1208 JOURNAL OF MICROELECTROMECHANICAL SYSTEMS, …

1208 JOURNAL OF MICROELECTROMECHANICAL SYSTEMS, VOL. 23, NO. 5, OCTOBER 2014

Magnetic-Actuated Stainless Steel Scanner forTwo-Photon Hyperspectral Fluorescence Microscope

Youmin Wang, Y. Daghan Gokdel, Nicolas Triesault, Lingyun Wang,Yu-Yen Huang, and Xiaojing Zhang

Abstract— We present a fluorescence two-photon laser scan-ning microscope that uses a soft-magnetic stainless steelmicroscanner to generate a 2-D Lissajous scanning pattern.The wide-scanning low-voltage driven gimbaled torsional stain-less steel microelectromechanical systems (MEMSs) scanneris designed and then fabricated using electrical dischargemachining. This technology offers unique advantages by allowinglarger mirror surface areas (4 mm × 5 mm) to enhance thefluorescence collection efficiency at low incoming signal level, andproviding a rapid prototyping and low-cost alternative to silicon-based MEMS devices, particularly when large displacementsand large field of view are required. A maximum total opticalscan angle (TOSA) of 20.6° at 112 Hz for a drive powerof 200 mW is required for the slow-scan movement, whereasthe fast-scan movement occurs at the resonance frequency of1268 Hz and has a TOSA of 26.6° using a drive power of400 mW. The soft-magnetic microscanner incorporated in thetwo-photon hyperspectral fluorescence microscope demonstratesits applicability in two-photon hyperspectral imaging of circulat-ing cancer cells, stem cells, and biological tissue with extrinsicfluorophores. [2013-0226]

Index Terms— Stainless steel microscanner, two-photonmicroscopy, hyperspectral imaging, electrical dischargemachining.

I. INTRODUCTION

COMPARED to the various microscopy techniquesemployed in these systems, two-photon laser scanning

microscope (TPLSM) has shown great promise as a versatilenon-invasive fluorescence imaging tool over the last twodecades [1]–[3]. TPLSM improves on conventional confo-cal fluorescence microscopy by using a near infrared (NIR)femtosecond laser pulses to excite fluorophores through thesimultaneous absorption of two photons within 10–15 fs[4], [5]. Taking the advantage of using a typically femtosecondlaser source, TPLSM reduces tissue photo-damage, increasespenetration depth [6], [7], and alleviates the tuning difficulty

Manuscript received July 17, 2013; revised February 21, 2014; acceptedFebruary 23, 2014. Date of publication May 6, 2014; date of current versionSeptember 29, 2014. This work was supported by the National Institute ofHealth. Subject Editor S. Merlo.

Y. Wang and L. Wang are with the Department of Electrical and ComputerEngineering, University of Texas at Austin, Austin, TX 78712 USA (e-mail:[email protected]; [email protected]).

Y. D. Gokdel, N. Triesault, Y.-Y. Huang, and X. Zhang are with theDepartment of Biomedical Engineering, University of Texas at Austin, Austin,TX 78712 USA (e-mail: [email protected]; [email protected];[email protected]; [email protected]).

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

Digital Object Identifier 10.1109/JMEMS.2014.2308573

of optical system by removing the pinhole required in con-focal microscopy [8]. In addition, with the determination ofabsorption spectrums of many extrinsic (e.g. FITC, DAPI,quantum dots) or intrinsic (e.g. collagen fiber, NADH, FAD)fluorophores labeling the biological structures and biochem-istry functions, the advent of two-photon microscopy (TPM)made it possible of revealing the tissue characteristic byspectral analysis using single wavelength NIR excitation formultiple fluorophores [2].

While broadly applied in various cancer diagnosis appli-cations [9]–[11], research work is currently focused on theminiaturization of TPM for use in minimally invasive handheldimaging systems. While TPM is still demanding more costlyparts at current stage comparing with other imaging modalitiessuch as confocal system, its unique features such as deeperpenetration depth, alleviated heating issue, and wider excita-tion spectrum for intrinsic fluorophores interest researches onthe miniaturization of it. A portable two-photon imaging probeused to view suspected tumor sites noninvasively could replacecostly and painful biopsy procedures and therefore improvetreatment, reduce processing cost, and lessen interpretationinaccuracy in remote areas that lack adequate clinical centersand healthcare facilities [12]. Various types of miniaturizedlaser scanning TPM schemes have been developed, includingscanning cantilever mechanisms utilizing fiber tips [13], [14],small lens [15], [16] and a microelectromechanical (MEMS)scanning micromirror [17]–[20].

