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Techniques for depth-resolved imaging through turbid media including coherence-gated

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2003 J. Phys. D: Appl. Phys. 36 R207

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INSTITUTE OF PHYSICS PUBLISHING JOURNAL OF PHYSICS D: APPLIED PHYSICS
J. Phys. D: Appl. Phys. 36 (2003) R207–R227 PII: S0022-3727(03)35485-3

TOPICAL REVIEW

Techniques for depth-resolved imaging

through turbid media including
coherence-gated imaging
C Dunsby and P M W French
Photonics Group, Physics Department, Imperial College London, Prince Consort Road,
London SW7 2BZ, UK
E-mail: paul.french@ic.ac.uk

Received 3 March 2003

Published 1 July 2003
Online at stacks.iop.org/JPhysD/36/R207

Abstract
This article aims to review the panoply of techniques for realising optical
imaging through turbid media such as biological tissue. It begins by briefly
discussing optical scattering and outlines the various approaches that have
been developed to image through scattering media including spatial filtering,
time-gated imaging and coherence-based techniques. The discussion
includes scanning and wide-field techniques and concentrates on techniques
to discriminate in favour of unscattered ballistic light although imaging with
scattered light is briefly reviewed. Wide-field coherence-gated imaging
techniques are discussed in some detail with particular emphasis placed on
techniques to achieve real-time high-resolution three-dimensional imaging
including through turbid media, providing rapid whole-field acquisition and
high depth and transverse spatial resolution images.

1. Introduction
1.1. Foreword
This article aims to describe the background and state-of-the-
art of coherence-gated imaging for potential applications in
high-speed three-dimensional profiling, biomedical imaging
and imaging through the atmosphere and seawater. It will focus
particularly on low coherence photorefractive holography,
which has been shown to provide real-time high-resolution
three-dimensional imaging, including through turbid media,
providing rapid whole-field image acquisition with high depth
and transverse spatial resolution. While there is an enormous
breadth of application for such technology, the most exciting
is surely biomedical imaging.
The investigation of optical techniques for the study
of biomedical systems is a rapidly developing field that
has seen a dramatic expansion in recent years, partly due
to the tremendous growth of the biomedical sciences and
biotechnology. Both medicine and biotechnology require
appropriate instrumentation to analyse and monitor biological
systems and many of the tools that have been developed
can be transferred from optical microscopic investigations
to macroscopic ‘in vivo’ diagnostics. The Holy Grail
for biomedical optics is a realization of a device akin
to the Star Trek ‘Tricorder’ that provides non-invasive
diagnostic capability through spectroscopic imaging, to
provide chemically specific, or ‘functional’ information
about biological tissue. This goal, often described as
‘optical biopsy’, inevitably requires image acquisition through
significant depths of biological tissue. Its realization,
however, presents a major scientific challenge since tissue
is extremely heterogeneous and interacts very strongly with
optical radiation. While it is this interaction that provides
the opportunity for functional biomedical imaging, the strong
absorption and scattering of the various tissue components
have historically restricted optical imaging to thin histological
tissue sections or to superficial tissues.
Absorption is a significant issue for optical imaging in
tissue but there is an absorption window for most biological
tissue between 650 and ∼1.4 µm. At longer wavelengths, the
absorption due to water is increasingly significant. Absorption

0022-3727/03/140207+21$30.00 © 2003 IOP Publishing Ltd Printed in the UK R207

Topical Review
will exponentially attenuate optical radiation according to:
−µa l
I = I0 e (1)
where I0 is the incident intensity and I is the transmitted
intensity after a distance l. µa is the absorption coefficient of
the medium (e.g. biological tissue). Its inverse is described
as the absorption length and represents the mean distance
travelled by a photon in the medium before being absorbed.
In the absorption window of biological tissue, the absorption
coefficient varies between ∼0.003–0.07 cm−1 , which is quite
acceptable for biomedical imaging. Semiconductor lasers and
the recently developed tunable solid-state laser technology can
now conveniently provide optical radiation in this spectral
region. Owing to the recent development of convenient, user-
friendly solid-state laser technology covering the near infra-red
(NIR), there has been a huge increase in the amount of research
activity in this area.
Having addressed the issue of absorption, the biomedical
optics community is devoting much of its effort to overcoming
the problem of scattering of optical radiation. In order
to form high (diffraction-limited) resolution images, or to
make accurate quantitative intensity measurements, it is highly
desirable to use light that has not been scattered. The intensity
of the unscattered component of an optical signal will be
attenuated according to:
I = I0 e−µs l (2)
where µs is the scattering coefficient of the medium. Its
inverse represents the mean distance a photon propagates
in the medium before being scattered and is described as
the scattering mean free path (MFP). For biological tissue
the scattering MFP is typically on the order of 100 µm and
so this is a severe problem for all but the thinnest tissue
samples. The ingenuity and resources deployed to tackle this
challenge are considerable, however, physics and engineering
communities have made very significant progress in the last
few years, particularly exploiting the revolution in laser and
imaging technology that has occurred over the last decade—
notably ultrafast and diode-pumped laser technology, solid-
state digital imaging technology (e.g. CCD) and advances in
image acquisition and processing capabilities.
Until the mid 1990s, the state-of-the-art optical source
technology available for biomedical optics and spectroscopy
was that of dye lasers, with their experimental complexities
and associated hazards, which were largely restricted to the
laboratories of laser experts. The demonstration of new tunable
solid-state lasers, however, brought unprecedented spectral
versatility with near turn-key operation to many users with
little or no laser experience. This means that laser spectroscopy
and novel imaging techniques could be applied to many new
fields including biomedicine. The advent of compact and
low-cost diode-pumped solid-state lasers mean that techniques
exploiting exotic sources such as ultrafast lasers can be feasibly
deployed in clinical or field-deployable instruments, rather
than only in laser laboratories. The ability of laser scientists
to control almost every property of the laser radiation provides
many new opportunities to acquire information about both
samples and the medium through which the radiation has
propagated. In particular, it will be seen that ultrashort pulse
lasers and ultrafast detector technology provide several means
to dramatically enhance images obtained through turbid media.
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While most of the research in this area has been directed
towards biomedical goals, the techniques are clearly applicable
to other turbid media and may well be rather more usefully
employed, e.g. for imaging through sea water, smoke or aerosol
media.
Since the world’s first femtosecond Ti : Sapphire laser
in 1987 [1], ultrafast solid-state laser sources have been
developed to provide spectral coverage from the visible to
∼1.6 µm in the NIR, based on Cr : YAG [2], Cr : Forsterite
[3], Pr : YLF [4], Cr : LiSAF and Cr : LiSGAF [5, 6]. Apart
from Pr : YLF, which is pumped in the band 450-480 nm
and lases at some 20 discrete wavelengths in the visible, all
these laser systems are tunable and may be directly diode-
pumped (Cr : LiSAF and Cr : LiSGAF) or may be pumped by
diode-pumped Yb doped fibre lasers [3]. Such diode-pumped
solid-state laser technology, together with ultrafast detectors
and instrumentation, provide many opportunities for both low
coherence and time-resolved imaging as will be discussed
in later sections. They also provide new opportunities for
fluorescence imaging, including fluorescence lifetime imaging
[7], coherent anti-Stokes Raman imaging [8] and ultraprecision
laser surgery using ultrafast laser ablation [9].
Recent work on biomedical imaging has built on
techniques developed using sophisticated but large and
expensive (e.g. ultrafast) lasers and developed more deployable
systems, e.g. using broadband LEDs, superluminescent diodes
(SLDs) or thermal light sources instead of femtosecond lasers
for coherence-gated imaging. This trend is likely to continue
with the development of compact fibre and diode based
sources. Thus, the widespread clinical deployment of optical
imaging techniques draws closer.
2. Imaging through scattering media
2.1. Overview
The problem of imaging an object through a scattering
medium is schematically represented in figure 1, which
illustrates how a conventional ‘shadowgram’ imaging system
may be impacted when the object is embedded in a turbid
medium. In figure 1(a), the photons travel in straight lines
and their intensity distribution is modified by the object and
recorded on the detector—typically a camera. Figure 1(b)
shows how this is degraded when the object is located in
a scattering medium. Some of the incident photons will
not be scattered but will continue to propagate in straight
lines: these are called the ballistic photons. The number
of unscattered photons, however, will decrease exponentially
with propagation distance and for any significant scattering
depth the ballistic signal will be swamped by multiply scattered
photons which will saturate a conventional detector and
obscure the image. In any real turbid medium, the number
of ballistic photons will also be reduced by absorption. For
relatively modest scattering depths it is possible to use filtering
techniques to block the multiply scattered photons and try
to form images with the ballistic light. This is the goal of
much of the work described here. In the context of ballistic
light imaging through biological tissue, Hee et al [10] have
estimated that, for radiation around 800 nm and considering the
maximum permissible intensity incident on living patients, the

