional view photograph of the endoscopic packaging is shown in Figure 7a, including a GRIN lens-based fiberbased collimator, electrothermal MEMS scanner, and optical and acoustic coupler. The new MEMSbased PA microendoscope can acquire high-resolution photoacoustic images of tissue specimens. The image performance of this new photoacoustic microendoscope has been demonstrated on a mouse ear, as shown in Figure 8c. Based on an advanced ultra-compact electrothermal MEMS 2D scanner, a novel miniaturized MEMS-based photoacoustic (PA) microendoscope has been recently developed through collaborative team work led by Xi and Xie [44]. This work has been the most advanced PA microendoscopic imaging system and is close to clinical application. The cross-sectional view photograph of the endoscopic packaging is shown in Figure 7a, including a GRIN lens-based fiber-based collimator, electrothermal MEMS scanner, and optical and acoustic coupler. The new MEMS-based PA microendoscope can acquire high-resolution photoacoustic images of tissue specimens. The image performance of this new photoacoustic microendoscope has been demonstrated on a mouse ear, as shown in Figure 8c.  Thus far, several miniaturized photoacoustic microendoscopes have been developed using electromagnetic [46][47][48] or electrothermal scanners [44]. To co-axially steer both laser and ultrasonic beams, a water-immersible electromagnetic MEMS scanner [48] has been custom-made to operate in the ultrasound coupling media. Due to the high detection sensitivity and broader bandwidth with very compact form factor, a micro-ring resonator [43,[49][50][51][52] has attracted more attention and been fully explored, such as the transparent micro-ring for microendoscope applications [51,52].

MEMS-Based Confocal Microendoscopy
Compared to other relatively new imaging modalities, confocal microscopy has been studied for decades. MEMS-based confocal microendoscopes were invented a long time ago since the first seminal work demonstrated by Kino and Dickensheets [53]. Later, researchers have focused on improving the lateral or axial resolution and depth imaging while miniaturizing the confocal microendoscopes using MEMS technologies. For instance, the new MEMS-based 3D confocal microendoscope with a tunable Z-focus has been developed by Xie's team [45] using an advanced electrothermal MEMS scanner with tunable objective lens mounted in the center of the moving Zaxis stage with large translational movement (>300 µm) at low voltage. As shown in Figure 8, the MEMS-actuated tunable objective lens is located at the distal end, which is very close to the tissue specimen. By being fully packaged in stainless steel tubing, the fiber-based microendoscope could potentially be applied for clinical applications on humans (Figure 8b).  Thus far, several miniaturized photoacoustic microendoscopes have been developed using electromagnetic [46][47][48] or electrothermal scanners [44]. To co-axially steer both laser and ultrasonic beams, a water-immersible electromagnetic MEMS scanner [48] has been custom-made to operate in the ultrasound coupling media. Due to the high detection sensitivity and broader bandwidth with very compact form factor, a micro-ring resonator [43,[49][50][51][52] has attracted more attention and been fully explored, such as the transparent micro-ring for microendoscope applications [51,52].

MEMS-Based Confocal Microendoscopy
Compared to other relatively new imaging modalities, confocal microscopy has been studied for decades. MEMS-based confocal microendoscopes were invented a long time ago since the first seminal work demonstrated by Kino and Dickensheets [53]. Later, researchers have focused on improving the lateral or axial resolution and depth imaging while miniaturizing the confocal microendoscopes using MEMS technologies. For instance, the new MEMS-based 3D confocal microendoscope with a tunable Z-focus has been developed by Xie's team [45] using an advanced electrothermal MEMS scanner with tunable objective lens mounted in the center of the moving Zaxis stage with large translational movement (>300 µm) at low voltage. As shown in Figure 8, the MEMS-actuated tunable objective lens is located at the distal end, which is very close to the tissue specimen. By being fully packaged in stainless steel tubing, the fiber-based microendoscope could potentially be applied for clinical applications on humans (Figure 8b). Thus far, several miniaturized photoacoustic microendoscopes have been developed using electromagnetic [46][47][48] or electrothermal scanners [44]. To co-axially steer both laser and ultrasonic beams, a water-immersible electromagnetic MEMS scanner [48] has been custom-made to operate in the ultrasound coupling media. Due to the high detection sensitivity and broader bandwidth with very compact form factor, a micro-ring resonator [43,[49][50][51][52] has attracted more attention and been fully explored, such as the transparent micro-ring for microendoscope applications [51,52].