Microelectromechanical system (MEMS) technologies areuniquely suited to combining actuators for guiding light invery small volumes with micro-optical elements for in vivobeam scanning and imaging. The scaling laws in opticsenable miniaturized imaging probes with functionality thatcannot practically be achieved with traditional technologies.MEMS design processes that integrate complementary metal-oxide-semiconductor (CMOS) circuitry on the same chip asthe MEMS device are widely seen as the solution to thisproblem, and have gained significant attention in recent years.Replacing the bulky galvanometers typically used as scanningunits with MEMS instrumentation is a key step allowingfor smaller and more portable laser scanning microscopes[21]–[23]. Various types of MEMS scanner actuation schemeshave been developed in categories of electrothermal [24],electrostatic [25], piezoelectric [26], and electromagnetic [27]actuations. Streamlined silicon based MEMS micromirrors,driven by an electrostatic comb drive, have been previ-ously demonstrated and applied in handheld confocal, optical

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WANG et al.: MAGNETIC-ACTUATED STAINLESS STEEL SCANNER 1209

Fig. 1. Schematic of two-photon fluorescence microscope based on the magnetic-actuated micro-scanner.

coherence tomography (OCT) systems [28]–[32]. However,typical electrostatic comb drive structures usually suffer fromdrawbacks such as high voltages for actuation, which limittheir use on medical devices of which safety restrictionsare carefully enforced. Compared to electrostatic actuation,magnetic actuation benefits from its larger scanning angle andsmaller driving voltage making it ideal for in vivo applicationswhere patient safety is a priority [33], [34]. To further maxi-mize the driving voltage versus scanning angle ratio, one routebeside numerous innovations on device structure is to seeknew material for better scanning imaging performance. Variousmaterials have been explored to enhance the MEMS scanneroptical scanning features [35]–[37]. However, most of themstill suffer from complicated fabrication process comparingwith standard semiconductor fabrication process for MEMSdevices, which restricts their mass use in a point-of-care med-ical device we desire. In this paper, we present a two-photonfluorescence microscope that uses a soft-magnetic stainlesssteel micro-scanner as its two-dimensional (2D) scanning unit.We have previously presented the preliminary results andcharacterization of the stainless steel scanner [38]. The novelstainless steel scanner utilizes a simple design and fabricationprocess of electrical discharge machining (EDM), which pro-vides a larger scanner surface area to enhance fluorescencelight collection efficiency for wide-field skin cancer detection,while requiring a low voltage (<10V) to drive the largescanning angle (>20°). The presented design of the EDMfabricated stainless steel scanner and the optical design ofthe MEMS integrated hyperspectral fluorescence TPM wascharacterized by imaging spike stained circulating tumor cells(CTC) and the mesenchymal stem cells (MSCs) attached onmicrocarrier beads (MCB) in a PEGylated fibrin gel.

II. PRINCIPLE OF OPERATION

The imaging process in TPLSM begins with the deliveryof an ultrafast infrared laser pulse train to the optical scan-ning device which steers the laser light onto the imaging

sample through various well-aligned optical imaging compo-nents. Typically, a visible-infrared dichroic mirror is placedat the intersection of illumination and detection arms toallow only visible fluorescence signal to pass through andbe detected by an opto-electro convertor. Fig. 1 depicts areal-time hyperspectral fluorescence based TPM which uses amagnetically actuated stainless steel micro-scanner as the lightsteering unit and a Ti-sapphire femtosecond laser (80 MHzMai Tai, Spectra Physics) as the light source. The illuminationTi-sapphire laser was selected to be centered at 800nm, whichis capable of exciting various fluorophores such as DAPI,fluorescein isothiocyanate (FITC), yellow fluorescent protein(YFP) and Alexa 568 [39], [40]. Although these fluorophoreshave different one-photon excitation (OPE) spectrums, due todifferent quantum mechanical selection rules, a fluorophore’stwo-photon excitation (TPE) spectrum is usually not equivalentto the double wavelengths of its OPE spectrum, allowingfor the TPE spectrum for various fluorophore’s to overlap.As shown in Fig. 1, the illumination beam coming from theTi-sapphire laser is reflected by a dichroic mirror with 775nmcutting-off wavelength (FF775, Semrock, Inc.), then deflectedby the stainless steel micro-scanner, and is focused onto thesample with the help of an objective lenses with variousmagnification levels. The forwarding illumination laser poweris controlled at 50mW before passing through the dichroicmirror. The power controlling was realized by placing by athin film femtosecond laser line polarizer (420-0126, EksmaOptics) on the imaging path mounted on a rotational stage.The polarizer can be rotated to approach the matching laserlight polarization to decrease the optical power.

