Miniature near-infrared dual-axes
confocal microscope utilizing a
Jonathan T. C. Liu, Michael J. Mandella, Hyejun Ra, Larry K. Wong, Olav Solgaard, and Gordon S. Kino
Edward L. Ginzton Laboratory, Stanford University, Stanford, California 94305, USA
Wibool Piyawattanametha, Christopher H. Contag, and Thomas D. Wang
James H. Clark Center for Biomedical Engineering & Sciences, Stanford University, Stanford, California 94305, USA
Received August 17, 2006; accepted October 3, 2006;
posted October 27, 2006 (Doc. ID 74170); published January 12, 2007
The first, to our knowledge, miniature dual-axes confocal microscope has been developed, with an outer di-
ameter of 10 mm, for subsurface imaging of biological tissues with 5–7 ?m resolution. Depth-resolved en
face images are obtained at 30 frames per second, with a field of view of 800?100 ?m, by employing a two-
dimensional scanning microelectromechanical systems mirror. Reflectance and fluorescence images are ob-
tained with a laser source at 785 nm, demonstrating the ability to perform real-time optical biopsy. © 2007
Optical Society of America
OCIS codes: 170.0180, 170.1790, 170.3880, 170.5810, 170.2680, 170.2520.
Conventional single-axis confocal microscopes per-
form optical sectioning with an axial resolution that
scales with NA according to an inverse square law.
For high-resolution imaging, a large NA is required,
which implies a short working distance and small
field of view (FOV) unless a large-diameter objective
is employed. Such objectives are difficult to miniatur-
ize, often requiring multiple elements to mitigate the
effects of aberrations due to high-NA focusing and
preobjective beam scanning.1Therefore a design con-
tradiction exists in building a miniature single-axis
confocal that images deeply over a large FOV with
high axial resolution. The dual-axes confocal archi-
tecture utilizes two low-NA beams, with intersecting
focal volumes, for illumination and light collection.2–4
Unlike single-axis confocals, where transverse reso-
lution is much superior to axial resolution, the dual-
axes confocal design provides a resolution that is
relatively balanced in all spatial dimensions. In addi-
tion, it has been shown that the dual-axes configura-
tion provides superior optical sectioning and rejection
of out-of-focus scattered light compared with a single-
axis design.5Low-NA lenses enable long working dis-
tances, which, along with the superior rejection of
scattered light afforded by the dual-axes configura-
tion, enable deep subsurface imaging in tissues. The
long working distance also provides room for an
aberration-free postobjective scanner to image over a
large field of view, which in this case is accomplished
by a two-dimensional (2D) microelectromechanical
systems (MEMS) scanner.
Figure 1 shows a simplified layout of the miniature
dual-axes confocal microscope. The design incorpo-
rates a parabolic reflector to ensure that any two col-
limated beams aligned parallel to one another will in-
tersect at their focus. The 10 mm diameter parabolic
reflector (Anteryon BV Eindhoven, The Netherlands)
has a focal length of 4.6 mm and a 1.9 mm diameter
hole at the center where an index-matching hemi-
sphere is placed. The fused-silica hemisphere en-
hances the resolution by 1/n, where n is the index of
aberrations.5A custom 2D MEMS mirror,6which
scans the illumination and collection beams in tan-
dem as they are focused by the parabolic mirror, di-
rects the beams through the hemispherical lens and
into the specimen.
The angles ? and ?, shown in Fig. 1, correspond to
the intersection half-angle of the beams and the free-
space NA of the individual beams, respectively. The
theoretical resolution (FWHM) may be calculated,5in
micrometers, assuming Gaussian beams truncated to
allow 99% power transmission, ?=0.785 ?m, ?
=0.128, ?=24°, and n=1.4:
to minimize various
n? cos ?= 2.2,
Collimated illumination beam. (2) Collimated collection
beam. (3) MEMS 2D scanner. (4) Parabolic reflecting sur-
face. (5) Index-matching hemisphere.
Miniature dual-axes confocal scan head optics. (1)
OPTICS LETTERS / Vol. 32, No. 3 / February 1, 2007
0146-9592/07/030256-3/$15.00© 2007 Optical Society of America
n? sin ?= 5.0.
Fiber-pigtailed collimators (Lightpath Technolo-
gies), designed for single mode use at telecom wave-
lengths (SMF-28 fiber), are used in this preliminary
prototype. Although the Lightpath collimators and fi-
bers are not designed for single mode transmission at
785 nm, we have observed single mode illumination
due to negligible mode mixing along the short fiber
lengths used (several meters). In addition, the use of
the 9 ?m core SMF-28 fiber improves the collection
efficiency for fluorescence imaging (larger pinhole),
albeit at the cost of a slight degradation in resolution
compared with the calculation in Eq. (1) for purely
single mode illumination and collection.
Figure 2 depicts various elements of the miniature
design. The collimators are placed in precision wire
electrical discharge machined v-grooves with centers
separated by 3.7 mm. A pair of 1° wedges (Risley
prisms) are positioned after each collimator. These
are rotated to provide angular adjustment of the
beams, aligning them parallel to each other as well
as parallel to the central axis of the parabolic mirror.
mounted at the center of a rectangular printed circuit
board (PCB), which is mounted at the end of an axial
sliding mechanism to enable imaging at various
depths within the sample. The circuit board acts as a
junction between external electrical wires from a
high-voltage amplifier (AgilOptics) and the MEMS
device itself, which is connected through wire bonds
onto bonding pads on the PCB. Oversized mounting
holes in the PCB allow for lateral adjustment of the
MEMS mirror to center the illumination and collec-
tion beams on the mirror surfaces. Driving signals
are generated with LabVIEW on National Instru-
ments wave form generators. Each dimension of the
MEMS mirror is actuated with two wave forms that
are equal but 180° out of phase.6–9
Figure 3 shows a scanning electron micrograph of
the MEMS device. Vertical electrostatic comb actua-
?3.2 mm?2.9 mm?