MEMS-Based Confocal Microendoscopy
Compared to other relatively new imaging modalities, confocal microscopy has been studied for decades. MEMS-based confocal microendoscopes were invented a long time ago since the first seminal work demonstrated by Kino and Dickensheets [53]. Later, researchers have focused on improving the lateral or axial resolution and depth imaging while miniaturizing the confocal microendoscopes using MEMS technologies. For instance, the new MEMS-based 3D confocal microendoscope with a tunable Z-focus has been developed by Xie's team [45] using an advanced electrothermal MEMS scanner with tunable objective lens mounted in the center of the moving Z-axis stage with large translational movement (>300 µm) at low voltage. As shown in Figure 8, the MEMS-actuated tunable objective lens is located at the distal end, which is very close to the tissue specimen. By being fully packaged in stainless steel tubing, the fiber-based microendoscope could potentially be applied for clinical applications on humans (Figure 8b).
Another interesting electrothermally-actuated MEMS fiber scanner has also been invented and fully integrated into the MEMS-based confocal microendoscope [54]. A SEM image of the compact electrothermal MEMS fiber scanner is shown in Figure 9b. As shown in Figure 9, the team from KAIST [54] has recently developed a novel scanning fiber-enabled ultra-thin confocal microendoscope which can be easily inserted into the miniature tool channel of the medical laparoscope, shown in Figure 9c. Another interesting electrothermally-actuated MEMS fiber scanner has also been invented and fully integrated into the MEMS-based confocal microendoscope [54]. A SEM image of the compact electrothermal MEMS fiber scanner is shown in Figure 9b. As shown in Figure 9, the team from KAIST [54] has recently developed a novel scanning fiber-enabled ultra-thin confocal microendoscope which can be easily inserted into the miniature tool channel of the medical laparoscope, shown in Figure 9c. Compared to the conventional single-axis confocal architecture [45], the novel dual-axis confocal (DAC) configuration offers superior dynamic range in the Z-axis with higher axial resolution. Based on the fully-scalable DAC optics architecture, miniaturization using 2D/3D MEMS scanners and micro-optics have been performed during the past ten years. Both electrostatic MEMS scanners and thin-film piezo-electrical (PZT: lead zirconate titanate)-based MEMS scanners have been developed for MEMS-based DAC microendoscopy.
A monolithic thin-film piezo-electrical MEMS scanner [55] (footprint in less than 3.2 mm by 3.0 mm), with both vertical (Z-axis) and lateral (X-axis or Y-axis) scanning capabilities, has been demonstrated for the first time to perform horizontal and vertical cross-sectional imaging. The schematic drawing (Figure 10a) illustrates the integration of the thin-film PZT-based MEMS scanner with multidimensional freedom inside the optical design of the DAC microendoscope. A photograph of the thin-film PZT based MEMS device, which provides large translational motion for Z-axis focus change (>200 µm) and wide tilting angle (> ± 5° mechanically) for lateral scanning, is shown in Figure  10b.  Compared to the conventional single-axis confocal architecture [45], the novel dual-axis confocal (DAC) configuration offers superior dynamic range in the Z-axis with higher axial resolution. Based on the fully-scalable DAC optics architecture, miniaturization using 2D/3D MEMS scanners and micro-optics have been performed during the past ten years. Both electrostatic MEMS scanners and thin-film piezo-electrical (PZT: lead zirconate titanate)-based MEMS scanners have been developed for MEMS-based DAC microendoscopy.
A monolithic thin-film piezo-electrical MEMS scanner [55] (footprint in less than 3.2 mm by 3.0 mm), with both vertical (Z-axis) and lateral (X-axis or Y-axis) scanning capabilities, has been demonstrated for the first time to perform horizontal and vertical cross-sectional imaging. The schematic drawing (Figure 10a) illustrates the integration of the thin-film PZT-based MEMS scanner with multidimensional freedom inside the optical design of the DAC microendoscope. A photograph of the thin-film PZT based MEMS device, which provides large translational motion for Z-axis focus change (>200 µm) and wide tilting angle (>± 5 • mechanically) for lateral scanning, is shown in Figure 10b.