(a) Incident Image-bearing
collimated light ballistic light
(camera)
Detector
Object
(b) Incident collimated Image-bearing
ballistic light
light
Tissue Object Scattered
diffuse light
Figure 1. (a) shows a simple ‘shadowgram’ imaging system, and
(b) illustrates how performance is impacted when the object is
located in a scattering medium such as biological tissue.
ballistic signal will fall below the shot noise limited detection
threshold after ∼36 MFP, i.e. after being attenuated by e36 .
This corresponds to a tissue depth of ∼4 mm. Note that
when considering other turbid media such as seawater, the
attenuation length due to absorption and scattering can be
significantly longer, e.g. 2 m for coastal seawater [11], and
so the corresponding maximum scattering depth for ballistic
imaging can be ∼80 m. Of course, when imaging through
media such as seawater, the constraints on the maximum
permissible incident intensity will be greatly relaxed compared
to biomedical imaging and so the ballistic imaging depth may
increase correspondingly, according to equations (1) and (2).
One interesting approach to increase the imaging depth
in biological tissue is to temporarily decrease the scattering
coefficient of the tissue by the application of appropriate
‘clearing agents’, e.g. glycerol for skin imaging [15]. This can
work because the scattering in biological tissue occurs mainly
at refractive index boundaries between internal structures.
The application and absorption of an appropriate clearing
agent can modify the local refractive index in the tissue
and therefore match the refractive index of the surrounding
tissue to that of the scattering structures. This can lead to a
reduction in the optical scattering coefficient and is termed
optical clearing [16]. Recently, this technique has been
demonstrated to enhance the penetration depth achievable
with optical coherence tomography (OCT) and to produce
additional contrast [17].
For many practical thicknesses of biological tissue,
however, the ballistic signal will fall below the (shot noise)
detection limit and all the detected photons will have
been scattered. One is helped, however, by the fact that
most practical turbid media, including biological tissue are
highly forward scattering. The anisotropy of scattering is
Topical Review
parameterized by ‘g’, which is the mean cosine of the scattering
angle. For biological tissue, g is typically in the range
0.7–0.9. This implies that a large fraction of the photons will
be only slightly deviated from their original direction upon
each scattering event. For scattering depths at which there is
effectively no ballistic light, there may be a significant number
of photons that have been only slightly deviated about the
ballistic direction. These photons, which follow a ‘snake-like’
path about the original direction, can provide information
about a reasonably well-defined path through the tissue. One
can consider this ‘snake-light’ signal to be approximately
exponentially attenuated according to [12]:

I = I0 e−µs l (3)
where µs is the transport scattering coefficient which is
given by
µs = µs (1 − g) (4)
One can consider its inverse, described as the transport
scattering length, to represent the distance a collimated beam of
light will propagate in the medium before becoming effectively
isotropic. Since g ∼ 0.9 for biological tissue, the transport
length is typically ten times longer than the scattering MFP.
Thus, one can estimate that snake light will be detectable after
transmission through no more than ∼360 MFP, corresponding
to ∼4 cm of tissue. After propagating more than two or
three transport lengths, however, most photons have undergone
strong scattering (i.e. out of their incident direction) and may
be described as diffuse.
Figure 2 illustrates different types of photon trajectories
through a scattering medium such as biological tissue and
indicates how a temporal impulse is modified by propagation
through a turbid medium. The ballistic light takes the shortest
path through the medium and so arrives at the detector
first. The earliest arriving scattered light is the forward
scattered snake light, for which the path length uncertainty
and associated transverse excursion increase with delay in
arrival at the detector. Strongly scattered diffuse light arriving
with increasing delay presents a corresponding increase in its
path length and transverse excursion uncertainty. In general,
one can obtain superior quantitative measurements or obtain
diffraction-limited image resolution if one can make use of
ballistic light. For intermediate scattering thickness one can
improve measurement accuracy or image resolution if one can
preferentially select the less scattered ‘snake light’. This may
be conveniently done by time-of-flight for transillumination
set-ups because the path lengths of the snake photons are
shorter than the more strongly scattered diffuse light. However,
improving spatial resolution and reducing uncertainty in the
optical path length by selecting earlier arriving light using a
narrower time gate will be at the expense of signal strength.
In practice, one must use a sufficiently wide time gate to
achieve an adequate signal-to-noise ratio at the detector within
an acceptable integration time. Under these constraints
Moon et al [13] have estimated that the best spatial resolution
achievable when imaging though a scattering medium with
diffuse light is given by ∼0.2 L where L is the thickness of the
scattering medium.
To select only the ballistic light and prevent the later
arriving light from saturating the detector, one requires an input
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Topical Review