A custom made software kit was developed in Java andLabVIEW for parallel driving signal generation, data acqui-sition, real-time image construction, and post processing forhyperspectral imaging. The software kit I/O port generatesa low voltage signal to modulate the flux generated by anelectrocoil to actuate both axes of the micro-scanner at thedevice’s resonant frequencies and create a Lissajous scanning


pattern on the tissue sample. The TPE fluorescence light isguided back by the scanner and reflected by the dichroicmirror before striking a volume phase holographic (VPH)grating. The dispersed light is detected by a 16-channel linearphotomultiplier (PMT) array (H11459, Hamamatsu Photonics,Quantum Efficiency ≈30%) where the spectral informationis acquired and analyzed by our custom developed softwarekit to generate the hyperspectral images at 10 frames persecond (FPS).

To create a closed-loop feedback control system, a positionsensitive device (PSD, S5990-01, Hamamatsu Photonics) wasincorporated into the forward illumination path in order todetermine location of micro-scanner in real time which allowsthe system to compensate for damping effects and improveimage stability. For torsional scanners, the energy loss dueto nonlinear viscous forces, i.e. air damping, and materialfatigue creates an unstable relationship between the appliedtorque and deflection angle. Also, stochastic perturbations,such as vibration, driving electronics jitter, thermal changes,and shock, can introduce mechanical misalignment into themirror position [41]. This feedback control system compen-sates for disturbances or imperfections in the micro-scannerwhich may cause the FOV to fluctuate to reduce imageblurring. Without a feedback system, the end user would needto estimate the amplitude, frequency, offset, and phase of theLissajous pattern in order to construct an image. The newsystem incorporating the PSD automates and simplifies theimage construction operation.

A portion of the forward illumination light is directed tothe PSD by a 5:95 beamsplitter which placed between themicro-scanner and the objective lens. The PSD will generatetwo sinusoidal patterns which depict the relative X and Yposition of the micro-scanner and help analyze the nonlin-ear behavior of the torsion suspensions and the oscillatorystability.

For data acquisition, the signal collected by the 16 channelPMT was recorded by a 16 analog input channel data acquisi-tion (DAQ) card (National Instruments PXIe-6358) at the sam-pling rate of 1M samples per second, and the two input channelDAQ card (National Instruments PCI-6111) was used recordthe PSD signal. The sampling rate was determined accordingto the consideration of the scanning speed of the scanner tocreate a 500 × 500 pixel map, at an imaging frame rate of10 fps. To help remove unwanted environmental noise from thePSD signal, a simple band pass circuit was implemented beforedata acquisition. A continuous digital pulse train triggeringsignal, with a frequency equal to acquisition rate, was routedto the programmable free interface (PFI) channel of bothDAQ cards in order to synchronize the outgoing micro-scanneractuation signal and the incoming 16 channel florescence dataand the PMT signal which represents the fluorescence datanonlinearly spaced across the visible spectrum.

The synchronized fluorescence and PSD data is sent fromLabVIEW to a Java based program via TCP/IP. The PSDsignal is passed through a digital filter in order to removefrequencies irrelevant to micro-scanner actuation. An auto-correlation algorithm is used to determine the phase delaybetween the known actuation signal and the filtered PSD

signal. The correct phase delay allows the Java program toplot the intensity values along the phase-corrected Lissajousscanning pattern to create an output window displaying all16 images.

III. DESIGN AND FABRICATION

To design a low-cost, durable, and high-performanceportable MEMS based TPM for real-time imaging, themicro-scanner needs to have the following desirable char-acteristics: (1) large optical scanning angle which translatesinto a larger FOV; (2) comparatively large mirror surfacearea and high-reflectance for better fluorescence light col-lection efficiency; (3) high fast-scan to slow-scan ratio toincrease the image resolution; (4) smaller total system sizeand power consumption to ameliorate the portability of themicroscope for in vivo use; (5) utilize low-cost and abundantstructural material. Stainless steel is an alternative micro-scanner fabrication material to conventionally used silicon.This material offers various inherent advantages like simpleactuation mechanisms with low actuation voltages, simpleand low-cost fabrication processes, and preferable mechanicalproperties [42]. Utilizing EDM technique which has precisiondown to 50um in the industry, micromirror device on themillimeter scale could be fabricated with the all-ferromagneticmaterial to ease actuation. In this work, we propose a 2Dgimbaled stainless steel micro-scanner where both the mirrorand the outer frame rotate about the fast and slow axes at theirrespective resonant frequencies to generate the Lissajous scan-ning pattern from the incoming laser beam for use in TPM.

