1478 IEEE PHOTONICS TECHNOLOGY LETTERS, VOL. 25, NO. 15, AUGUST 1, 2013

3-D Confocal Laser Scanning Microscopy Based ona Full-MEMS Scanning System

Lin Liu and Huikai Xie

Abstract— We have developed a confocal microscope usingMEMS devices for laser scanning in all three axes. This full-MEMS laser scanning system is equipped with a MEMS lensscanner for deep scanning and a MEMS mirror for two-axislateral scan. The MEMS lens scanner can move an objective lensaxially by 515 µm at only 3.2 V. The two-dimensional MEMSmirror can achieve ± 20° optical scan angle at 4.5 V. Two-dimensional and three-dimensional confocal reflectance imagesof microparticles embedded in PDMS and acute rat brain tissueare obtained. The axial and lateral resolutions are measured as9.0 and 1.2 µm, respectively.

Index Terms— MEMS mirror, MEMS lens, confocalmicroscopy, 3-D scan, thermal actuation.

I. INTRODUCTION

CONFOCAL laser scanning microscopy (CLSM) hasgained extensive applications in biomedical imaging,

industrial inspection and metrology for its superior resolution,high contrast and powerful optical-sectioning capacity [1].CLSM relies on a pinhole to screen out the out-of-focus blurso that the information at specific depths of the sample canbe acquired, enabling optical sectioning and 3-D imaging [1].If CLSM is extended to endoscopic imaging, the diagnosisof precancerous lesions can be performed in vivo and nonin-vasively instead of excisional biopsy. The major challenges,however, are the miniature light scanners that can performlarge-range scan under low voltage. 3-D scanners are requiredto fully employ the optical sectioning capacity of CLSM andto generate 3-D confocal images.

MEMS technology is a promising solution. Confocal micro-scopes based on miniature MEMS scanners have been exten-sively studied [2]–[9]. Maitland et al. and Shin et al.both reported confocal microscopes employing 2-D electro-static MEMS mirrors for lateral scan but no depth scan[2], [3]. The authors reported a confocal microscope witha MEMS lens scanner for depth scan [4], but a motor-controlled stage was used for lateral scan. Several groupshave reported MEMS-based confocal microscopes capableof 3-D scan, but the depth scans were obtained by motor-driven sliding stages [5]–[7]. The instability from frictionand the limitations of size, speed and cost are the potentialissues of motor-based depth scan. Jeong et al. proposed a3-D confocal microscope using a 2-D electrostatic MEMSmirror for lateral scan and a 1-D electrostatic MEMS mirror

Manuscript received February 13, 2013; revised April 23, 2013; acceptedJune 2, 2013. Date of publication June 20, 2013; date of current versionJuly 12, 2013. This work was supported by the U.S. National ScienceFoundation under Award #1002209.

The authors are with the Department of Electrical and Computer Engi-neering, University of Florida, Gainesville, FL 32611-6130 USA (e-mail:linliu86@ufl.edu; hkxie@ece.ufl.edu).

Color versions of one or more of the figures in this letter are availableonline at http://ieeexplore.ieee.org.

Digital Object Identifier 10.1109/LPT.2013.2267794

Fig. 1. Schematic design of the MEMS-based 3-D CLSM.

for depth scan. The lateral scanner was driven at 190 V and100V, and the axial scanner was driven at resonance and 180V[8]. Gorecki et al. developed a 3-D confocal microscope bystacking an electrostatic microlens for x-y-axis scan and anelectrostatic microlens for z-axis scan [9]; a z-scan range of100 μm was achieved at 50 V but the lateral scan range wasonly 30 μm even at 200 V.

Here we present a CLSM based on a miniature full-MEMS3-D scanning system. The lateral scan is performed by a 2-DMEMS mirror with large scan angle under low driving voltage.The depth scan is accomplished by a MEMS lens scanner withlarge tunable depth at low voltage. This letter integrates the3-D full-MEMS scan to confocal microscopy and demon-strates the capability and feasibility of this scan system inconfocal imaging application. With florescent imaging incor-porated in the near future, this technology is promising forhigh-resolution in vivo imaging.

II. 3-D CONFOCAL SCANNING MICROSCOPE

A. Imaging System Design

The schematic diagram of the 3-D CLSM system is shownin Fig. 1. The collimated laser beam ( λ = 638 nm) emittedfrom a laser diode (Blue Sky Research) passes through aspatial filter. Then the light is collimated to a 1 mm-diameterbeam. After passing through a beam splitter, the light beamis scanned in x- and y- directions by a MEMS mirror. Afterthat, a MEMS lens scanner with an integrated objective lens(Thorlabs) focuses the beam into the sample, and meantimescans the objective in z-direction. The light returned from thesample is collected by the objective lens and descanned bythe MEMS mirror and the beam splitter. Finally, the lightis focused to the pinhole by a lens, picked up by an APD(Hamamatsu), and acquired into a PC via a data acquisitioncard (DAQ). The pinhole diameter is 25 μm. The system isnon-telecentric since the objective is axially scanned by theMEMS lens scanner that results in varying distance betweenthe MEMS mirror and the objective. The initial distancebetween the mirror plate and the end surface of the objectiveis about 2.0 mm.

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LIU AND XIE: 3-D CLSM BASED ON A FULL-MEMS SCANNING SYSTEM 1479

Fig. 2. (a) SEM of a lens scanning platform. (b) Photograph of an assembledlens scanner. (c) SEM of bimorph actuators. (d) SEM of a MEMS mirror.(e) SEM of a LSF actuator.

B. MEMS Scanners

The enabling devices are MEMS scanners. The MEMSlens scanner has a scanning platform (Fig. 2(a)) with anobjective lens assembled on it (Fig. 2(b)). The platform has anopening in the center for light to pass through. The platform issymmetrically supported by four electrothermal lateral-shift-free (LSF) actuators [10]. Good stability and robustness areachieved by making bimorph beams wide and connecting alarge array of bimorph beams (Fig. 2(c)) to the platform. Theobjective is an aspheric lens with a diameter of 2.4 mm, a backfocal length of 0.88 mm and a NA of 0.55. The objective lensis glued to the platform by UV glue. The platform is elevatedupward at about 800 μm after the device release due to theresidual stresses in the bimorph beams. The elevation of theplatform drops to about 550 μm after the lens is assembled.The footprint of the lens scanner is 3.3 mm × 3.3 mm. Thelens scanner generates a vertical displacement of 515 μmat only 3.2 V (Fig. 3(a)). The frequency response of theassembled lens scanner is shown in Fig. 3(b) with the firstresonance peak at about 30 Hz. The MEMS mirror, as shownin Fig. 2(d), has a 1 mm× 1 mm mirror plate supported by fouridentical and symmetrically arranged LSF actuators (Fig. 2(e)).The footprint of the MEMS mirror is 2 mm × 2 mm. Themirror scans ± 20° (optical) at 4.5 V (Fig. 3(c)). The resonantfrequency of the MEMS mirror is 475 Hz (Fig. 3(d)). Boththe MEMS lens scanner and mirror were fabricated using acombined surface- and bulk-micromachining process [10].

III. IMAGING EXPERIMENTS AND RESULTS

A. Imaging System Characterization

The axial and lateral resolutions have been characterized,as shown in Fig. 4(a) and Fig. 4(b). The axial resolutionwas measured by moving a mirror in z-direction with amotorized linear stage through the focal plane of the objectiveand recording the change of the power level detected by theAPD. The FWHM axial resolution was found to be 9.0 μm.The lateral resolution was measured by laterally translatinga reflective edge on the focal plane and the 10%–90% edgewidth was measured as 1.2 μm. The effective NA of thesystem is 0.35 due to the fact that the laser beam does notfully fill the objective. The theoretical axial resolution and

Fig. 3. (a) Z displacement vs. voltage of the assembled lens scanner.(b) Frequency response of the assembled lens scanner. (c) Optical scan anglevs. driving voltage of the MEMS mirror. (d) Frequency response of the MEMSmirror.