Ballistic, “snake” and diffuse scattered light

Collimated lig
light “Snake” light
Ballistic light
Diffuse light

Detector

t t

Incident signal Transmitted signal

Scattering
medium

Figure 2. Schematic of TPSF observed when an temporal impulse is modified by propagation through a scattering medium.

pulse duration and time gate of ∼100 fs. To select the snake (a) Incident Image-bearing
light it is necessary to use a time gate shorter than ∼10 ps. The collimated light ballistic light
total envelope of the distribution of transmitted photon arrival
times is described as the temporal point spread function (TPSF)
and typically extends over several nanoseconds. Measuring
the TPSF and extracting statistical parameters such as the
mean time-of-flight make it possible to determine the average
optical path length and hence make average measurements
Scattering medium
of tissue absorption between a source and detector. Fitting
the TPSF to a model of photon transport provides a means Time gate
(low coherence)
to determine more information concerning the absorption and
scattering properties of thick biological tissue samples. The (b)
statistical models of photon transport range, with different
degrees of approximation, from full Monte Carlo simulation
of photon propagation though radiative transport theory to the
diffusion equation. Essentially one considers the statistical
behaviour of large numbers of photons with a distribution
of propagation paths. In this way one can make ‘spatially
averaged’ measurements or, using arrays of sources and Low coherence
detectors, one can tackle ‘the inverse problem’ to derive images gate
by calculating what distribution of absorption and scattering
Figure 3. Time-gated imaging through turbid media in
properties would have produced the measured (diffuse) light
(a) transmission and (b) reflection.
signals. This is an extremely challenging goal but one that
is being aggressively pursued, particularly for mammography
and imaging of brain function. well-resourced groups—although this situation is changing
There are thus three regimes of optical imaging through with the advent of low-cost time-correlated single photon
scattering media: ballistic light imaging, which provides the counting (TCSPC) technology [14]. Snake-light imaging has
highest (diffraction-limited) resolution based on rectilinear received the least attention and is least understood. It is often
photon propagation but which is only applicable for thin approached as a limit of ballistic or diffuse imaging but there
scattering samples; diffuse light imaging, which may be used is very little theoretical work in this area since it has proved
with the thickest scattering samples, provided that there is difficult to find a useful analytic model of weakly scattered
some detected signal, but which relies on a statistical treatment light.
of photon propagation and which provides relatively poor
resolution and snake-light imaging, which is an intermediate 2.2. Imaging in transmission or reflection
regime encountered when imaging through media that are
predominantly forward scattering. Ballistic light imaging has Applying the same technique to imaging through a scattering
received the most attention, being the most straightforward medium in transmission or reflection can produce significantly
to implement and readily modelled by geometric optics. different results. Figures 2 and 3(a) pertain to transmission
It is being applied to many diverse applications including images. By considering figure 3(a), one can see that, in
imaging of microstructures, remote sensing and biomedical principle, an ideal time-gated detector may be used to select the
optics. Diffuse imaging is primarily targeted at biomedical ballistic photons that arrive first and reject all of the scattered
applications such as mammography and brain imaging. There photons that inevitably have longer trajectories. Thus, a
is an extensive literature on the underlying theory but the cost diffraction-limited two-dimensional image may be formed
and complexity of the necessary instrumentation has limited with no degradation due to scattered light. If the sample is
experimental research activity to a relatively small number of too thick to permit ballistic light imaging, then the earliest