Fig. 2(a) and (b) depict the 2D torsional gimbaledmicro-scanner structure, the magnetic actuation scheme andschematic of the coil used for magnetic actuation, respec-tively. The design parameters are listed in Table I. A smallexternal permanent magnet creates a strong DC external fieldused to magnetize the ferromagnetic material (stainless steelmaterial type 420) into its saturated state thus creating afixed magnetization vector M that remains in-plane due to themicro-scanner’s anisotropic design. The function generatorthat is controlled by the PC shown in Fig. 1, provides a fre-quency dependent current input I(ω) to the driving electrocoilwhich creates an H-field to actuate the steel micro-scanner.

A conceptual drawing about the employed magnetic actua-tion scheme is shown in Fig. 2. The applied H-field magne-tizes the ferromagnetic material and creates a magnetizationvector M that remains in plane due to the shape anisotropyand whose magnitude and direction is dependent on theapplied external field. North and south poles of the magne-tized ferromagnet experience forces F1 and F2. Assuming acircular mirror shape with a diameter D and thickness tm, themagnitude of these two forces can be simply computed by

F = m Dtm H cosϕ (1)

A closed-in view of magnetic actuation between the coil andthe scanner is depicted in Fig. 3 in which a cantilever beammaking an out-of-plane bending movement together with anelectro-coil providing N · I ampere-turn magnetomotive force(MMF) and a permanent magnet with a magnetic path lengthof Lmag are present.


Fig. 2. Scanner Design and Operations. (a) Computer aided design (CAD) for the stainless steel scanner. (b) Schematic representation of forces and torquesunder non-uniform H-field for the proposed steel scanner [43].

TABLE I

STAINLESS STEEL SCANNER DESIGN PARAMETERS

Assuming the fringing fields are negligible in the system,the current applied to the electro-coil creates a magnetic fieldwhich is equal to

H ∼= N I

L(2)

This magnetic field H, provides a flux ϕ in the magneticcircuit which is illustrated in above figure. In lumped elementmodeling [44], the line integral of H around a suitable closedpath is called the magnetomotive force and it is symbolizedas FMM. In this magnetic circuit FMM becomes equal to [45].

FM M = N I = Hmag Lmag + HgapLgap + Hair Lair (3)

where Hmag is the H-field in the permanent magnet, Hgap isthe H-field in the gap between the actuator and the electro-coiland Hair is the H -field in between the permanent magnet andthe far-side of the electro-coil as illustrated in Fig. 3(a). At thispoint, it is important to note that Lgap is the distance between

the permanent magnet and the electro-coil. It is a function thatchanges with the bending cantilever �z.

Lgap = z0 − �z (4)

and Lair is the effective distance between the permanentmagnet and the far-side of the electro-coil. To a first orderapproximation, it is equal to

Lair ∼= 2z0 + Lcoil (5)

It is a known fact that the flux in a magneticcircuit is always the same. Thus one can say that in thismagnetic circuit, φ = φmag = φair = φgap The flux throughany cross-sectional surface S in the magnetic circuit can bedefined as

φ =∫

SB.d A = Bair A (6)

Therefore, it is possible to write down the magnetic fluxdensities in term flux φ as below

Bmag = φ

Amag; Bair = φ

Aair; Bgap = φ

Agap(7)

where Amag, Aair and Agap are the areas of the relatedregions as illustrated in Fig. 3(a), respectively. Here, Agapis approximately equal to Aair. Having also known that inthe permeable material, Bmag = μmag Hmag and in the airBair = μ0 Hair where μ0 is the permeability of the free space,one can rewrite the following,

FM M = N I = φ

μmag AmagLmag + φ

μ0 AairLgap(�z)

+ φ

μ0 AairLair (8)

Combining above equations, the magnetomotive forcebecomes equal to

FM M = N I = φ

[Lmag

μmag Amag+ Lcoil + 3z0 − �z

μmag Aair

](9)

In this equation, the term in square brackets give thereluctances of the magnet and the total air gap in series. It canbe said that since the leakage flux in neglected, this equation


Fig. 3. (a) Schematic drawing of magnetic actuation setup. (b) Schematic of the coil used for magnetic actuation. (c) Magnetic circuit equivalent.(d) Electro-mechanical analogy.

gives the upper limit of φ. The above magnetic actuationsystem can be represented as a magnetic circuit diagram asshown in Fig. 3(d). In this circuit, Ampere’s circuital law canbe applied as an analog to Kirchoff’s voltage law. Therefore,we can rewrite above equation in a more compact form

FM M = N I =∑

k

RkφK = φ(Rmag + Rair ) (10)

where Rmag and Rair are equal to

Rmag = Lmag

μmag Amag(11)

And

Rair = Lcoil + 3z0 − �z

μ0 Aair(12)

where FMM, Rk and φk are the analog of electromotive force,resistance and current passing through the circuit, respectively.In Fig. 3(d), the mechanical equivalent circuit of the system isillustrated. In other words, the displacement of the actuator �zis directly related to the applied current I of the electro-coilwith above equations.

