In vivo brain imaging using a portable 2.9 g
two-photon microscope based on a
microelectromechanical systems scanning mirror
Wibool Piyawattanametha,1,2Eric D. Cocker,1Laurie D. Burns,1Robert P. J. Barretto,1Juergen C. Jung,1
Hyejun Ra,3Olav Solgaard,3and Mark J. Schnitzer1,4,*
1James H. Clark Center for Biomedical Engineering and Sciences, Stanford University, Stanford,
California 94305, USA
2National Electronics and Computer Technology Center, Pathumthani, Thailand 12120
3Edward L. Ginzton Laboratory, Stanford University, Stanford, California 94305, USA
4Howard Hughes Medical Institute, Stanford University, Stanford, California 94305, USA
* Corresponding author: firstname.lastname@example.org
Received March 19, 2009; accepted April 29, 2009;
posted June 29, 2009 (Doc. ID 108932); published July 23, 2009
We present a two-photon microscope that is approximately 2.9 g in mass and 2.0?1.9?1.1 cm3in size and
based on a microelectromechanical systems (MEMS) laser-scanning mirror. The microscope has a focusing
motor and a micro-optical assembly composed of four gradient refractive index lenses and a dichroic micro-
prism. Fluorescence is captured without the detected emissions reflecting off the MEMS mirror, by use of
separate optical fibers for fluorescence collection and delivery of ultrashort excitation pulses. Using this mi-
croscope we imaged neocortical microvasculature and tracked the flow of erythrocytes in live mice. © 2009
Optical Society of America
OCIS codes: 180.4315, 170.2520, 170.1470, 170.5810, 170.3890, 110.2760.
An important aim of recent research on nonlinear op-
tical microscopy has been to create miniaturized
laser-scanning microscopes and endoscopes for bio-
medical investigation and clinical purposes. Such de-
vices would propel a range of applications benefiting
from portable instrumentation and should ultimately
be more economical than tabletop instrumentation.
Among key potential applications are imaging mi-
crovascular function or cellular dynamics in live or
behaving animals , minimally invasive diagnostics
, and image-guided therapeutic intervention .
Promising nonlinear contrast modalities for use in
miniaturized microscopes include two-photon fluores-
cence [1,4,5], second-harmonic generation [2,6], co-
herent anti-Stokes Raman scattering , and stimu-
lated Raman scattering .
Crucial design issues with miniaturized nonlinear
imaging devices concern laser-scanning mechanisms.
Several mechanisms have been explored for two-
photon imaging, including cantilever fiber-scanners
operating at resonant [9,10] or non-resonant [11–13]
frequencies, as well as microelectromechanical sys-
tems (MEMS) scanning mirrors [3,6,14,15]. Cantile-
ver scanners generally have longer lengths than
MEMS scanners and if operated at resonance also re-
strict the choice of scanning frequencies. MEMS
scanners allow batch fabrication, generally at least
modest abilities to zoom and pan across the imaging
field, and flexibility in the adjustment of scan rates.
To date, two endoscopes for two-photon imaging
based on MEMS scanners have been described [3,16],
both of which are larger than the microscope we
present below. One endoscope involved a double-clad
optical fiber and routed fluorescence signals in reflec-
tion off the MEMS mirror , which aids miniatur-
ization but reduces robustness to light scattering
when imaging deep in optically dense tissue. The re-
port on the other endoscope included only spatially
filtered images . Neither device was shown capable
of imaging in live subjects or to have sufficient sensi-
tivity for fast physiological measurements. Here,
we describe a 2.9 g microscope based on a two-
dimensional MEMS scanner and the use of this in-
strument for tracking erythrocyte flow in live mice.
We constructed a portable two-photon microscope
that is 2.9 g in mass and ?2.0?1.9?1.1 cm3in vol-
ume. A hollow-core bandgap fiber (Blaze Photonics)
FWHM) from a tunable Ti:sapphire laser (Tsunami,
Spectra-Physics) to the microscope. In this fiber the
pulses incur negligible self-phase modulation ,
which arises in conventional single-mode or double-
clad fibers with the 0.01–1 nJ pulse energies com-
monly used for two-photon excitation. Group-velocity
dispersion in the bandgap fiber vanishes at ?800 nm,
so we tuned the laser emission to ?790–810 nm to
obviate dispersion precompensation .
The light delivered to the microscope passes
through an aspherical collimating lens (LightPath)
and reflects off the MEMS scanner Figs. 1a–d. The
scanner is 1 mm?1 mm, has a gimbal design, and
contains six banks of electrostatic vertical comb ac-
tuators for steering the laser beam in two angular di-
mensions (Fig. 1b). We batch fabricated scanners of
this type on a double-side polished, double-silicon-on-
insulator wafer using four deep reactive ion-etching
steps . A double-sided printed circuit board (Figs.
1b and 1c) holds the scanner die and attaches to its
voltage control wires.