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arriving scattered light (snake light) may be detected and used
to form an image uncorrupted by the later arriving scattered
light.
The reflection geometry does not enjoy this potential
complete rejection of scattered light, although imaging in
reflection does inherently provide depth resolution via confocal
imaging and/or time-gated detection (using the time-of-flight
of the photons, in the same way that SONAR provides
depth information). Ideally, a time-gated detector used in
reflection mode will acquire photons that have undergone
a single backscattering event from a given depth in the
sample. Unfortunately, even for ballistic light imaging at
a shallow depth, there may always be multiply scattered
photons with trajectories that cause them to arrive at the
detector in the same time slot as ballistic photons from a
deeper depth. This is represented in figure 3(b) (green and
pink photon trajectories). Thus, time-gating alone can never
exclude all scattered light, although the narrower the time
gate, the fewer unwanted multiply scattered photons will be
detected. Note that, as discussed later, coherence provides
an additional means of discriminating against the unwanted
multiply scattered photons. When imaging through thicker
samples, it is necessary to increase the width of the time gate
in order to acquire sufficient signal and then corruption of the
desired signal by unwanted scattered light becomes rapidly
worse. This is the situation for snake-light imaging, which is
usually therefore implemented in a transmission geometry.
Unwanted detection of scattered photons can degrade
images in two ways. In figure 3(b) the green photon trajectory
illustrates inter-pixel cross-talk, which leads to degradation of
transverse spatial resolution and to speckle noise in coherent
imaging systems. It occurs when a multiply scattered photon
arrives at a wide-field imaging detector in the wrong transverse
pixel but right time slot and so still gets registered by the
detector. It does not occur in ballistic transmission imaging
systems that reject all (later arriving) scattered light but it is an
important issue for snake-light and diffuse light transmission
imaging systems with wider time gates. For wide-field
reflection imaging geometries, this inter-pixel cross-talk can
be a major problem since it can occur for even ballistic imaging
systems and becomes increasingly more problematic for snake
light and diffuse light imaging systems.
The pink photon trajectory shown in figure 3(b) illustrates
in-line scattering, when multiply scattered photons return to
their original trajectory to produce axial or longitudinal cross-
talk. Thus, photons scattering from shallower depths than the
image plane may be multiply scattered to arrive in the same
time slot and in the same pixel as ballistic photons. This
axial cross-talk can occur in all imaging systems operating
in reflection and is impossible to eliminate.
To summarize, the deleterious effects of unwanted
multiply scattered light are much more severe for imaging
in a reflection, rather than a transmission, geometry. For
this reason, a given imaging technique is likely to be
able to image through a significantly thicker scattering
medium in transmission. In particular, ballistic transmission
imaging systems can, in principle, reject all scattered light.
Transmitted light imaging systems do not, however, provide
depth information and so three-dimensional imaging must
be realized using tomographic techniques, in a manner
Topical Review
similar to x-ray computer-aided tomography (CAT) scans.
This approach, which is computationally intensive, has
been successfully demonstrated for a coherence detection
imaging system [18] and is regularly employed for diffuse
light imaging, as discussed in section 2.3.4.2.1. For many
practical applications, however, imaging in transmission is not
convenient or practical because the scattering medium is too
thick (e.g. for ballistic or snake-light imaging) or too distant
(for remote imaging). Also, the inherent optical sectioning
provided by the reflection imaging geometry is attractive for
many applications, particularly in biomedicine where high-
resolution three-dimensional images are desirable for optical
biopsy.
2.3. Imaging techniques that reject scattered light
2.3.1. Introduction: ballistic or snake light? All techniques
discussed in this section aim to improve the quality of images
acquired through scattering media by rejecting some or all the
scattered light. In this sense, they are all ballistic light imaging
techniques but most of them can also acquire images using
light that is only weakly scattered. The change from ballistic
to snake-light imaging is gradual. One can acquire the highest
resolution diffraction-limited images using ballistic photons or
one can sacrifice image quality to penetrate to greater depths
through a scattering medium. The acceptable compromise
will be determined by the specific application. In general,
the regime of weakly scattered light is not well understood and
is the subject of much current interest.
2.3.2. Spatial filtering
2.3.2.1. Whole-field spatial filtering. Perhaps the most
straightforward means to image through a scattering medium
is to employ some form of spatial filtering in a conventional
imaging system in order to improve the signal-to-noise ratio
(S/N). This exploits the observation that when a photon is
scattered, it usually changes its direction and, in a collimated
imaging system, most photons are scattered to higher spatial
frequencies.
A simple collimating grid, as shown in figure 4(a)
preferentially transmits only those photons travelling parallel
to the direction of incidence. This technique is widely
used in x-ray imaging through thick tissue sections where it
provides an enhancement of the S/N of up to ∼5. X-ray
photons, however are only weakly scattered by biological
tissue (scattering MFP ∼5 cm) and only a few are multiply
scattered before detection.
For optical radiation, a more practical alternative to the
collimating grid is a spatial filter in the Fourier plane [19, 20],
as shown by figure 4(b). This simple technique is effective
for reflection imaging to shallow scattering depths of up to
∼5 MFP (10 MFP in the round trip) and rather longer in the
transmission geometry. Of course the use of a spatial filter
in a wide-field imaging system will increasingly limit the
achievable spatial resolution of the image, as the filter becomes
stronger in order to reject more scattered light. In practice,
it is sensible to decide upon the target spatial resolution and
then use a spatial filter to limit the scattered light detected as
far as possible. Most systems for imaging through scattering
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(a) Incident collimated
light
Scattered
diffuse light
(b) Incident collimated
light
f f
Figure 4. Wide-field spatial filtering by (a) collimating grid and (b) Fourier plane spatial filter.
media exploit a spatial filter somewhere—although this is not
always acknowledged. Also, many techniques do not actually
prevent scattered light from reaching the detector by spatial
filtering but exploit it to obtain their desired images. Examples
include wide-field coherence-gated imaging techniques that
require object beam wavefronts to be matched to those of
a reference beam [21] and nonlinear optical systems with
phase-matching requirements such as in a parametric amplifier
system [22].
Ultimately, the efficacy of spatial filtering when imaging
through strongly scattering media is limited by the many
multiply scattered photons with trajectories parallel to the
incident direction that compromise the image by both axial
cross-talk, resulting from in-line scattering and inter-pixel
cross-talk from scattered photons with trajectories parallel to
their incident direction. Taking spatial filtering to its limit, one
can dispense with wide-field imaging altogether and apply the
maximum spatial filtering to eliminate inter-pixel cross-talk in
a (confocal) scanning system, as discussed in the next section.
2.3.2.2. Scanning (confocal) imaging systems. By acquiring
image pixels sequentially and scanning in two or three
dimensions to record an image, it is possible to completely
eliminate inter-pixel cross-talk and greatly ameliorate the
detrimental effects of scattered light. This approach is widely
used in ballistic and diffuse light imaging and two possible
implementations are shown in figure 5.
It is well known that the confocal scanning microscope
[23] offers superior resolution compared to the conventional
whole-field microscope [24], particularly in the longitudinal
direction, and, when used in reflection, provides a three-
dimensional imaging facility that has become the instrument
of choice for imaging objects embedded in a semi-transparent
medium. The current drive to towards optical biopsy and
functional imaging has led many researchers to try to apply
microscopy directly to subjects and several groups have
applied the confocal microscope to imaging through scattering
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Collimating grid
Ballistic and
in-line scattered
light + inter-pixel
cross-talk
Fourier spatial filter
f f
(a)
Optical Optical
fibre fibre
Ballistic light
and in-line
scattered light
(b)
Ballistic light
and in-line scattered light
Figure 5. Sequential pixel acquisition trough turbid media using
confocal scanning in (a) transmission and (b) reflection.
media, including through biological tissue in vivo (e.g. [25]).
Reasonable high-resolution (mainly ballistic) light images
have been obtained to depths of 300–500 µm, depending
on the type of tissue, since the confocal microscope is a
highly effective spatial filter. Ideally the incoming radiation
is focused to a single voxel and only light backscattering
(or fluorescing) from that voxel is collected. Scattered light can
blur the focused beam outside the target volume, however, and
unwanted photons from other voxels can be scattered back into
trajectories that will be collected by the microscope. Scattering
of the incoming beam before focus is perhaps the most serious
constraint on imaging depth—particularly when using high
numerical aperture (NA) objectives. Spherical aberration is
also an issue that compromises the achievable resolution and
becomes more significant with increasing NA [26].
Nonlinear microscopy, for which the signal is proportional
to the square or cube of the incident intensity, has been
shown to increase the achievable imaging depth. Multi-photon
microscopy, which records the fluorescence signal arising
from an intensity-dependant two [27] or three [28] photon