Although new thin-film PZT-based MEMS scanners show promising technical advantages over conventional MEMS devices, their micro-machining processes are still challenging due to the complexity of preparation and patterning of thin-film piezo-electrical materials. On the other hand, the traditional electrostatic MEMS scanner [56][57][58][59][60] has recently been fully explored with unique mechanical flexure designs to meet the requirements from 3D confocal microendoscopic imaging systems [61][62][63][64][65]. As shown in Figure 11, a novel monolithic electrostatic MEMS scanner with switchable lateral and vertical scanning capabilities have been successfully demonstrated with a compact footprint (<3.2 mm × 3.0 mm) for DAC microendoscopes. The new electrostatic scanner is based on the parametric resonance working principle with an in-plane comb-drive configuration. Through design optimization, the driving voltage can be close to 40 V, which is safe for human patients. With cross-sectional depth imaging, MEMS-based DAC microendoscopes may potentially be used for molecular contrast agent-based multi-color fluorescence imaging [66][67][68][69] for colorectal cancer early detection in the human gastrointestinal tract. demonstrated for the first time to perform horizontal and vertical cross-sectional imaging. The schematic drawing (Figure 10a) illustrates the integration of the thin-film PZT-based MEMS scanner with multidimensional freedom inside the optical design of the DAC microendoscope. A photograph of the thin-film PZT based MEMS device, which provides large translational motion for Z-axis focus change (>200 µm) and wide tilting angle (> ± 5° mechanically) for lateral scanning, is shown in Figure  10b.  Although new thin-film PZT-based MEMS scanners show promising technical advantages over conventional MEMS devices, their micro-machining processes are still challenging due to the complexity of preparation and patterning of thin-film piezo-electrical materials. On the other hand, the traditional electrostatic MEMS scanner [56][57][58][59][60] has recently been fully explored with unique mechanical flexure designs to meet the requirements from 3D confocal microendoscopic imaging systems [61][62][63][64][65]. As shown in Figure 11, a novel monolithic electrostatic MEMS scanner with switchable lateral and vertical scanning capabilities have been successfully demonstrated with a compact footprint (<3.2 mm × 3.0 mm) for DAC microendoscopes. The new electrostatic scanner is based on the parametric resonance working principle with an in-plane comb-drive configuration. Through design optimization, the driving voltage can be close to 40 V, which is safe for human patients. With cross-sectional depth imaging, MEMS-based DAC microendoscopes may potentially be used for molecular contrast agent-based multi-color fluorescence imaging [66][67][68][69] for colorectal cancer early detection in the human gastrointestinal tract. By combining two separate electrostatic MEMS scanners, lateral (XY) and vertical (Z-axis) scanners [70], respectively, a new 3D MEMS scan engine-based DAC microendoscope with multicolor achromatic optics design could perform real-time 3D volumetric imaging in the tissue specimen for both clinical applications and system biology studies on live rodents. Furthermore, monolithic multiple degree-of-freedom or a 3D thin-film PZT-actuated micro-stage [8,9,10] will also potentially provide the 3D imaging without increasing the optical design complexity. As alternative approaches for miniature confocal system design, tunable optics-based [71] and micro-grating-based spectral encoded confocal microendoscopes [72] can realize depth imaging and en-face imaging with fewer scanning components.

MEMS-Based Multiphoton Microendoscopy
Not only being used in OCT and confocal system, electrostatic MEMS scanners have already demonstrated their critical roles for miniaturized multiphoton microendoscopic imaging system development since the first prototype was demonstrated by Piyawattanametha in 2006 for mice brain in vivo imaging [73,74]. Extended applications [75][76][77] have also been studied using the electrostatic MEMS 2D scanner-enabled multiphoton microendoscope, including femtosecond laser-based microsurgery [77].