As a result a net force FNET = F2−F1 is exerted on themirror as shown in Fig. 2(b) [46]. This resulting net forcestemming from the H-field gradient is pulling the magnet to

the coil as a function of the distance between the centers of themagnet and the coil. This net force triggers the structure to anunwanted out-of-plane pumping mode. However, the structureis specifically designed as a double clamped torsional beamwith a central mass. Thus, thanks to its geometry, structureis inclined to make a torsional movement at its fundamentalmode and moreover, at that operation frequency the out-of-plane pumping movement has a quite low displacement whichcan be easily neglected compared to the torsional movement.

This implication suggests that we can simply ignore theeffect of FNET while actuating desired torsional steel scanners.However, there exists also torque component acting on thestructure, which tends to minimize the overall energy in anactuator system by aligning the magnetization with the fieldlines of the external magnetic field. Torque created in counter-clockwise direction can be calculated as [47].

T = F1�Lm

2cosϕ + F2

�Lm

2cosϕ (13)

where ϕ is the angle between the easy-axis of the scanner andthe upper-surface of the electro-coil and Lm is the length ofthe magnetic material, in this case the inner mirror major axis.If we combine above equations, torque T becomes

T = M H V cosϕ (14)


Fig. 4. Finite-element analysis of the micro-scanner showing the torsional movement for both the slow and fast-scanning axis at 112 and 1292 Hz respectively.

where V is the volume of the magnetic material. The torque Tis dependent on both magnetization M and applied externalH-field. It is important to note that these two parameters arealso functions of the electro-coil driving current (t) = Isin(ωt).If the offset level of the applied electro-coil current is zeroand if a magnetic DC field created by a permanent magnet isabsent in the system, then the torque T created by a small,non-saturation external H-field can be approximated to [48]

T = [M H V/2](1 − cos(2ωt))cosϕ (15)

This equation tells us that created torque has a DC com-ponent and an AC component whose value is changing withcos(2ωt). It is important to note that the frequency of the ACcomponent is twice the drive current frequency ω. It meansthat in order to trigger the structure into its designed funda-mental resonant mode, a coil driving frequency that is exactlyhalf of the designed value must be used.

In order to increase the torque and eventually the dis-placement, related M and H values of the system must beincreased. A permanent magnet creating a strong DC externalfield (increase in H-field) can be used to magnetize theferromagnet into saturation (increase in the magnetization M)in the actuation of the scanners. Another approach is to usea bigger ferromagnet to increase the volume V. Dependingon the case and used bulk material; this approach requiresto use a thicker magnetic layer on the scanner or to simplyuse a bigger ferromagnet providing a higher value of V. If apermanent magnet is preferred to magnetize the ferromagnetas in the case, the torque equation becomes

T = MS(H sin(ωt))V cosϕ (16)

Torque T and eventually the actuation is now bidirectionalsince the direction of Ms does not change at all becauseof the added permanent magnet in the system. Moreover,the frequency of the forcing function becomes equal to theelectro-coil driving frequency. When the frequency of the ACcurrent driving the electrocoil matches the resonance peaks ofthe vibration modes of the micro-scanner, the displacement isenhanced by the mechanical quality factors of the respectivemotion.

Finite-element analysis (FEA) results of the designed micro-scanner are shown in Fig. 4 for slow and fast scan axes,respectively. Outer frame generates an angular displacementabout the slow-scan axis at the resonant frequency of 125.4 Hzcorresponding to the refresh rate of the 2D Lissajous scanningwhereas the inner mirror generates a scan-line that is orthog-onal to the slow-scan mode at approximately 1223.7 Hz.

Standard EDM process is used to fabricate the designedstructure. After fabricating and defining the shape of thescanner, a 5 μm thick SU8 photoresist is spin coated onto theinner mirror in order to decrease the roughness of the steelsurface. Following this, a 50nm thick Ti is sputtered onto theSU8 mold as an adhesive layer. Then a 300 nm thick Al issputtered onto the Ti layer to enhance the reflectivity of theinner mirror surface, and the RMS roughness reached as muchas 5% for the Al film deposited. A holder for the scanner isdesigned and fabricated using three dimensional (3D) printingtechnologies from acrylonitrile butadiene styrene (ABS) ther-moplastic material as shown in Fig. 5. The fabrication of thescanner system is finalized with the placement of the off-the-shelf electrocoil and the permanent magnet as shown inFig. 5(b).