absorption, has been shown to provide increased depth
penetration into scattering media (e.g. [29]). The increase
in penetration depth is partly due to the use of longer
wavelength excitation radiation, which is less attenuated in
biological tissue, and partly to the fact that the scattering
only has a significant impact on the collected (fluorescence)
light, since the nonlinear excitation provides an effective
point source localization and only fluorescence light is
collected—so scattered excitation light does not produce
cross-talk—although attenuation of the excitation signal is a
problem. It is also proposed that multi-photon microscopy
is less compromised by spherical aberration [30]. Of course
multi-photon fluorescence imaging is restricted to fluorescent
samples. Other nonlinear processes, however, have also
been shown to offer similar benefits in scattering media.
In particular third harmonic microscopy [31] is showing
promise as a nonlinear three-dimensional imaging modality,
and, since it relies only on a refractive index change to generate
the intensity-dependant third harmonic signal, it can be applied
to a wide range of samples. In a similar manner to multi-photon
absorption microscopy, the signal is at a different wavelength
from the incoming radiation and so scattering of this latter
‘pump’ beam is not a problem. The penetration depth of
nonlinear microscopy is limited by the ability to achieve the
required intensity level in the focal plane. To some extent,
the attenuation due to scattering can be offset by increasing the
input power but eventually a further limit is set by the maximum
permissible intensity level at the surface of the tissue. Using
regenerative amplifier technology, the penetration depth of two
photon fluorescence is estimated to be improved by two to three
scattering MFPs over that achievable using a Ti : Sapphire laser
oscillator alone [32].
These nonlinear microscopy techniques exploit ultrafast
lasers to provide pulse trains exhibiting high peak power but
low average power. Another important property of modern
ultrafast lasers, however, is their short pulse duration and
low timing jitter and this can be exploited to further reject
scattered light through time-gating. The combination of
confocal imaging as a means of rejecting scattered light and
time-gating, particularly low coherence time-of-flight gating,
has provided the highest penetration depths of ballistic light
imaging in scattering media.
2.3.3. Time-gating
2.3.3.1. Introduction. It should be immediately apparent
from figure 3 that time-gating offers a means to discriminate
against scattered light either by itself or in combination with
spatial filtering. When imaging in transmission, ballistic
photons will always take the shortest path between source and
detector and so time-gating can be very effective. However,
many of the time-gates used in practice are not sufficiently
fast to select only the ballistic light, for scattering media
that are sufficiently thin to permit ballistic light imaging, this
typically requires a temporal discrimination faster than 1 ps.
Such temporal discrimination is possible using low coherence
interferometry and some nonlinear correlation techniques, but
is not achievable with electronic detectors. For this reason,
incoherent time-gated detectors, which typically have temporal
responses of tens to hundreds of picoseconds, usually collect
Topical Review
some weakly scattered snake light in addition to the ballistic
component and the resolution is consequently degraded.
2.3.3.2. Incoherent time-gates. Incoherent time gating may
be realized using fast electronic instrumentation, such as streak
cameras, fast avalanche photodiodes or photomultipliers or
gated optical image intensifiers (GOI), or it may be realized
using intensity-dependant nonlinear optical gates based on
harmonic generation, parametric amplification, the optical
Kerr effect and stimulated Raman scattering.
Typically photodiodes and photomultipliers exhibit
response times of the order of tens to hundreds of picoseconds
and so are not effective at rejecting scattered light—although
they may be used to characterize it for diffuse light imaging
(see section 2.4). Historically, one of the most important
time-gated detectors applied to this field has been the electro-
optic streak camera, which exhibits high sensitivity and a
linear response that can be faster than 1 ps. In principle,
these are two-dimensional detectors, offering one spatial
dimension and one time dimension but, when applied to
imaging through scattering media, they are normally used as
single pixel detectors, owing to the cross-talk along the spatial
dimension corresponding to the entrance slit. Streak cameras
are too expensive and complicated to use in most real-world
applications but they provide an excellent opportunity to study
the TPSF of a pulse upon traversing a scattering medium
and have been used by several groups for this purpose (e.g.
[33, 34]). Streak cameras have particularly been applied to
studying the earliest arriving scattered light (e.g. [33]) and the
impact of the time gate on the achievable image resolution
[35]. The TPSF can give information about both the scattering
and absorbing parameters of the medium being probed and,
by scanning the source and detector, images can be acquired
through thick scattering media such as tissue samples of several
centimetres thickness (e.g. [36–39]). Unfortunately, the need
for long integration times to detect weak signals, combined
with the requirement for scanning pixel-by-pixel, makes the
image acquisition times prohibitively long when using streak
cameras or other single pixel detectors.
An interesting alternative electronic detector that provides
whole-field time-gated images is the GOI. This is a
microchannel plate intensifier that may be rapidly switched on
and off, effectively providing a fast shutter with an exposure
time that can be as short as 70 ps. To date this technology
has mainly been applied to wide-field fluorescence lifetime
imaging (e.g. [7]), although it has been used for whole-
field time-gated imaging through turbid media including
seawater [40], through which it provided images of 0.675 cm
bars through 4.5 m of turbid water corresponding to 13 MFP
attenuation lengths (in the round trip). Recently, a GOI has
been used to record images through samples of biological tissue
of several centimetres thickness, corresponding to hundreds
of MFP [41]. The images presented are formed from the
earliest arriving scattered light that can be detected and are
of relatively poor resolution, primarily due to inter-pixel
cross-talk. Recently, several groups have started to use
the GOI as a multi-channel time-gated detector for diffuse
light imaging. This approach involves using the GOI to
simultaneously measure the TPSF profiles from multiple fibre
probes simultaneously [42].
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Topical Review
Incident signal
t t
Scattering
medium
Proportional to the
ballistic signal
Nonlinear
medium
Figure 6. Incoherent time-gated imaging through turbid media using a nonlinear optical correlation gate.
With both streak cameras and GOIs, it is possible to
achieve depth-resolved imaging but the depth resolution will be
limited by the gate time—with 100 ps corresponding to 1.5 cm
depth resolution in air. To realize three-dimensional imaging
with micron resolution it is necessary to use time gates of less
than 100 fs. This is not possible with electronics but may be
achieved using intensity-dependent nonlinear optics, in which
the time gate is the same duration as the optical pulse used.
The principle of nonlinear optical time-gating, which may be
implemented for single pixel detection or for whole-field time-
gated imaging, is shown in figure 6. Essentially only that
part of the signal TPSF that is temporally coincident with the
reference pulse is detected because the reference pulse opens an
intensity-dependent nonlinear ‘shutter’. This technique may
also be described as time correlation using nonlinear optics.
Commercially available ultrafast lasers, typically based
on Ti : Sapphire, routinely provide pulses of 100 fs duration
and research systems have produced pulses as short as 6.5 fs
[43, 44]. Thus, it is possible to realize time gates that might
select only the ballistic light. Historically, however, this area
of research was first addressed using picosecond pulses, often
from Nd : YAG based laser systems. Perhaps the most obvious
nonlinear time gate is cross-correlation by second harmonic
generation (SHG), since this is closely allied to second
harmonic autocorrelation, which is the standard technique
for measuring ultrashort pulses. This technique works by
combining the desired ballistic light with the reference pulse
in a nonlinear crystal and generating a second harmonic signal
that is proportional to the product of their intensity profiles.
The reference pulse essentially samples the TPSF according to
their relative delays. It had been applied to three-dimensional
imaging by ‘range-gating’ [45] and has been demonstrated
to realize ballistic light imaging in transmission through a
phantom of 28 MFP scattering thickness using a single pixel
scanning configuration [46]. These approaches are usually
single channel pixel-scanning techniques although there has
been a recent demonstration of single-shot acquisition of two-
dimensional x–z image slices using a titled reference beam
in an SHG set-up incorporating cylindrical lenses [47]. As
discussed in section 2.3.2.2, nonlinear microscopy, in which
the sample itself acts as the nonlinear medium, also provides
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“Snake” light
Ballistic light
Diffuse light
Transmitted signal
Raman Frequency Picosecond
doubling
generator Nd: YAG laser
crystal
Incident
signal
t
Scattering Transmitted signal
medium
Amplified Raman time gate
ballistic signal
Raman
amplifier
Figure 7. Principle of nonlinear optical time-gating using
amplification by stimulated Raman scattering.
a means to image through scattering media but this does not
exploit time-gating to reject scattered light.
Amplification by stimulated Raman scattering has also
been used as a nonlinear optical shutter (figure 7). The
efficiency with which an intense pulse is Raman scattered
to its first (or higher) Stokes-shifted wavelength is greatly
enhanced by the presence of ‘seed’ radiation at that Stokes
wavelength, which beats with the intense ‘pump’ pulse to
drive the material vibration that is the origin of the Raman
scattering. If, in a Raman active medium, the signal radiation
TPSF of the light emerging from a scattering medium is
combined with an intense pump pulse (at a wavelength that
is one Stokes shift lower than the signal wavelength), then
that portion of the signal TPSF that is temporally coincident
with the pump pulse will be amplified—with a gain coefficient
that can be ∼109 . Using 30 ps 2.5 mJ pump pulses in Raman
cells filled with H2 , this technique has been demonstrated at the
Naval Research Laboratories, Washington DC, to permit wide-
field time-gated imaging at 5 Hz repetition rate with a spatial
resolution of 150 µm though 33 MFP of scattering medium
in transmission [48] and has provided three-dimensional
imaging through 17 MFP (round trip) scattering thickness with
 Topical Review