Recently, handheld and endoscopic multiphoton microscopes have been developed with custom-made electrostatic MEMS 2D scanners [78] and Er-doped fiber laser [79,80]. For example, as shown in Figure 12, a new MEMS based two-photon fluorescent microendoscope [78] with a compact distal end is packaged in the stainless steel tube. The 2D MEMS resonant gimbal-based scanner can perform a lateral scan around the X-and Y-axes. With administration of Hoechst (nucleic acid stain), in vivo fluorescence imaging has been demonstrated in the distal colon of CDX2P-NLS By combining two separate electrostatic MEMS scanners, lateral (XY) and vertical (Z-axis) scanners [70], respectively, a new 3D MEMS scan engine-based DAC microendoscope with multi-color achromatic optics design could perform real-time 3D volumetric imaging in the tissue specimen for both clinical applications and system biology studies on live rodents. Furthermore, monolithic multiple degree-of-freedom or a 3D thin-film PZT-actuated micro-stage [8][9][10] will also potentially provide the 3D imaging without increasing the optical design complexity. As alternative approaches for miniature confocal system design, tunable optics-based [71] and micro-grating-based spectral encoded confocal microendoscopes [72] can realize depth imaging and en-face imaging with fewer scanning components.

MEMS-Based Multiphoton Microendoscopy
Not only being used in OCT and confocal system, electrostatic MEMS scanners have already demonstrated their critical roles for miniaturized multiphoton microendoscopic imaging system development since the first prototype was demonstrated by Piyawattanametha in 2006 for mice brain in vivo imaging [73,74]. Extended applications [75][76][77] have also been studied using the electrostatic MEMS 2D scanner-enabled multiphoton microendoscope, including femtosecond laser-based microsurgery [77].
Recently, handheld and endoscopic multiphoton microscopes have been developed with custom-made electrostatic MEMS 2D scanners [78] and Er-doped fiber laser [79,80]. For example, as shown in Figure 12, a new MEMS based two-photon fluorescent microendoscope [78] with a compact distal end is packaged in the stainless steel tube. The 2D MEMS resonant gimbal-based scanner can perform a lateral scan around the X-and Y-axes. With administration of Hoechst (nucleic acid stain), in vivo fluorescence imaging has been demonstrated in the distal colon of CDX2P-NLS Cre;adenomatosis polyposis coli (CPC;Apc) mouse model, which mimics human colorectal cancer diseases, as shown in Figure 13. A single-frame from a video sequence is shown in Figure 13a while the post-processed image after averaging (5 frames) is shown in Figure 13b. Compared to the images of H and E slides, the sub-cellular high-resolution microscopic imaging system could potentially provide histology-like imaging. diseases, as shown in Figure 13. A single-frame from a video sequence is shown in Figure 13a while the post-processed image after averaging (5 frames) is shown in Figure 13b. Compared to the images of H and E slides, the sub-cellular high-resolution microscopic imaging system could potentially provide histology-like imaging.  Due to the footprint size of the electrostatic MEMS scanner, the distal end of the microendoscopic scanhead could not be easily miniaturized to less than 2.0 mm. However, the bulk PZT tube-based fiber scanner could potentially be fabricated with an ultra-thin wall and an outer diameter less than 1.5 mm so that the piezo tube fiber scanner enabled multiphoton microscope's distal end could be very small. Fiber scanner-based miniaturized multiphoton microscope was first demonstrated by Helmchen and Denk in 2001 for in vivo imaging on rodents' brains [81]. In addition, similar to the very small piezo tube-based fiber scanner [82,83], a bulk piezo sheet-based 2D raster-mode fiber scanner has also been investigated for multiphoton microendoscopic label-free imaging on unstained tissue specimens [84]. diseases, as shown in Figure 13. A single-frame from a video sequence is shown in Figure 13a while the post-processed image after averaging (5 frames) is shown in Figure 13b. Compared to the images of H and E slides, the sub-cellular high-resolution microscopic imaging system could potentially provide histology-like imaging.  Due to the footprint size of the electrostatic MEMS scanner, the distal end of the microendoscopic scanhead could not be easily miniaturized to less than 2.0 mm. However, the bulk PZT tube-based fiber scanner could potentially be fabricated with an ultra-thin wall and an outer diameter less than 1.5 mm so that the piezo tube fiber scanner enabled multiphoton microscope's distal end could be very small. Fiber scanner-based miniaturized multiphoton microscope was first demonstrated by Helmchen and Denk in 2001 for in vivo imaging on rodents' brains [81]. In addition, similar to the very small piezo tube-based fiber scanner [82,83], a bulk piezo sheet-based 2D raster-mode fiber scanner has also been investigated for multiphoton microendoscopic label-free imaging on unstained tissue specimens [84].