tors provide gimbaled torsional motion in two or-
thogonal dimensions to allow a 2D raster scan. The
inner axis possesses a mechanical resonance fre-
quency of 3.5 kHz whereas the outer axis exhibits a
resonance at 1.25 kHz. The inner axis of the scanner
is driven sinusoidally at its resonance frequency, en-
abling a large deflection angle to be obtained with a
reduced-amplitude driving wave form. The scan
range of this fast inner axis is 800 ?m in our proto-
type. The slow outer axis is driven with a sawtooth
wave form, modified with a smooth turn around to
prevent mirror ringing. The slow axis scan range of
the MEMS mirror used here is 100 ?m, correspond-
ing to an optical deflection of ±1.0° at a voltage of
110 V. Additional details on the MEMS fabrication
and operation have been described elsewhere.6–9
Axial resolution is measured to be 7.5 ?m by trans-
lating a plane mirror in the z-direction and recording
the FWHM. A reflectance image of a U.S. Air Force
(USAF) target is shown in Fig. 4, in which the lines
in element 4 of group 7 are resolvable (with a 5.5 ?m
line-space period). For these studies, the maximum
laser intensity on the sample is 1 mW at 785 nm. The
edges of the field of view are dark since the image-
section plane is curved, with the center of the field of
view imaging 25 ?m deeper than at the edge.
For fluorescence images, a near-infrared dye from
Li-Cor Biosciences is used (IRDye 800CW) with an
absorption maximum at 774 nm and an emission
maximum at 789 nm in water. The dye is dissolved in
saline at a concentration of 0.1 ?g/?L ?90 ?M?.
Fresh colon biopsy samples are obtained from the
Palo Alto Veterans Affairs Hospital with informed pa-
tient consent. Colon specimens are soaked in the dye
solution for 1 min before being irrigated with water
to remove excess dye. An emission filter from Sem-
795.2 to 1771 nm, is used to remove the laser back-
ground prior to detecting the fluorescence signal with
a Hamamatsu PMT (H7422-50).
witha passband from
drawings. The outer diameter for this device is 10 mm,
with all essential optics and optical paths contained within
the central 5 mm diameter. (1) Fibered collimators. (2) Ris-
ley alignment prisms. (3) MEMS PCB. (4) Parabolic reflec-
tor. (5) Axial sliding mechanism to control imaging depth.
(6) Removable end cap holding the parabolic reflector.
Miniature dual-axes microscope package design
scanner. Dimensions of the MEMS chip are 3.2 mm
?2.9 mm, with each mirror surface measuring 650 ?m
?600 ?m. (1) Outer torsional spring. (2) Outer axis electro-
static comb actuators. (3) Inner torsional spring. (4) Inner
axis electrostatic comb actuators.
Scanning electron microscopy image of the 2D
February 1, 2007 / Vol. 32, No. 3 / OPTICS LETTERS
A proof-of-principle fluorescence image of a normal
human colon, at a depth of 20 ?m beneath the mu-
cosal surface, is shown in Fig. 4(b). The image is an
average of five frames acquired at 30 frames/s. Note
that small colonocytes surrounding the three circular
crypt lumen are resolvable.
Several improvements are planned for future pro-
totypes. Optimized MEMS scanners will be used,
with larger deflection angles, enabling increased
fields of view. Future mirrors will be evaporatively
coated with a layer of aluminum to improve the re-
flectivity of the two mirror surfaces.9In addition, cus-
tom gradient-index collimators will be utilized with
larger beam diameters than our current collimators.
As a result, the NA of the beams will be increased,
providing an improvement in resolution according to
Eq. (1), as well as an improvement in collection effi-
ciency (scales with the square of NA). These improve-
ments in optical efficiency will enable improved im-
aging at greater tissue depths in the future.
We have demonstrated what we believe to be the
first design, construction, alignment, and utilization
of a miniature dual-axes confocal reflectance and
fluorescence microscope at 785 nm. While the outer
diameter of this device is 10 mm, all of the essential
optics and optical paths lie within a central 5 mm di-
ameter. A future 5 mm device will be attained by uti-
lizing alternative alignment techniques. The even-
tualgoal isto perform
microendoscopy with a device that may be conve-
niently deployed through the instrument channel of
an endoscope. Together with the use of small fluores-
cent molecules targeted against biomarkers of dis-
ease, such a device could revolutionize the practice of
endoscopy by enabling real-time optical biopsy for the
early and accurate detection of cancer and for guid-
ing therapeutic procedures.
This work was funded in part by grants from the
National Institutes of
CA109988. This work has also been supported by
funding through the Center for Biophotonics, a Na-
tional Science Foundation Center managed by the
University of California, Davis (PHY 0120999). J. T.
C. Liu is supported by a Canary Foundation/
American Cancer Society postdoctoral fellowship for
early cancer detection. The authors thank Shai
Friedland and Roy Soetikno for technical support. J.
Liu’s e-mail address is email@example.com.
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obtained at 30 frames/s, demonstrating resolvable lines in
element 4 of group 7 (5.5 ?m period). FOV=350 ?m
?100 ?m. (b) Dual-axes fluorescence image of normal co-
lonic mucosa at 785 nm, showing circular crypts and sur-
rounding colonocytes. The image is an average of five
frames acquired at 30 frames per second. Imaging depth
=20 ?m at the image center. FOV=800 ?m?100 ?m.
(a) Dual-axes reflectance image of a USAF target,
OPTICS LETTERS / Vol. 32, No. 3 / February 1, 2007