<5 mm depth resolution [49]. It should be noted that this
implementation used relatively powerful lasers to achieve these
results. Such technology would be inappropriate for many
applications, particularly in biomedicine but may be acceptable
for remote imaging applications. Aside from the complexity,
the chief drawbacks to this technique are the limited aperture
of the Raman amplifiers, which limits the achievable spatial
resolution and the fact that the scattered light is at the same
wavelength as the time-gated signal and so contributes to noise
at the detector. Spontaneous Raman scattering also contributes
to the noise and determines the minimum detectable signal.
The depth resolution could be improved by using shorter
pulses, as are commercially available today.
Another nonlinear optical effect that has been applied
to whole-field time-gated imaging through turbid media is
parametric amplification. Parametric generation, in which a
pump photon in a nonlinear medium splits into two photons
(described as the ‘signal’ and the ‘idler’) of lower energy, can
be considered as the inverse of SHG. Parametric amplification
describes this process when it is seeded by an appropriate
weak signal at a lower wavelength, in a manner analogous to
amplification by stimulated Raman scattering. In degenerate
parametric amplification, one pump photon splits into two
photons of twice the wavelength (see figure 8). This has
been exploited to selectively amplify the early-arriving light
from a signal TPSF at 780 nm when imaging in transmission
through a scattering medium of 20 MFP scattering thickness
using 2 mJ, ∼400 fs pulses at 20 Hz [22]. As noted in
section 2.3.2.1, the limited angular acceptance imposed by
the phase-matching requirements provided a strong spatial
filter that supplemented the rejection of scattered light by the
time gate. The images were acquired pixel-by-pixel using a
confocal scanning configuration that, combined with the low
pulse repetition rate, resulted in a long image acquisition time.
Parametric amplification has also been applied to whole-field
image acquisition through a scattering medium of 22 MFP
thickness, achieving a resolution of 20 µm using signal/pump
pulses of 55 ps/35 ps at 1064 nm/532 nm at 10 Hz repetition
rate [50]. Also, imaging of 9 mm bars through 4 cm of chicken
breast (∼400 MFP) has been achieved [51]. Recently, this
technique for time-gated imaging through turbid media has
been implemented with a laser system operating at 1 kHz pulse
repetition rate [52].
The optical Kerr effect provides one of the oldest
techniques for ultrafast time-gating [53] and has also been
exploited for time-gated imaging, in conjunction with spatial
filtering in the Fourier plane [54]. The optical Kerr effect
is exploited such that a powerful reference pulse induces
birefringence in a Kerr active medium placed between two
crossed polarizers. An image-bearing signal is directed
through this arrangement and only the component that is
temporally and spatially overlapped with the intense reference
pulse experiences an intensity-dependent polarization rotation
(due to the optical Kerr effect) such that it is transmitted
through the final polarizer. This time gate may be employed
in the image plane or in the Fourier plane. Because of the
requirement for high intensity across the whole of the signal
to be transmitted, it is often impractical to use a Kerr gate in
the image plane with a significant field of view. In the Fourier
plane, however, the relatively tightly focused reference beam
can act as a spatial filter and preferentially select the ballistic
or scattered light components.
2.3.4. Coherence-gated imaging
2.3.4.1. Introduction. The coherence properties of laser
radiation provide a powerful means to discriminate against
scattered light exploiting the fact that ballistic light retains
coherence with a reference beam whereas scattered light can
become effectively incoherent. Stetson proposed coherence
gating as a means of imaging through fog in 1967 [55] and
demonstrated c.w. holography of a ceramic pitcher that was
imaged through soapy water. Later Caulfield [56] refined
the technique to incorporate a simple time gate element
into the experiment—anticipating modern techniques that use
low coherence sources for high-resolution imaging through