Based on the extensive experience on the multiphoton imaging system development, the team Due to the footprint size of the electrostatic MEMS scanner, the distal end of the microendoscopic scanhead could not be easily miniaturized to less than 2.0 mm. However, the bulk PZT tube-based fiber scanner could potentially be fabricated with an ultra-thin wall and an outer diameter less than 1.5 mm so that the piezo tube fiber scanner enabled multiphoton microscope's distal end could be very small. Fiber scanner-based miniaturized multiphoton microscope was first demonstrated by Helmchen and Denk in 2001 for in vivo imaging on rodents' brains [81]. In addition, similar to the very small piezo tube-based fiber scanner [82,83], a bulk piezo sheet-based 2D raster-mode fiber scanner has also been investigated for multiphoton microendoscopic label-free imaging on unstained tissue specimens [84].
Based on the extensive experience on the multiphoton imaging system development, the team led by Li at Johns Hopkins University has recently developed a novel piezo tube-based fiber scanner-enabled miniaturized two-photon and second harmonic imaging system [85]. Aimed for label-free functional histology in vivo, the new fiber-optic scanning two-photon endomicroscope mainly consists of several key components, including a flexible double cladding fiber (DCF) for laser excitation and harvesting emission light, GRIN lens, and the very small piezo tube-based fiber scanner. A miniaturized custom-made objective with longitudinal focal shift has been developed by collaborating with GRINTECH (GmbH, Jena, Germany). A phase diffractive grating is sandwiched between two GRIN elements, as shown in Figure 14b.  The novel fiber optic multiphoton microendoscope developed by Liang [85] performs both twophoton fluorescence (2PF) and second harmonic generation (SHG) label-free structural imaging in vivo on small animal and human patients. As shown in Figure 15a,b, the overlay of intrinsic 2PF and SHG images have been acquired ex vivo from mouse liver. The emission signal was detected through two spectral channels: 496-665 nm (green, 2PF signal) and 435-455 nm (red, SHG signal). Figure 15c,d show the in vivo two-photon auto-fluorescence images of the mucosa of mouse small intestine, while the two detection channels are 417-477 nm for NADH (green) and 496-665 nm for FAD (red). Timelapse SHG images of a cervical collagen fiber network have been acquired through intact ectocervical epithelium of cervices dissected from preterm-birth mouse models Figure 15e    The novel fiber optic multiphoton microendoscope developed by Liang [85] performs both two-photon fluorescence (2PF) and second harmonic generation (SHG) label-free structural imaging in vivo on small animal and human patients. As shown in Figure 15a,b, the overlay of intrinsic 2PF and SHG images have been acquired ex vivo from mouse liver. The emission signal was detected through two spectral channels: 496-665 nm (green, 2PF signal) and 435-455 nm (red, SHG signal). Figure 15c,d show the in vivo two-photon auto-fluorescence images of the mucosa of mouse small intestine, while the two detection channels are 417-477 nm for NADH (green) and 496-665 nm for FAD (red). Time-lapse SHG images of a cervical collagen fiber network have been acquired through intact ectocervical epithelium of cervices dissected from preterm-birth mouse models Figure 15e and normal pregnant mice (Figure 15f) at gestation day 15.
Electrostatic comb-drive actuated MEMS scanner and piezo fiber scanners have demonstrated their great potential in the development of multiphoton microendoscopes. In addition to those two actuation mechanisms, electrothermal MEMS scanners [86,87] have also been proposed for fiber scanning in the multiphoton imaging system although it may not be quite ready for clinical applications yet. two spectral channels: 496-665 nm (green, 2PF signal) and 435-455 nm (red, SHG signal). Figure 15c,d show the in vivo two-photon auto-fluorescence images of the mucosa of mouse small intestine, while the two detection channels are 417-477 nm for NADH (green) and 496-665 nm for FAD (red). Timelapse SHG images of a cervical collagen fiber network have been acquired through intact ectocervical epithelium of cervices dissected from preterm-birth mouse models Figure 15e and normal pregnant mice (Figure 15f) at gestation day 15. Electrostatic comb-drive actuated MEMS scanner and piezo fiber scanners have demonstrated their great potential in the development of multiphoton microendoscopes. In addition to those two actuation mechanisms, electrothermal MEMS scanners [86,87] have also been proposed for fiber scanning in the multiphoton imaging system although it may not be quite ready for clinical applications yet.

