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OPTICS LETTERS / Vol. 31, No. 7 / April 1, 2006
Confocal reflectance theta line scanning
microscope for imaging human skin in vivo
Peter J. Dwyer and Charles A. DiMarzio
Department of Electrical Engineering, Center for Subsurface Sensing and Imaging Systems (CenSSIS),
Northeastern University, 360 Huntington Avenue, Boston, Massachusetts 02115-5000
James M. Zavislan
Institute of Optics, University of Rochester, Wilmot Building, Rochester, New York 14627
William J. Fox
Lucid, Inc., 2320 Brighton-Henrietta Town Line Road, Rochester, New York 14623
Milind Rajadhyaksha
Dermatology Service, Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York 10022
Received August 30, 2005; revised November 9, 2005; accepted November 30, 2005; posted January 12, 2006 (Doc. ID 64513)
A confocal reflectance theta line scanning microscope demonstrates imaging of nuclear and cellular detail in
human epidermis in vivo. Experimentally measured line-spread functions determine the instrumental optical section thickness to be 1.7± 0.1 ␮m and the lateral resolution to be 1.0± 0.1 ␮m. Within human dermis
(through full-thickness epidermis), the measured section thickness is 9.2± 1.7 ␮m and the lateral resolution
is 1.7± 0.1 ␮m. An illumination line is scanned directly in the pupil of the objective lens, and the backscattered descanned light is detected with a linear array, such that the theta line scanner consists of only seven
optical components. © 2006 Optical Society of America
OCIS codes: 110.0180, 120.3890, 120.4570, 170.1790, 180.1790, 180.5810.
Point scanning confocal reflectance microscopy is a
well-developed technology for optical sectioning
within human skin in vivo.1–5 Nuclear and cellular
detail in the epidermis and collagen and microcirculation in the dermis are imaged with a resolution
that compares well to that of histology. However,
point scanning confocal microscopes require raster
scanning in two dimensions, which results in large
and complex optoelectromechanical configurations.
Confocal theta line scanning based on detection with
a line detector offers a fundamentally simpler
method: scanning is required in only one dimension
and descanning only once, which significantly simplifies the instrumentation.
In this Letter, we illustrate the design and performance of a simple confocal theta line scanning microscope. The instrument consists of only seven components, that provides optical sectioning, resolution,
and contrast that are comparable to those of point
scanning for imaging human epidermis in vivo. Our
design is based on two existing techniques: line scanning through a divided objective lens pupil that was
originally developed by Koester6 and theta microscopy that was developed by Stelzer and Lindek7 and
Webb and Rogomentich.8 Figure 1 shows the laboratory prototype. Illumination is with a diode laser at
the near-infrared wavelength of 830 nm. The collimated beam is focused to two orthogonal lines using
a cylindrical lens and one portion of a divided objective lens pupil. The primary line is formed at the focus of the objective lens and is used for imaging. The
secondary line (not used for imaging) is approximately 1.6 mm from focus due to the fact that the cylindrical lens is placed approximately 10 mm in front
0146-9592/06/070942-3/$15.00
of the pupil of the objective lens. The primary illumination line is scanned by the first facet of a prismatic
mirror that is mounted on an oscillating galvanometric scanner (MiniSAX, GSI Lumonics, Billerica,
Mass.) that is driven by a sawtooth ramp signal. In
principle, we scan directly in the pupil of the objective lens. In practice, however, the prismatic scanner
is physically close to, but not exactly in, the pupil.
Therefore, the illumination beam slightly “walks”
across the pupil. There is, however, no vignetting at
the edges of the field of view because the 10 mm di-
Fig. 1. Confocal theta line scanning microscope. The primary line is formed in the y direction (perpendicular to the
page) at the focus of the objective lens and is used for imaging. The ratio u / r determines sectioning, lateral resolution, and contrast, and was chosen to be 0.5.
© 2006 Optical Society of America
April 1, 2006 / Vol. 31, No. 7 / OPTICS LETTERS
943
The section thickness was measured by the standard
method of axially translating a ␭ / 20 flat mirror surface through focus and measuring the detected power
versus axial position 共z兲. The FWHM of the axial LSF
plot represents the section thickness. Full-thickness
human epidermis specimens were harvested from
discarded fresh surgical excisions under Institutional
Review Board approval and placed on the mirror to
measure LSFs under actual tissue-induced scattering
and aberration conditions.
Figure 2 is an experimental plot of an axial confocal theta LSF under nominal instrument conditions
and through full-thickness (approximately 75 ␮m)
human epidermis. The figure shows the influence of
tissue-induced scattering and aberrations on the
axial confocal theta LSF. With a slit of 5 ␮m width in
front of the detector, the nominal instrumental secFig. 2. Experimental plot of an axial confocal theta linespread function (LSF).
ameter beam, with walk of ±0.9 mm, sufficiently
overfills the illumination portion (7 mm) of the objective lens pupil.
The objective lens is custom made (Lucid, Inc.,
Rochester, N.Y.) for the needs of clinical imaging applications: low magnification of 10⫻, high numerical
aperture (NA) of 0.8, water immersion, correction for
the 1 mm thick coverglass window, and a field of view
of 1 mm. The focal length 共fobj兲 is 17.5 mm, and the
scan angle is ±1.6°, producing a total scanned field
(fobj times scan angle) of ±0.5 mm. Illumination
power of 5–10 mW on the skin does not damage tissue and is adequate for imaging epidermis and superficial dermis in vivo.
The illumination scan fills a portion of the pupil of
the objective lens, while the diametrically opposite
portion is used to collect light that is backscattered
from the tissue. Physically, a divider strip separates
the pupil of the objective lens into an illumination
pupil and a detection pupil (Fig. 1). The backscattered light is descanned by the second facet of the
prismatic mirror and focused by a biconvex lens
through a slit onto a complementary metal oxide
semiconductor line detector (Fig. 1). The complementary metal oxide semiconductor line detector (LIS
1024, Photon Vision Systems, Cortland, N.Y.) consists of an array of 1024 pixels, each 7 ␮m long
⫻ 125 ␮m wide. Slit widths of 5 to 100 ␮m were used
in front of the detector for section thickness measurements and imaging experiments, where a width of
5 ␮m corresponds to the diffraction-limited line halfwidth (i.e., lateral resolution) in the plane of the detector. Images are captured with a frame grabber
(Digital Meteor II, Matrox, Dorval, Quebec, Canada).
An image consists of 1024 共lines兲 ⫻ 1024 共pixels兲, displaying a field of view of 1 mm.
Experimental measurements of confocal theta linespread functions (LSFs) were made to determine optical section thickness and lateral resolution. The
confocal theta LSFs were measured under both
diffraction-limited (nominal instrument) conditions
and scattering and aberrating (deep within human
dermis, through full-thickness epidermis) conditions.
Fig. 3. Images of human epidermis in vivo. Scale bars,
50 ␮m.
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OPTICS LETTERS / Vol. 31, No. 7 / April 1, 2006
tion thickness is 1.7± 0.1 ␮m, and through fullthickness epidermis 9.2± 1.7 ␮m, for 30 measurements each. The scattering and aberration due to the
epidermis reduce the sectioning by a factor of approximately five deep in the dermis. However, within
the epidermis itself, the sectioning is sufficiently high
for imaging nuclear and cellular detail and is comparable to that of confocal point scanners.3–5
Lateral resolution was measured by the standard
method of imaging a chrome-on-glass edge (Ronchi
Ruling, F38-566, Edmund Scientific, Barrington,
N.J.). The lateral resolution was measured, again,
under nominal instrument conditions and through
full-thickness human epidermis. When a Gaussian
spot scans across an edge, the FWHM lateral resolution has been calculated to be 0.94 times the 10%–
90% points on the integrated irradiance or detected
power profile.4 With a slit of 25 ␮m width in front of
the detector, the nominal lateral resolution is
1.0± 0.1 ␮m, and through full-thickness epidermis,
1.7± 0.1 ␮m, for 10 measurements each.
Images of human skin in vivo are shown in Fig. 3.
Nuclei and cells are seen in all the layers of the epidermis, demonstrating optical sectioning that corroborates the FWHM measurements from the axial
LSFs. In the upper epidermis (Fig. 3A), the stratum
corneum (SC), granular (GR), and spinous (SP) cell
layers are visualized. In these layers, the nuclei appear dark (arrows) within bright and grainy cellular
cytoplasm. Deeper in the epidermis (Fig. 3B), smaller
basal cells are seen with dark nuclei (arrows), arranged in ring-shaped clusters (arrowheads). Basal
cells appear brighter due to the pigment melanin.3
These images appear similar to those obtained with
confocal point scanners.1–5
In conclusion, confocal theta line scanning microscopy provides optical sectioning and lateral resolution that is sufficiently high to image nuclear and cellular detail in human epidermis in vivo. The
sectioning, resolution, and contrast are comparable
to those provided by current confocal point scanners
within the scattering and aberrating conditions of
human skin.
This research was supported, in part, by the National Institutes of Health/National Cancer Institute
(award 1R43CA93106 and 2R44CA93106) and by the
National Science Foundation Partnerships in Education and Research Program (award EEC-012931). P.
J. Dwyer’s e-mail address is [email protected].
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