Frequency Femtosecond
doubling
laser
crystal

Incident
signal

t
Scattering
Transmitted signal
media

Parametric pump
Amplified idler wave (time gate)
component Signal
Nonlinear medium wave
(degenerate OPA
Type II phase-matching)
Polarizer

Figure 8. Principle of nonlinear optical time-gating using amplification by parametric amplification.

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Topical Review
scattering media. Probably, the most widely employed
coherence-gated technique today is that described as OCT,
which combines scanning confocal microscopy with coherent
heterodyne detection. There are, however, many different
approaches to coherence-gated imaging, some of which are
described in the following section. Broadly speaking, they
can be divided into single-pixel scanning techniques, which
may be applied in transmission or reflection, or whole-
field imaging techniques such as coherent optical heterodyne
detection or holography. In essence, all coherence-gating
techniques rely on some type of heterodyne detection to detect
weak ballistic signals and many techniques achieve shot-noise
limited detection. Techniques such as OCT work in the
time domain while holography can be considered as a spatial
heterodyne system. Each approach has specific advantages
and there is usually a trade-off between sensitivity, image
acquisition rate and complexity.
2.3.4.2. Coherent optical heterodyne techniques. This
section deals with techniques that preferentially select
unscattered light based on its coherence with a reference beam
and exploit heterodyne detection in the time domain.
2.3.4.2.1. Imaging using single channel coherent detection:
coherent detection imaging. If one considers the confocal
imaging system represented by figure 5(b), one can see that
only those scattered photons that are collinear with the desired
ballistic signal will be collected. Typically at least three
scattering events will be required for a photon to recover
its initial ballistic trajectory and so this will be a very small
fraction of the total photon flux. Nevertheless, when imaging
to tissue depths of more than 3–5 MFP (300–500 µm of tissue),
the in-line scattered light can swamp the ballistic signal in
a confocal microscope. If an interferometer is added to the
detection system, then scattered photons may be rejected on
the basis that they do not contribute to the fringe pattern that
results from the interference with a reference [57].
Optical heterodyne detection has been implemented in
a transmission geometry with a single frequency c.w. laser
source, exploiting the temporal coherence of the signal
to provide heterodyne detection of the interference signal
together with the directional ‘antenna’ property provided by
the spatial coherence [21]. By employing a CAT system to
combine a series of single-pixel transmission measurements
through different projections of the sample, images have been
achieved through a range of biological and other scattering
samples [58] including chicken egg, chicken bone, human teeth
and opaque industrial products [59], with spatial resolution
down to 50 µm (e.g. [60]). This technique has been described
as ‘coherent detection imaging’ (CDI).
When the coherence length of the imaging radiation is
long compared to the sample scattering MFP, photons that
scatter back into the original direction can still contribute to the
detected heterodyne signal since they can still interfere with
the reference beam. This can be greatly alleviated by using
broadband radiation of low temporal coherence. In this case,
an interferometric signal is only detected when the pathlength
of the signal and reference photons are matched to within the
coherence length of the source. Effectively low coherence
interferometry provides a time gate since scattered photons
whose pathlength fail this criterion will not contribute to the
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heterodyne signal (although all scattered photons incident at
the photodetector are a source of background noise). Enhanced
performance of CDI has been demonstrated with broadband
radiation and, in confocal transillumination imaging systems,
the use of low coherence radiation has been demonstrated to
provide superior performance [61, 62].
2.3.4.2.2. Imaging using single channel coherent detection:
OCT. As well as better discriminating against scattered light,
however, low coherence interferometry also provides a ranging
capability when used in reflection. The depth is given
by the time-of-flight of the detected photons and the depth
resolution by half the coherence length. Implementing low
coherence interferometry in a confocal microscope provides
three-dimensional imaging with very strong discrimination
against scattered light. This approach is widely described
as OCT [63] and has been extensively developed for in vivo
biomedical imaging in the eye and in strongly scattering
tissues. For reviews of OCT see [64, 65], also there is now
a comprehensive reference work on OCT available [66].
Typically, the reflected light is collected by a single mode
fibre and input into a Michelson interferometer. Here, the
signal beam is interfered with a reference beam that has a
constantly varying delay, as shown in figure 9. Interference
occurs only when the reference arm length matches the path
length travelled by the ballistic component to within the
coherence length of the source. Scanning the reference arm
therefore allows different depths within the sample to be
sectioned. By measuring the amplitude of the interference
fringes a reflected signal versus depth plot can be made.
Then by scanning in two dimensions a three-dimensional
image may be formed. A heterodyne detection scheme can
be implemented using the Doppler shift introduced into the
reference arm when rapidly scanning the pathlength (e.g. by
moving the mirror). This allows very low coherent light
levels to be detected, and such systems have shown a dynamic
range of >100 dB. It should be noted, however, that any
scattered photons that do reach the detector will impair the
dynamic range of detection. In OCT, a single mode fibre
is typically used to deliver and detect the light, which adds
the advantages of a confocal detection to prevent much of the
scattered photons from reaching the detector. Furthermore, the
entire interferometer can be realized in optical fibre with a 3 dB
fused fibre coupler taking the role of the 50/50 beamsplitter.
This leads to a very robust, compact and sensitive system.
Low coherence
light input
Optical
Fringe
fibre
detector
Sample
Optical arm
fibre
x
Reference arm x
Figure 9. Optical coherence tomography.