IV. EXPERIMENTAL RESULTS

Fabricated structure is shown in Fig. 5 along with its ABSplastic holder. The steel scanner that is shown is 2 cm by 2 cmin length, with the inner mirror 6 mm in semi-major axis and4.8 mm in semi-minor axis respectively. The plastic holder wasdesigned to ensure the micro-scanner’s mounted securely andto reduce the vibrational power loss, which could be deliveredto the micro-scanner as scanning actuation power. Fig. 5(b)summarizes the entire scanning actuation system containingthe electrocoil, plastic holder.

The peak displacements and corresponding resonance fre-quencies of the actuator for different drive levels are plottedin Fig. 6(a), while the single-axis resonant scanning is shownin Fig. 6(b). Actuated under the resonant frequencies, themicromirror was scanning with a Lissajous fashion as shownin Fig. 6 (c). As can be seen from this figure, there is a linearrelationship between the drive voltage and the displacement,


Fig. 5. Micro-scanner and Device Assembly. (a) Fabricated 2 cm × 2 cm stainless steel scanner mounted on an ABS holder. (b) Actuation module togetherwith the mounted stainless steel scanner in the bench-top TPM characterization system.

Fig. 6. Characterization of the Micro-scanner. (a) Voltage dependence of the scanning amplitude and the resonant frequency of the steel scanner. (b) Slow-scanline acquired at 112 Hz. (c) Lissajous pattern of the laser beam scanning path generated from laser beam reflected the scanning dual axis stainless steel mirror.

as expected as the drive level is increased, electrical springsoftening effect is observed [49], which results in a small2% downward shift in the resonance frequency. Fig. 6(b)exemplifies the acquired scan line at 112Hz and Fig. 6(c)shows the Lissajous scanning pattern when both axes actuated.The bright vertical line in the center is due to the deformationand uneven tilting of the mirror surface. Techniques such asrim & cross-rib structures [37] are expected to be incorporatedto minimize the dynamic mirror deformation.

Fig. 7(a) and (b) plots the mechanical transfer characteristicsof the tested device as a function of the operation frequencyfor slow and fast scan movements, respectively. The slow-scanoperation takes place at 112Hz whereas the fast-scan motiongenerates a scan line at 1268Hz with a constant drive powerof 200mW.

The hyperspectral fluorescence imaging performance of themicro-scanner based TPM is demonstrated by imaging ofspike stained CTCs and the MSCs attached on micro beads

in a PEGylated fibrin gel. During cancer metastasis, CTCsare shed from the primary malignant melanoma site into thesurrounding vasculature which the cells may seed additionaltumor sites and dramatically worsen the patient’s cancerprognosis. Thus CTCs are widely recognized as indicators ofthe patient’s cancer disease status [50]. However, CTCs areusually rare and are extremely difficult to detect. NumerousCTC screening methodologies have been explored and somedistinguish themselves as novel miniaturized point-of-careapproaches [51], but nearly all require a painful and time-consuming isolation, enrichment and characterization process.Carrying the advantages of high selectivity, small size, andlow photo-toxicity, MEMS based TPM integrated with CTCmicrofluidic devices shows promise and may prove itself as thenext groundbreaking point-of-care CTC screening technique,echoing recent endeavors on optofluidic cell analysis [52].MEMS enabled portable hyperspectral TPM would create amuch more efficient CTC detection system due to the device’s


Fig. 7. Total optical scanning angles of the gimbaled steel scanner versus drive frequency. (a) Outer frame. (b) Inner mirror.

Fig. 8. (a) Stainless steel MEMS micromirror based TPM imaging calibration using COLO 205 cancer cell line after CTCs screening with a microfluidicdevice. Scale bar is 20μm. (b) Control image from Olympus BX51 desktop fluorescent microscope for comparison.

inherent speed and compact size allowing it to be incorporatedinto a point-of-care device. The device’s accuracy would onlybe dependent on the selectivity of two-photon fluorescenceimaging, which is enhanced and facilitated by various extrinsicfluorophores staining techniques. Here, the fluorescence TPMutilizes with a water-immersion multi-photon objective lens(CFI Fluor 60XW, N.A. = 1, Nikon Corp.) with the MEMSmicromirror for imaging. As a result a FOV of 200μm ×400μm and lateral resolution of 1μm was achieved for thefluorescence TPM, with an imaging rate of 10 FPS. Accordingto the Gaussian profile descriptive equation Zr = nπr2

0/λwhere r0 denotes the lateral resolution and Zr meaning theRayleigh range of the Gaussian beam, the axial resolution isexpected to be 2.94μm. Fig. 8 was acquired from the 540nmwavelength window of the hyperspectral fluorescence imagingof cultured cancer cell line of COLO 205 after the screeningexperiment. Though imaging sensitivity is expected to furtherenhance through utilizing a higher quantum efficiency photodetector, the preliminary characterization results illustratedhere from the spike CTCs depicts the great potential of a fasthyperspectral screening imaging system as a new diagnostictool for fast CTCs calibration after screening.

Three 3D physiological and morphological analyses areessential in verifying the cellular phenotype in tissue engi-neering [53]. MEMS enabled TPM provides a portable andconvenient solution for the tissue 3D physiological and mor-phological fluorescence characterization. Fig. 9 demonstratesthe hyperspectral depth-sectional characterization of the phys-iological and morphological features of the MSCs attachedon a MCB in a PEGylated fibrin gel. As the characterizationtissue model, the human mesenchymal stem cells are attachedto the MCB array. 2D hyperspectral images of 16 wavelengthswere imaged by the MEMS enabled TPM at depths at 0μmto 206μm from the top of the MCB, with the depth sequencegap of 1 μm. Fig. 9(a) provides the orthogonal view ofthe MSCs attached on MCB, while Fig. 9(b)–(e) providethe cross-sectional view of the sample at multiple depths.All the figures of Fig. 9 are the composite of the imagesacquired from the 480 nm and 540 nm wavelength windowsof the hyperspectral fluorescence MEMS TPM. No obviousoverlapping was observed for the emission wavelength bandsfor DAPI and Calcein AM. The blue components notate theDAPI stained cell nuclei area, while the green componentsnotate the Calcein AM stained whole cell plasma area.


Fig. 9. MEMS TPM images acquired from and the MSCs attached on a MCB in a PEGylated fibrin gel. (a) Three-dimensional rendering of a sequence of207 lateral MEMS TPM images of the MSCs in isometric view. Sequence depth gap is 1 μm. (b-e) Selection of lateral images from an imaging depth of 42,58, 63 and 78μm in the x-y planes. Field of view in all lateral images is 0.6mm × 1mm. Scale bars are 100μm.

The microcarrier bead assays with human MSCs were usedin the angiogenesis assays in a fibrin-based in vitro model.While morphological characterization is critical to determinewhether desired cell types were achieved for tissue engineer-ing, the successful morphometric measurements and quantifi-cation of the assay demonstrates the validity of using MEMSTPM to fulfill this requirement. Moreover, with the power oflarge imaging range, high resolution, low photo-toxicity andsmall form factor, MEMS TPM is ideal, if not essential, toreal-time monitor the process of tissue-development on site.

V. CONCLUSION

To reduce the scanning driving voltage requirements andexplore new materials for MEMS micromirror fabrication, anovel stainless steel micro-scanner with large mirror surfacearea and easy fabrication profile was designed, fabricated andincorporated in a hyperspectral fluorescence TPM system. Thestainless steel micromirror requires a driving voltage lowerthan 10V while providing large scanning amplitude (>20°).Large imaging field of view was thus required by the stainlesssteel micromirror based microscope with an imaging rate of10 FPS. Future miniaturization of the portable TPM probeusing the stainless steel MEMS micromirror has been shown tobe a possible and valid approach from the optical imaging per-formance characterization of the MEMS enabled TPM. Vari-ous biological tissue samples, including spike CTCs and MSCsattached to MCB with extrinsic fluorophores were used for thecharacterization and evaluation for the imaging performanceof MEMS enabled hyperspectral TPM. Beyond traditional useof MEMS TPM in endoscopic cancer diagnosis and imagingguided femtosecond laser surgery, the successful imaginginvestigation of these samples opens up the applicationsof MEMS TPM for point-of-care CTC fast screening, prefer-ably in an optofluidic fashion, and real-time morphologicalmonitoring of tissue development on site. The right next-stepaction we need to perform from the current research, is theincorporation of the device into the handheld imaging system

and seeking even smaller version of the device. Oriented bythe needs in these emerging fields, MEMS TPM could finditself integrated in various forms, fitting into those applicationswith demonstrated appealing features of higher resolution,deeper imaging into tissue and lower phototoxicity in a furtherminiaturized version.

ACKNOWLEDGMENT

We are grateful to Julie Rytlewski and Dr. Laura Suggsfrom the University of Texas at Austin for their preparationsMSCs gel samples and their knowledge input on tissue engi-neering applications. We thank Elaine Ng and Oanh NgocHoang from UT BME for their assistances in CTC samplepreparation.