Fluorescence Wide-Field Endoscopy
Since its first application in the scanning-probe microscopes, miniaturized piezo tube based fiber scanner have demonstrated its great potential in several optical imaging modalities, such as OCT [34,36], or multiphoton [82,88]. By taking advantages of its ultra-thin form factor, the piezo tube has also been used in the scanning fiber endoscope (SFE) for wide-field imaging with both reflective [2] and fluorescent modes [89]. Compared to other MEMS scanner-based fluorescence imaging systems [90], the SFE-based endoscopic imaging system could have a much smaller outer diameter. Recently, Savastano and Zhou have demonstrated multimodal laser-based angioscopy [91] for structural, chemical, and biological imaging of atherosclerosis using the miniature catheter imaging system. As shown in Figure 16a, the scanning fiber endoscope excites tissues by scanning blue (424 nm), green (488 nm), and red lasers (642 nm) in a spiral pattern. Backscattered (reflectance) light and the fluorescent signal is collected by a ring of multimode fibers located in the periphery of the scanner housing and shaft and conducted to a data acquisition computer for image reconstruction. The optical system can be packaged with an outer diameter of 2.1 mm (left) or 1.2 mm (right) endoscopes, as shown in Figure 16b.

Fluorescence Wide-Field Endoscopy
Since its first application in the scanning-probe microscopes, miniaturized piezo tube based fiber scanner have demonstrated its great potential in several optical imaging modalities, such as OCT [34,36], or multiphoton [82,88]. By taking advantages of its ultra-thin form factor, the piezo tube has also been used in the scanning fiber endoscope (SFE) for wide-field imaging with both reflective [2] and fluorescent modes [89]. Compared to other MEMS scanner-based fluorescence imaging systems [90], the SFE-based endoscopic imaging system could have a much smaller outer diameter. Recently, Savastano and Zhou have demonstrated multimodal laser-based angioscopy [91] for structural, chemical, and biological imaging of atherosclerosis using the miniature catheter imaging system. As shown in Figure 16a, the scanning fiber endoscope excites tissues by scanning blue (424 nm), green (488 nm), and red lasers (642 nm) in a spiral pattern. Backscattered (reflectance) light and the fluorescent signal is collected by a ring of multimode fibers located in the periphery of the scanner housing and shaft and conducted to a data acquisition computer for image reconstruction. The optical system can be packaged with an outer diameter of 2.1 mm (left) or 1.2 mm (right) endoscopes, as shown in Figure 16b. A description and summary of performance for several different MEMS scanning mechanisms that are currently being developed for in vivo endomicroscopy are summarized in Table 1.  A description and summary of performance for several different MEMS scanning mechanisms that are currently being developed for in vivo endomicroscopy are summarized in Table 1.

Conclusions
In this review, we present a review of the latest advancements of MEMS actuator-based optical microendoscopy. High precision in manufacturing coupled with various optical/mechanical functionalities derived from MEMS fabrication techniques make these components well suited to integrate into many optical based microendoscopy. Nonetheless, it is crucial to gain understandings of other underlying principles, such as life-cycle, sizes, speed, material properties, force, operating ranges, and power consumptions to achieve optimum performance before uniting all components altogether. In addition, clinical considerations, such as usage simplicity and ergonomics, cannot be overlooked as those parameters are used to dictate the overall designs and selections of MEMS actuators for optical microendoscopy. Overall, electrostatic-based actuation is one of the most popular actuators employed in endoscopic-based imaging despite the fact that it provides both relatively moderate actuation force and limited scanning ranges. However, the advantages are their ease of fabrication, lower complexity of integration to endoscope packages, and relative fast scanning speed to help reduce motion artifacts. All in all, MEMS actuator-based optical microendoscopy has been showing great promise to deliver high-performance imaging on par with traditional microscopy in aiding medical diagnosis procedures.

Conflicts of Interest:
The authors declare no conflict of interest.

Abbreviations
The following abbreviations are used in this manuscript: