Iournal of Microscopy. Vol. 170. Pt 3 , June 1993. pp. 229-236.
Received 10 July 1992: accepted 1 December 1992
Orthogonal-plane fluorescence optical sectioning: three-
dimensional imaging of macroscopic biological specimens
A. H. VOIE,*t D. H. BURNS* & F. A. SPELMAN*?
'Center for Bioengineering and j'Regiona1 Primate Research Center, University of Washington.
Seattle. W A 98195. U.S.A.
Key words. Fluorescence, imaging, optical sectioning, three-dimensional
reconstruction, cochlea.
Summary
An imaging technique called orthogonal-plane fluorescence
optical sectioning (OPFOS) was developed to image the
internal architecture of the cochlea. Expressions for the
three-dimensional point spread function and the axial and
lateral resolution are derived. Methodologies for tissue
preparation and for construction, alignment, calibration
and characterization of an OPFOS apparatus are presented.
The instrument described produced focused, high-resolu-
tion images of optical sections of an intact, excised guinea-
pig cochlea. The lateral and axial resolutions of the images
were 1 0 and 26 pm, respectively, within a 1.5-mm field of
view.
Introduction
Three-dimensional (3-D) reconstructions of biological
specimens and systems provide an important way to
study, measure and analyse form and function. Most
often, a 3-D reconstruction is obtained by extracting
information from a series of 2-D histological sections of
an object. Thin histological sections are mounted on
microscope slides and can be imaged with either optical
or electron microscopes. The image can provide extra-
ordinarily fine detail. However, histological sections have
serious disadvantages for the purpose of 3-D reconstruction
(Castleman, 1979). One is the loss of register that occurs
when the sections become separated during sectioning.
Although resulting errors can be reduced by use of fiducials,
complete correction is not possible. Another problem is the
geometric distortion that can result from processing of the
slices. Finally, serial sectioning results in a single set of
parallel-plane slices. For some structures, this may not yield
a convenient means to extract data for accurate 3-D
reconstruction because there is a loss of sampling resolution
where the boundary of a structure of interest is nearly
parallel to the slicing planes. An example would be the
structures in the spiral-shaped cochlea (Santos, 1987).
0 1993 The Royal Microscopical Society
Important structures in the cochIea are best observed
from a number of angular orientations. This is not possible
with histological methods.
Recently, optical imaging techniques have been devel-
oped for obtaining a thin section within a specimen. Optical
sectioning is the process of moving a thick specimen
through the focal plane of an imaging detector to produce
an image of a thin region of that specimen (Castleman,
1979: Agard, 1984). There are many advantages to this
technique. Optical sectioning maintains the register
between sections. Also, the geometrical distortion inherent
in histological techniques is avoided. The image of an
object viewed in this manner will contain information
from structures both within and outside the focal
region, and so will appear blurred. The unfocused
component of the image needs to be removed or mini-
mized in order to resolve the in-focus component. Simil-
arly, greater depth discrimination is achieved by removal
of the unfocused information. The unfocused information
may be removed optically by use of techniques such
as confocal microscopy (Brackenhoff et al., 1989; Carlsson
& Liljeborg, 1989; Wilson, 1989), or by image pro-
cessing (Castleman, 1979; Agard, 1984; Agard et al.,
1989).
The literature does not discount the possibility of using a
confocal system to section a macroscopic biological speci-
men opticallly. The main disadvantages with this aproach
are the high cost of such a system and the difficulty of its
alignment owing to instrumentation tolerances. Addition-
ally, low-light-level conditions, as with fluorescence micro-
scopy, increase the image-acquisition time, and this
precludes imaging in real-time.
Image deblurring by digital image processing methods
has several disadvantages. One is the relatively poor spatial
resolution compared with that obtained from histological
sections (Agard et al., 1989). Also, image processing
precludes the practical viewing of the sections in real-
time. Reconstruction of spiral structures remains proble-
230 A . H. V O I E E T A L .
Fig. 1. Orientation of the illumination axis, imaging axis and speci-
men for OPFOS imaging.
matic, since existing deblurring methods require parallel,
regularly spaced images (Agard et al., 1989).
Another approach to optical sectioning involves the use
of a planar illumination beam (Root, 1985; White, 1987).
Planar illumination has been used in other imaging modalities
such as flow cytometry and flow visualization (Merzkirch,
1974). The illumination beam is focused into a plane with a
cylindrical lens, and aligned to be co-planar with the depth of
field of the imaging detector. The beam is thus orthogonal to the
imagmg axis. The region where both the imaging system and
laser illumination focus coincide is the system focal zone.
We developed an imaging system that employs planar
illumination to excite fluorescent dye so that we could
produce images of optical sections of excised cochleas. The
motivation for developing this system was to obtain suitable
2-D data to enable an accurate 3-D reconstruction of the
cochlea. As shown in Fig. 1, planar illumination passes
through the cleared and fluorescently labelled specimen.
Dye on structures within the thin focal zone emits
omnidirectional light, forming an image on the sensing
element of the camera. Only dye on structures within the
focal zone emits light because no excitation occurs outside
this region. Since no light originates from outside the focal
region, the resulting image contains no out-of-focus
component. This imaging technique has been dubbed
orthogonal-plane fluorescence optical sectioning (OPFOS).
The aim of this article is to describe the OPFOS technique
and demonstrate its usefulness. First, the theoretical
framework is developed, followed by calibration and
characterization of the apparatus. An example of an
OPFOS image of an excised cochlea is shown. Finally, a 3-
D reconstruction of one of the spiral canals, produced from
the digitally recorded images, is presented.
List of symbols
To enable readers to understand the following descriptions
more easily, the symbols used in this paper are listed below.
measure of the planar illumination thickness
along the z-axis
measure of the planar illumination length along
the x-axis
f-number of the lens
intensity distribution function of the laser illumi-
nation beam
3-D point spread function of the imaging system
2-D image formed at back focal plane of the
imaging lens
wave number of the image-forming light
refractive index of the clearing solution
3-D structure in object space
2-D pixellation function
width of a square pixel that has been calibrated to
object-space dimensions
lateral resolution for a diffraction-limited optical
system
imaging lens blurring function
illumination beam boundary function in the x-z
plane
coordinates of an arbitrary point in space
acceptance angle of the imaging lens
illumination wavelength in air
Theory
The coordinate system and notation conventions used here
are similar to those employed by Castleman (1979) and
Agard (1984). The orientation of the axes is shown in Fig.
1. The origin is taken to be the centre of the focal zone. The
x- and y-axes of the image plane are scaled and aligned to
project onto the focal plane axes. The implicit simplification
is that the image magnification is unity. Also implied is that
no coordinate inversion occurs between the focal plane and
the image plane. This allows the subscripts to be dropped
from the notation, simplifying the expressions.
For spatially invariant blurring, image formation through
an optical system can be expressed as
where I(x, y) is the image formed with the imaging lens
focused at z = 0 , O(x, y, z) is the 3-D structure in object
space, h(x, y, z) is the 3-D point spread function (PSF) of the
system, and * is the convolution operator. An image results
from a convolution of the object with the PSF of the system.
Three-dimensional PSF
The PSF of an optical system is the product of the
illumination intensity distribution, g(x, y, z), and the
imaging lens blurring function, s(x, y, z) (Hiraoka et al.,
1990; Nakamura & Kawata, 1990). The PSF of the system
 ing

I
Collimating lens

Fig. 2. Schematic diagram of the optical components assembled for

OPFOS. The imaging lens does not contact the clearing solution.

can be expressed as

Fig. 3. The focusing characteristics of the cylindrical lens that pro-

In most optical imaging techniques, the illumination is
coaxial with the imaging axis and uniform upon or through
the object. In these cases the illumination intensity
distribution is constant, and thereby the PSF reduces to
s(xly, z). However, the illumination used in OPFOS imaging
is spatially non-uniform and is transverse to the imaging
axis. The illumination intensity distribution is determined
by the optical components of the illumination system.
Illumination system
An optical layout of the illumination system is shown in Fig.
2. The light source is a laser whose wavelength excites the
fluorescent dye in the specimen. A cylindrical lens with a
suitable f-number focuses the collimated illumination beam
to approximately planar dimensions. The f-number of the
cylindrical lens is the lens focal length divided by the
diameter of the expanded and collimated beam. The
illumination system is aligned so that the intensity
distribution is co-planar with the depth of field of the
imaging detector.
Illumination intensity distribution
The illumination beam is characterized as an intensity
distribution in a progression of planes normal to the x-axis.
The shape of the spot is circular up to the cylindrical lens.
The cylindrical lens focuses the beam so as to compress the
spot in the z-direction, but not in the y-direction. The shape
of the spot approaches a line at the focal plane of the
cylindrical lens at x = 0.
In the focal zone, the laser spot has a Gaussian intensity
distribution. In the x-z plane, the variation in the intensity
can be expressed as
duce the boundary function w ( ~ )The. thickness of the beam waist
is 2a, and the focal distance of the beam is 2b.
(Siegman, 1986). The spatial extent, or boundary, of the
spot is defined as the location at which the intensity falls to
l/e2 of its maximum (in this case, x-axis) value. The locus
of these points in the x-z plane is the beam boundary
function, w(x). The boundary function describes the shape
of the beam and is dependent on the focusing characteristics
of the cylindrical lens. The shape of w (x) is hyperbolic (Fig.
3) and is described by
The parameters a and bin Eq. (4) describe the dimensions
of the nearly planar illumination region. The thickness of
the beam waist is a function of the laser wavelength, A, and
the f-number of the cylindrical lens. This thickness is 2a.
where
The beam thickness increases by a factor of J 2 at an x
value of b on either side of the waist. The focal region of the
beam is defined to be between +b and -b. The focal distance
of the beam is the length in x of the focal region, and this
length varies with the square of the waist thickness. The
focal distance is 2b, where
with n being the refractive index of the clearing solution. It
should be noted that Eq. (5) is independent of n. This is
because wavelength and f-number are offsetting functions
of the refractive index in Eq. (5). Since there is one more
factor of X than f-number in Eq. (6), n appears in its
numerator.
232 A . H. VOIE ET AL.
Imaging system
Ideally, for diffraction-limited optical components, the lens
blurring function is described by
2 (kr sin a )
s= [
krsina ] formalin. The bulla and vestibular system were trimmed
(Inoue, 1986). is a first-order Bessel function, k is the
wave number (equal to 27~/X,where X is the wavelength of
the image-forming light), a is the acceptance angle of the
lens and r = (x2 + y2)1/2. This is the well-known Airy
pattern formed in the image plane by a point source in the
focal plane of the lens. The radius of the central bright spot
of s(x, y, z ) is r,, where
0.61X
r, = -.
n sin CY
In a diffraction-limited imaging system, the lateral resolu-
tion is r,.
However, for digital imaging systems, the effect of
pixellation must be taken into account. This can be done
by convolving the continuous-plane image with a pixella-
tion function, p(x, y). The resultant image is then described
by
For simplicity, p(x, y) is taken to be a square with sides of
length go. The value of p(x, y) is unity within the square
and zero otherwise.
Image resolution
The lateral resolution of the image depends on the size of po
relative to r,,. If po is less than ro/2, then the Nyquist
criterion for spatial sampling is satisfied (Inoue, 1986). In
that event, Eq. (9) approximates Eq. (1). However, for low-
magnification digital imaging systems where this criterion
is not met, the lateral resolution limit is 2po rather than ro.
A consequence of pixel-limited lateral resolution is an
increased depth of field. This is due to the fact that depth of
field varies inversely with lateral resolution (Sheppard,
1988).
In most optical sectioning techniques, the axial resolution
limit is the imaging depth of field (Hiraoka et al., 1990). In
the OPFOS method, however, the illumination beam is
focused to a thickness that is less than the imaging depth of
field. This ensures that the resulting image is entirely in
focus. Consequently, the axial resolution of the image is
equal to the thickness of the illumination beam, given by
Eq. (4). The axial resolution limit is 2a, defined in Eq. (5).
Methods
Specimen preparation
Cochleas were excised from guinea-pigs that had been
deeply anaesthetized and perfused with neutral buffered
away with dental burrs and the cochlea was cemented to a
small nylon sphere. The specimen was decalcified in
tetrasodium salt of ethylenediaminetetraacetic acid
(EDTA) and dehydrated in ethyl alcohol. It was cleared in
a solution of five parts methyl salicylate and three parts
benzyl benzoate (Haq, 1988). The cleared cochlea was
treated with fluorescent dye by immersion in a dye bath.
The bath was prepared by dissolving rhodamine isothio-
cyanate (RITC) in ethyl alcohol to a concentration of 1 mg/
ml. This solution was then diluted with clearing solution to
a final dye concentration of 5 x mg/ml. The RITC
absorbs at 550 nm and emits at 585 nm (Haugland, 1981).
Specimen holder
A custom-made specimen holder was designed for mount-
ing the cochlea. It was fabricated from a block of Delrin. The
chamber had clear sides to allow the laser illumination
beam to pass through unimpeded. This created an interior
chamber that was open on top so that it could be filled with
clearing solution. The purpose of the clearing solution was
to maintain the relative transparency of the cochlea during
the imaging process. The imaging system lens does not
contact this fluid. A rotation shaft extended into the
chamber and was orientated in the y-axis direction. The
shaft was attached to an optical shaft encoder. This
provided a digital display of the rotation angle in 0.7"
increments. A four-part chuck was built into the end of the
shaft to hold the specimen for imaging. To position the
sample, the specimen holder was attached to a stage that
could be translated with 5-pm precision along the three
coordinate axes.
Design criteria
The primary design criterion for the OPFOS system was to
obtain images of the canal cross-sections of guinea-pig
cochleas. These cross-sections were estimated to be 1.5 mm
across at the widest point. Therefore, the components of
illumination optics were chosen to provide a beam whose
focal distance was 1-5mm and whose thickness was
,< 30 pm over this length.
Illumination optics
The illumination source was a 1.25-mW green helium-
neon laser with a wavelength of 543.5 nm. Using Eqs. (5)
 O R T H O G O N A L - P L A N E FLUORESCENCE O P T I C A L S E C T I O N I N G
and ( 6 ) . we calculated that an f/26.7 cylindrical lens
would meet the design criteria. Specifically, the focal
distance was calculated to be 1.53mm, with a waist
thickness of 18.5 pm and a maximum thickness of 26.2 pm.
Imaging system
The imaging lens was an f l 0 . 9 5 Navitron macro lens with
a 25-mm focal length. This lens was chosen for its high
light-gathering capacity. The image was formed on the
sensor of a Javelin CCD video camera. A 560-nm-long pass
filter was positioned on the lens to block scattered laser light
from contributing to the image. Images were digitized with
a Gateway 2000 486 computer, equipped with a
640 x 480 pixel frame grabber from Imaging Technology.
'
Image acquisition and processing was done with Optimas
(Bioscan).
Calibration
A rectangular calibration grid was imaged to determine the
pixel dimensions. This grid was placed in the sample holder
and immersed in clearing solution. Incident incandescent
light was used to illuminate the grid. The image of the grid
was produced by the scattered light. In the calibration
procedure, two pixels in the image were selected with a
mouse. The object-space distance between these two points
was specified and the dimensions of the pixels were then
computed. Grid vertices were used as calibration points. The
pixel x- and y-dimensions were 5.08 x 5-25pm, for an
aspect ratio of 0.968. These dimensions were used to assign
x- and y-coordinate values to the pixels. These screen
coordinates were then used for further measurements.
Alignment
A small, pointed stainless-steel rod was used as an
alignment tool. This rod was placed in the sample holder
with its point orientated toward the camera. With the laser
beam just grazing this rod tip, a small image of the spot was
formed from the scattered light. The pointed rod was
translated in the x- and y-directions; the illumination plane
was considered aligned with the camera focal plane and the
horizontal stage translation plane when the radius and
intensity of the spot remained constant within the camera's
field of view.
Alignment of the translation stages with the axes of the
image-processing coordinates was also done with the
pointed rod. The point was translated by fixed amounts in
the x- and y-directions and the change in screen
coordinates was noted. Alignment in the z-direction was
determined as follows. The camera was positioned to
produce an in-focus image of the rod tip. The screen
coordinates of the tip image were recorded. The rod was
233
Reflected
liaht
Fig. 4. Configuration for beam intensity profile measurement.
then translated vertically by a fixed amount. The lens and
camera were translated along the imaging axis until the rod
tip image was again in focus. The screen coordinates of the
tip image were again noted to ascertain the lateral shift
with vertical translation. Translation was performed in both
positive and negative directions to measure the effect of
micrometer backlash. Micrometer backlash was less than
could be measured with pixels that were 5 pm across, and
therefore was considered negligible.
Results and discussion
Illumination beam dimensions
The thickness of the illumination beam was measured to
determine the axial resolution of the OPFOS image. A 45"
prism was placed in the sample holder and immersed in
clearing solution. The laser illumination was focused onto
the prism, as illustrated in Fig. 4. The refractive index of the
glass prism was similar to that of the surrounding clearing
solution. Most of the illumination intensity, therefore, was
transmitted through the prism. A small fraction of the
illumination intensity was reflected by the prism in the
direction of the imaging optics. The prism was positioned so
that its inclined face transected the waist of the illumination
beam. The resulting image appeared as a thin stripe
orientated in the y-direction. The stage was translated in
the x-direction and the waist of the beam was taken to be
the setting for which the width of the stripe was a
minimum. The intensity profile was then measured across
the stripe. The beam thickness was determined by
measuring the width of the profile at which its value was
l/e2 of the maximum intensity. A key advantage of using a
prism to measure beam thickness was that an intensity
profile could be determined from a single image. This
substantially simplified the process of locating the beam waist.
 Measured values

0.8 -

u
Q,
.N 0.4-

0 5 10 15 20 25 30 35 40 45
I.lm
Fig. 5. Normalized intensity profiles of the laser spot measured
along the z-axis at the focus of the cylindrical lens. The solid curve
represents theoretical values predicted from Eq. (2). The dashed
line indicates l/e2, which defines the beam boundary. Beam thick-
ness is determined by measuring the distance over which the pro- Fig. 7. An OPFOS image of a guinea-pig cochlea showing modiolus
6le exceeds this boundary value. The predicted thickness was (A), auditory nerve (B), basilar membrane (C), organ of Corti (D)
18.5 pm and the measured thickness was 20.0 pm. and Reissner's membrane (E). Dashed lines border the laser focal
Figure 5 shows the measured intensity profile of the zone. The dimensions of this region are 1.5 mm in the horizontal
and vertical directions. Lateral resolution in this region is 10pm:
illumination beam at the focus of the cylindrical lens. Also
axial resolution is 30 pm.
shown is the profile estimated from Eq. (3); the agreement is
reasonable. The measured beam thickness was 20.0pm, boundaries of the illumination beam in the x-z plane (Fig.
compared with an estimated thickness of 18.5 pm. 6). Also shown in Fig. 6 is the estimated beam boundary;
Beam width measurement was repeated after the prism very good agreement was achieved.
was translated 750pm in the x-direction on either side of
the cylindrical lens focus. On the side toward the laser, the
measured beam thickness was 28.3 pm; on the other side of
the focal point, the beam thickness was 27.9pm. The
estimated beam thickness at both these distances was
26.1 pm. These measurements were used to depict the

I='
Fig. 8. Normalized intensity profiles of the illumination beam mea-
I sured at the focus of the cylindrical lens. The solid curve (profile A)
-20 4 represents values from a beam that had not passed through any
-750 -500 -250 0 250 500 750
specimen material. A cleared cochlea was placed in the beam path
Distance (prn) along X axis
to assess its effect on the beam intensity profile. The dashed curve
Fig. 6. Boundaries of the laser illumination beam in the x-z plane (profile B) represents the beam profile at the cylindrical lens focus
of the focal zone. Solid lines represent measured values: dashed with the cochlea in the light path. The width of profile B was
lines represent theoretical values. 21.7pm, compared with 20.0pm for profile A.

Fig. 9. Three-dimensional reconstruction of the scala tympani of a
guinea-pig cochlea.
OPFOS image of intact cochlea
Figure 7 is an OPFOS image of a mid-modiolar section
through a guinea-pig cochlea. The dashed lines border a
square region of interest that measures 1.5 x 1 . 5 mm. The
region of interest corresponds with the location where the
beam is focused to the dimensions described above. The
direction of the laser light is from left to right. Thin, dark
horizontal bands can be seen in the image. These artefacts
are due to the illumination beam passing through regions of
higher dye concentration, causing a shadow to appear.
These shadows do not interfere with image interpretation
because the specimen may be rotated.
Many fine cochlear structures are visible in the region of
interest. The central core of the cochlea is the modiolus, in
which the auditory nerve can be seen. The basilar
membrane, the organ of Corti and the thin Reissner's
membrane can be seen in the canal cross-sections.
Reissner's membrane is two cell layers thick, or about
20 pm across.
Effect of local variations in refractive index
The clearing solution permeates the tissue of the specimen,
causing the refractive index of the specimen to approach
that of the surrounding clearing solution. However, slight
differences in refractive indices may exist between the tissue and
the solution, and within the tissue itself. These differences can
result in geometric distortion of the OPFOS image.
The effect of the specimen on the imaging process was
examined by repeating the beam intensity profile measure-
ment at the beam focus with a cleared and stained cochlea
in the laser light path. The assumption was made that if the
presence of the cochlea resulted in little distortion to the
illumination-focusing characteristics, then there would be
little distortion introduced to the light from fluorescent
structures as well. Figure 8 shows the results of these
measurements. The normalized intensity profile at the beam
focus with no cochlea in the light path is labelled profile A.
Measurements made after placing the cochlea in the light
path resulted in profile B. The width of profile B is 2 1.7pm,
compared with 20.0 pm for profile A. This demonstrates that
tissue absorbance and scattering of the illumination beam
have only a slight effect on the intensity profile of the beam.
Three-dimensional reconstruction
The lower of the three canals in the OPFOS image is the
scala tympani. Its basal terminus (not shown in Fig. 7) is the
round window membrane. The cochlea was rotated about
the y-axis to image the cross-section of the scala tympani
from the round window membrane to its apical terminus.
The outline of the canal was traced and digitized for each
image. The coordinates of these digitized points were
corrected for stage translation and shaft rotation. This
correction restored the true spatial relation between the
digitized points in the successive images. Points were
connected between the successive images to complete the
3-D reconstruction of the scala tympani (Fig. 9).
Quantitative measurements
Several physical parameters may be measured directly or
calculated from the 3-D reconstruction data. These
quantitative measurements can provide important informa-
tion for understanding cochlear architecture and function.
For example, the scala tympani centroid and cross-sectional
area were computed from the reconstruction data. Figure
1 0 shows the canal cross-sectional area plotted against
centroid arc length. It is clear that cross-sectional area
decreases as a function of centroid arc length, and that the
rate of change of this parameter is greatest in the first turn
of the canal. Viewing the data in this manner can provide
insights in such areas as fluid dynamics and cochlear
implant design.
Conclusions
We have described an imaging technique that is con-
236 A . H . V O I E ET A L .
Arc length (mm)
Fig. 10. Cross-sectional area of the scala tympani as a function of
the canal centroid arc length.
ceptually uncomplicated and relatively inexpensive to
construct. The OPFOS technique allows convenient
imaging of a much larger specimen than is generally
possible by other optical sectioning methods. It provides a
long working distance and a wide field of view. Addition-
ally, the specimen may be translated and rotated during the
imaging process. Because of this there is little loss of
sampling resolution for a complex structure between
images. This is particuarly useful for making quantitative
measurements and 3-D reconstructions of complex biologi-
cal structures.
The depth range of this imaging technique is limited
primarily by the extent to which the clearing solution can
render a specimen transparent. The success of the clearing
process is in turn dependent on the decalcification step.
Large bony structures (e.g. vertebrae) could be expected to
be more difficult to decalcify than smaller bony structures
such as the cochlea. Specimens that contain little calcium
could be expected to clear even more easily, so it may be
possible to image larger dimensions.
Acknowledgments
This research was supported by National Institutes of
Health grants RR00166 and DC002 74 and by a grant from
the Bloedel Hearing Research Center.
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