Fig. 4. (a) Axial resolution measurement. (b) Lateral resolution measurement.(c) Reflectance image of group 7 elements in a USAF resolution target. Thefield-of-view is about 120 μm × 120 μm. The smallest elements are 2.2 μmwide.

lateral resolution are 0.81 μm and 6.75 μm, respectively.The degradation is mainly due to some misalignments in thesystem. Fig. 4(c) is an en face 2-D image of the elements inGroup 7 of a USAF resolution target acquired by the CLSMsystem.

B. Confocal Imaging Results

2-D and 3-D confocal reflectance images have been takenon a PDMS sample with micro-particles embedded inside.The micro-particles are alloy particles with the sizes rangingfrom around 1 μm to tens of microns. The actuator paircontrolling the x-scan was differentially driven with a ramp

1480 IEEE PHOTONICS TECHNOLOGY LETTERS, VOL. 25, NO. 15, AUGUST 1, 2013

Fig. 5. (a) Photographs of the PDMS sample. (b)-(e): 2-D confocalreflectance images of the sample at different depths. (f) shows the stack of(b) ∼ (e) with some particles labeled. (g) A 3-D volume-rendered image ofthe micro-particles embedded in PDMS.

Fig. 6. (a) A 2-D confocal reflectance image of the rat brain tissue slice.(b) A 3-D volume-rendered image of the brain tissue slice.

waveform of 0 ∼ 4.2 V at 500 Hz. The scan in x-direction isdriven near the resonance peak for large scan angle and forhigh scan speed. The actuator pair for y-direction scan wasdifferentially driven with a ramp waveform of 0 ∼ 3.5 V at1 Hz. The frame rate is 1 frame/sec. All the actuators ofthe lens scanner were simultaneously driven with a rampwaveform at 1 mHz for the z-direction depth scan. Fig. 5shows a picture of the PDMS sample (a) and 2-D reflectanceconfocal images acquired with the CLSM at different depthsof the sample ((b)–(e)). The driving voltages of the MEMSlens scanner from (b) to (e) were 0.5, 0.6, 0.7 and 0.8 V,corresponding to the axial displacements of 25, 35, 45 and55 μm, respectively. As shown in Fig. 5(f), different particles

appeared and disappeared at different imaging depths. Fig. 5(g)is a 3-D image of the sample reconstructed by stacking the2-D image slices. The imaging volume is 120 μm × 120 μm× 270 μm.

Confocal imaging experiments have also been performed ona 150 μm thick slice of acute brain tissue. Fig. 6(a) shows a2-D en face reflectance image acquired by the CLSM systemwith an image size of 120 μm × 120 μm. Fig. 6(b) is avolume-rendered 3-D confocal reflectance image of the acutebrain tissue with an image volume of 120 μm × 120 μm ×400 μm.

IV. CONCLUSION

In conclusion, we have developed a CLSM using MEMSdevices for both lateral and axial scan. The full-MEMS 3-D scanning system has been demonstrated as a viable wayto achieve large-scan-range 3-D confocal imaging at lowvoltage and small size. Both 2-D and 3-D confocal reflectanceimages of micro-particles and acute brain tissue have beenobtained by this system. The axial and lateral resolutions are9.0 μm and 1.2 μm, respectively. The future studies willbe focused on integrating this full-MEMS 3-D scanner intoan endoscopic imaging probe to realize 3-D in-vivo confocalimaging. Miniature objective lens module design will also beinvestigated to further improve the resolution and FOV ofthe imaging system. The imaging speed can be improved byemploying faster lateral scanning micromirrors.

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