After reflecting off the scanner, the illumination
enters a micro-optical assembly (made to our design
by GRINTECH, GmbH) of four gradient refractive in-
dex (GRIN) lenses and a 2-mm-wide dichroic micro-
prism (short pass, ?700 nm) that separates the IR
August 1, 2009 / Vol. 34, No. 15 / OPTICS LETTERS
0146-9592/09/152309-3/$15.00© 2009 Optical Society of America
excitation and visible emission (Fig. 1d). Two of the
GRIN lenses, a 2 mm diameter scan lens (0.5 NA,
0.14 pitch, Ag doped) and a 1.8 mm diameter tube
lens (0.2 NA, 0.22 pitch, Li doped), serve together as
a beam expander. The illumination under fills the
back aperture of the objective, a 1 mm diameter
GRIN lens (0.58 NA, 0.23 pitch, Th doped), and is fo-
cused to the specimen with a lower NA than that for
fluorescence collection. The working distance from
the tip of the objective to the specimen plane is
280 ?m in air.
The full aperture of fluorescence emissions col-
lected by the objective returns through the tube lens,
the microprism, and a GRIN collection lens (0.5 NA,
0.27 pitch, Ag doped) that projects the emissions onto
a multimode polymer fiber (1.96 mm diameter core,
0.51 NA, Edmund Optics). This fiber guides the light
to a remotely placed photomultiplier tube. Our detec-
tion configuration, in which emissions are not routed
back though the scanner, differs from that of several
prior fiber-optic two-photon microscopes that used a
double-clad fiber for both illumination delivery and
fluorescence collection and thus required emission
The microscope’s housing, base plate, and internal
imaging head are composed of carbon-fiber filled
polyetheretherketone (PEEK), a conductive plastic
chosen to minimize static charge accumulation that
could damage the scanner. The base plate permits
mounting position. The housing allows axial adjust-
ments in the position of the imaging head, for coarse
focusing. For fine focusing, a dc micromotor (Faul-
haber) drives a shuttle, made of PEEK, that adjusts
the axial position of the bandgap fiber. The shuttle’s
in the microscope’s
range of movement is 1.5 mm, yielding 270 ?m of fo-
cal adjustment in the specimen.
We generally used a raster-scanning pattern, with
one axis of the scanner oscillating at resonance and
the other at the frame rate of 1–15 Hz. In dc opera-
tion, the maximum optical angular ranges for the in-
ner and outer axes of the scanner are about ±5° and
±4.3°, respectively (Fig. 2a). In ac operation, scan
rates can be adjusted from near dc to over the me-
chanical resonant frequencies
0.56 kHz for the inner and outer axes, respectively
To characterize the microscope’s resolution, we im-
aged 100 nm diameter fluorescent beads. The FWHM
of bead images in cross section yielded transverse
and axial resolutions
10.3±0.3 ?m (mean±s.e.m.; n=3 beads), respectively
(Figs. 2c and 2d). These values are comparable to
those we reported for a 0.48 NA resonant fiber-
scanning two-photon microendoscope . Images had
400?135 pixels, and the maximum field of view was
295 ?m?100 ?m.
To demonstrate in vivo usage, we studied neocorti-
cal microvasculature in adult mice anesthetized dur-
ing imaging by intraperitoneal injection of ketamine
and xylazine (80–100 and 16–20 mg/kg body mass,
respectively). We made a 2.5 mm diameter cranioto-
my (stereotactic coordinates: −2 mm relative to
lambda, −2.5 mm lateral) and injected into a tail vein
200 ?L of fluorescein isothiocyanate-dextran dye
(MW 2,000,000) diluted in saline solution ?58.3 mg/
mL? to brightly label the blood plasma. After placing
of 1.08 kHzand
of 1.29±0.05 ?m and
control the MEMS scanner and focusing micromotor. b, The
scanner die is wirebonded onto electrodes on the printed
circuit board (PCB). c, Computer-aided-design model of the
microscope, in a cut-away view. d, Laser illumination (red
a, Portable two-photon microscope. Electrical lines
voltage, and b, frequency response function for inner (blue
solid curve) and outer (red dashed curve) axes of a 1 mm
?1 mm MEMS scanner. For each axis time-varying signals
were applied to one of the two opposing comb banks. c, Lat-
eral and d, axial resolutions were determined as the
FWHM of curve fits (Airy function squared, c, and Gauss-
ian, d) to normalized, cross-sectional images of 100 nm di-
ameter fluorescent beads. Data from three beads (circle,
square, and diamond symbols) and fits from one bead (solid
curves) are shown.
(Color online) a, Optical deflection angle versus dc
OPTICS LETTERS / Vol. 34, No. 15 / August 1, 2009
the microscope atop the mouse’s head, we imaged
vessels near the neocortical surface (Figs. 3a–3c).
To observe microcirculation, we switched to a line-
scanning mode in which the outer axis was scanned
at resonance ?560 Hz? but the inner axis was held
fixed. This allowed tracking of individual erythro-
cytes in vessels in which flow was parallel to the line
scanning. Individual erythrocytes appeared as dark
diagonal streaks against the labeled blood plasma in
the resulting plots of space ?y? versus time ?t? (Figs.
3d–3f). The streaks’ slopes ?dy/dt? yielded velocity
steeper streaks indicating faster velocities (Fig. 3d).
In the examples of Figs. 3d–3f, flow speeds were
±s.d.), respectively, within ranges reported previ-
ously for mouse  and rat  neocortical capillar-
In summary, we created a portable, 2.9 g two-
photon microscope based on a two-dimensional
MEMS scanner and made the initial demonstration
of physiological measurements using a MEMS-based
two-photon imaging device. Future applications of
MEMS scanners likely include physiological studies
in alert animals or human subjects, as well as cellu-
lar level diagnostics. Our approach of using a MEMS
mirror for laser scanning should also be applicable to
other nonlinear optical contrast modalities.
This work was supported by grants to M. J. S. from
the Office of Naval Research (ONR), the National Sci-
ence Foundation (NSF), the NSF Center for Biopho-
tonics, the National Institutes of Health (NIH) Nano-
medicine Development Center for Optical Control of
Biological Function, and the Packard and Beckman
Foundations, NSF and Stanford Graduate Research
fellowships (L. D. B.), training grants from NIH
(E. C. and R. P. J. B.), and NECTEC (W. P.).
1. F. Helmchen, M. S. Fee, D. W. Tank, and W. Denk,
Neuron 31, 903 (2001).
2. M. E. Llewellyn, R. P. Barretto, S. L. Delp, and M. J.
Schnitzer, Nature 454, 784 (2008).
3. C. L. Hoy, N. J. Durr, P. Chen, W. Piyawattanametha,
H. Ra, O. Solgaard, and A. Ben-Yakar, Opt. Express
16, 9996 (2008).
4. B. A. Flusberg, J. C. Jung, E. D. Cocker, E. P.
Anderson, and M. J. Schnitzer, Opt. Lett. 30, 2272
5. W. Göbel,J.N. Kerr,
Helmchen, Opt. Lett. 29, 2521 (2004).
6. L. Fu, A. Jain, H. Xie, C. Cranfield, and M. Gu, Opt.
Express 14, 1027 (2006).
7. F. Legare, C. Evans, F. Ganikhanov, and X. S. Xie, Opt.
Express 14, 4427 (2006).
8. C. W. Freudiger, W. Min, B. G. Saar, S. Lu, G. R.
Holtom, C. He, J. C. Tsai, J. X. Kang, and X. S. Xie,
Science 322, 1857 (2008).
9. C. J. Engelbrecht, R. S. Johnston, E. J. Seibel, and F.
Helmchen, Opt. Express 16, 5556 (2008).
10. M. T. Myaing, D. J. MacDonald, and X. Li, Opt. Lett.
31, 1076 (2006).
11. R. Le Harzic, M. Weinigel, I. Riemann, K. König, and
B. Messerschmidt, Opt. Express 16, 20588 (2008).
12. J. Sawinski and W. Denk, J. Appl. Phys. 102, 034701
13. H. Bao, J. Allen, R. Pattie, R. Vance, and M. Gu, Opt.
Lett. 33, 1333 (2008).
14. W. Piyawattanametha, R. P. Barretto, T. H. Ko, B. A.
Flusberg, E. D. Cocker, H. Ra, D. Lee, O. Solgaard, and
M. J. Schnitzer, Opt. Lett. 31, 2018 (2006).
15. T. M. Liu, M. C. Chan, I. H. Chen, S. H. Chia, and C. K.
Sun, Opt. Express 16, 10501 (2008).
16. W. Jung, S. Tang, D. T. McCormic, T. Xie, Y. C. Ahn, J.
Su, I. V. Tomov, T. B. Krasieva, B. J. Tromberg, and Z.
Chen, Opt. Lett. 33, 1324 (2008).
17. W. Göbel, A. Nimmerjahn, and F. Helmchen, Opt. Lett.
29, 1285 (2004).
18. B. A. Flusberg, A. Nimmerjahn, E. D. Cocker, E. A.
Mukamel, R. P. Barretto, T. H. Ko, L. D. Burns, J. C.
Jung, and M. J. Schnitzer, Nat. Methods 5, 935 (2008).
19. D. Kleinfeld, P. P. Mitra, F. Helmchen, and W. Denk,
Proc. Natl. Acad. Sci. USA 95, 15741 (1998).
A.Nimmerjahn, and F.
over eight frames acquired over 2 s at 4 Hz. d–f, Line im-
ages taken by driving only the scanner’s outer axis, at its
resonant frequency ?560 Hz?. Flowing erythrocytes appear
as dark streaks in relief. Flow velocities were found from
the slopes of the dark streaks at the central region of the
line scan. Illumination power was 27 mW at the sample.
Scale bar in a also applies to b and c. Scale bar in e also
applies to f.
a–c, Images of neocortical capillaries, averaged
August 1, 2009 / Vol. 34, No. 15 / OPTICS LETTERS