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Youmin Wang is currently a Staff R&D Engineerat Himax Display Inc. He obtained his Ph.D. fromthe University of Texas at Austin in 2013, under thesupervision of Dr. John X.J. Zhang at the Laboratoryof Micro and Nano-scale Manipulation, Imaging andSensing. He received his B.E. degree in ElectricalEngineering from Shanghai Jiao Tong University in2008 and his M.S. degree in Electrical and ComputerEngineering from UT Austin in 2010. His researchinterests include: Micro Electro-mechanical System(MEMS) design and fabrication, portable and hand-

held medical imaging instrumentations, confocal reflectance and fluorescencemicroscopes, laser-tissue interaction mechanisms and various micro-scannerapplications.


Y. Daghan Gokdel received his B.S. degree in Elec-trical Engineering from Bilkent University, Ankara,Turkey, in 2004 and the M.S. and Ph.D. degrees allin electrical engineering from Bogazici University,Istanbul, Turkey, in 2007 and 2011, respectively,with a focus on microsystems. In 2012 he was aPostdoctoral Research Associate with University ofTexas at Austin, Department of Biomedical Engi-neering. He is currently an Assistant Professor ofElectrical and Electronics Engineering with IstanbulBilgi University, Turkey where he is also the found-

ing Director of Microsystem Laboratory. His research interest includes design,fabrication and characterization of MEMS.

Nicolas Triesault received his B.E. from the Bio-medical Engineering Department of the Universityof Texas at Austin in 2013 and is now working atINT Inc. as a software engineer.

Lingyun Wang received the B.S. degree in elec-tronics and information system from East ChinaNormal University, Shanghai, China, in 2000, theM.S.degree in electrical engineering from Universityof Alaska Fairbanks, Fairbanks AK, USA, in 2002and the Ph.D. degree in electrical and computerengineering from University of Texas at Austin,Austin, TX, USA, in 2012. He is currently withthe Printing and Personal System Organization ofHewlett-Packard as MEMS process engineer in Cor-vallis, OR, USA. His research interests include

antenna, numerical modeling, ionosphere scintillation analysis, nanophotonics,photonic crystal devices, nanomaterial processing, and integrated optoelec-tronics for biomedical imaging.

Yu-Yen Huang obtained his Ph.D. in the Depart-ment of Biomedical Engineering, the Universityof Texas at Austin in 2013. He is currentlya Research Associate in the Thayer EngineeringSchool, Dartmouth College. His research interestsinclude the development of the nano/micro-electro-mechanical system (NEMS/MEMS) based devicefor optical imaging and biological applications. Hismain research is focused on the design and fabrica-tion of the immunomagnetic microfluidic screeningsystem for circulating tumor cells (CTCs) detection

and analysis, which could improve the recognition and monitoring of pre-cancer of breast cancer.

Xiaojing Zhang received his Ph.D. degree fromStanford University, CA, USA, in 2004. He wasa Research Scientist at Massachusetts Institute ofTechnology (MIT), Cambridge, before joining thefaculty at University of Texas of Austin in 2005.He is currently an associate professor at the Uni-versity of Texas of Austin (UT Austin) in theDepartment of Biomedical Engineering, with jointaffiliations with Institute for Cellular and MolecularBiology (ICMB), Microelectronics Research Centerand Texas Materials Institute. His research focuses

on exploring bio-inspired nanomaterials, scale-dependent biophysics, andnanofabrication technology, towards developing new diagnostic devices andmethods on probing complex cellular processes and biological networkscritical to development and diseases. He has published more than 120 peerreviewed papers and proceedings, presented more than 45 invited seminarsworldwide, and filed over 15 U.S. patents (five patents issued). His researchfindings have been highlighted in many public media, and were licensed to twocompanies: CardioSpectra, Inc., and Nano Lite Systems, Inc. He has organizedmany major conferences in the area of MEMS/BioMEMS, nano technologiesand biomedical engineering. In addition to being the principle investigator ofmany major grants from U.S. federal agencies such as NIH, NSF, and DARPA,he also received many prestigious awards, including: the Wallace H. CoulterFoundation Early Career Award for Translational Research in BiomedicalEngineering in 2006, the British Council Early Career RXP Award in 2008,NSF Faculty Early Career Development Program (NSF CAREER) Award in2009-2014, DARPA Young Faculty Award in 2010, and an invitee to attendU.S. National Academy of Engineering, Frontiers of Engineering (NAE-FOE)program in 2011, the NAE Frontiers of Engineering Education (NAE FOEE)program in 2012, and subsequently China-America Frontiers of EngineeringSymposium (CAFOE) program in 2013.

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