OCT can reach the shot-noise detection limit, which
corresponds to a penetration depth of ∼18 MFP (36 MFP in
transmission), corresponding to ∼2 mm of biological tissue
[67]. In practice, however, OCT is limited by ‘longitudinal
speckle’, i.e. photons which have scattered back into their
original path with a time delay appropriate for detection but
which have not been singly backscattered from the target depth
in the sample. This in-line, in-time scattered light often limits
the useful penetration depth of OCT in strongly scattering
tissue to ∼8–10 MFP.
OCT is probably the most mature coherent technique
for imaging through scattering media. It has been used to
image a range of biological tissues, both in vitro and in vivo,
from the retina to the gastro-intestinal tract. However, the
need for scanning to produce two-dimensional images means
that image acquisition times are limited (3.7 µm transverse
resolution images can be formed in ∼2.5 s [68]). Images of
a xenopus heart beating [69] have been demonstrated. Here,
the rapid scanning required to increase the achievable frame-
rate was realized using a piezo-electric fibre stretcher capable
of scanning over 3 mm at 600 Hz [70]. Fibre stretchers are
limited due to polarization and hysteresis effects and so high-
speed OCT is now usually implemented using rapid depth-
scanning delay lines utilizing a scanning mirror in the Fourier
plane of a lens imaging a diffraction grating to provide phase
and/or group delay [71]. This arrangement can provide a scan
velocity of 6 m s−1 at 2 kHz over a range of 3 mm in an OCT
set-up. This technique has enabled video-rate OCT imaging of
a beating xenopus embryo heart at frame-rates up to 32 frames-
per second [72]. OCT has also been extended to include
polarization sensitive detection [73], measurements of sample
velocity through the induced Doppler shift [74] and spectrally
resolved detection [75].
Clearly as the scan speed is increased, the integration
time for each pixel decreases and so increasing the image
frame-rate for OCT requires advances in both scanning speed
and in the average power of the low coherence radiation
source. The use of confocal detection with single mode
fibres requires a spatially coherent source with low temporal
coherence and high average power. This is difficult to achieve
since high power is usually associated with gain narrowing in
lasers. Mode-locking is one way to circumvent gain-narrowing
and typically a mode-locked femtosecond solid-state laser
(Ti : Sapphire or Cr : Försterite) is employed to provide average
powers of 100 mW and spectral widths of 50–100 nm, yielding
depth resolutions as low as ∼2 µm. Such femtosecond
lasers have only recently become commercially available
and are relatively large, complex and expensive. For the
ultimate resolution, the femtosecond pulses may be used
to generate supercontinua in microstructured optical fibres,
thereby achieving sub-micron coherence lengths [76]. Near-
micron level axial resolution has also been demonstrated using
this method at the more deeply penetrating centre wavelength
of ∼1.1 µm [77, 78]. In real-world applications this complex
technology is often replaced by SLDs that typically provide up
to a few milliwatts average power coupled into a single mode
fibre with spectral widths of ∼20–40 nm to provide a depth
resolution of ∼10–20 µm [79]. This is the approach taken in
commercial OCT systems used for retinal imaging.
There are at least two other approaches to provide spatially
coherent radiation with a low temporal coherence length.
Topical Review
One is the rapid scanning of a frequency tunable source, to
synthesize the required coherence function. Using a grating-
tuned external cavity semiconductor laser, a linewidth of
25 nm was achieved with 35 mW average power in 100 ms
[80]. A similar approach has also been implemented
using a rapidly tuned Cr : Försterite laser [81]. Clearly,
such techniques are limited if one wishes to achieve very
fast image acquisition. An alternative approach is based
on a broadband c.w. diode-pumped solid-state lasers that
incorporates intracavity spatial dispersion of the radiation
frequency spectrum. This technique, which provides high
average power spatially coherent broadband radiation without
the complexity of mode-locking, is scaleable in both average
output power and spectral width and has been demonstrated
to provide 10's nm spectral width and up to 100 mW average
power [82].
Typically OCT acquires and displays data in ‘XZ-scan’
mode, i.e. one transverse dimension and one depth dimension.
This is often appropriate for biomedical imaging applications
as it corresponds to a typical histological section. For many
other applications, however, an en face image is required
(XY-scan), corresponding to a transverse image section at a
fixed depth (Z). This has been realized by developing a rapid
transverse confocal scanning system [83] and can provide en
face images at a few hertz frame-rate. For faster imaging, one
must reduce or remove the requirement to acquire the pixels
(or voxels) in series and develop whole-field imaging systems
with parallel pixel acquisition. This will entail using two-
dimensional imaging detectors such as CCD cameras. The
next section outlines techniques to acquire ‘en face’ (XY)
wide-field depth-resolved images and section 2.3.4.4 describes
approaches to acquire transverse scan (XZ) images in parallel.
2.3.4.2.3. Multiple channel coherent detection: wide-field
optical heterodyne imaging. The limited image acquisition
rate of OCT that is imposed by the requirement to (confocally)
scan the image, pixel-by-pixel, has led to many proposals
for speeding up the acquisition rate. In general, this
is realized using a multiple channel (wide-field) detector,
such as a CCD camera instead of a single photodetector,
although there has been significant work towards developing
arrays of ‘smart detectors’ that are essentially multiple OCT
systems performing heterodyne envelope detection in parallel
[84]. Currently, an array of 58 × 58 pixels has been
achieved and this technique has also demonstrated video-
rate volumetric imaging [85]. Full field coherent heterodyne
(interferometric) detection, however, is usually implemented
using CCD cameras to record the interference signal and
the use of low coherence light in a reflection geometry
provides depth-resolved imaging. The desired whole-field
coherent signal may be extracted from the acquired ‘coherent
signal + incoherent background’ images by recording a
series of acquisitions with different phase delays (φ1 − φ2 )
between the object and reference beams and combining them
appropriately in post-processing.
Unfortunately, the lack of the confocal filter in wide-
field coherence-gated imaging means that much more scattered
light will fall on the detector and contribute to a background
on the integrated signal. Also, the dynamic range of CCD
cameras is much lower than that of the photodiodes (100 dB)
usually employed for OCT. Another consideration is that CCD
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Topical Review
detectors can not adequately sample rapidly changing signals,
so they are not able to provide synchronous detection at
kilohertz and higher frequencies to reduce 1/f noise. Rather,
CCD cameras may be used with relatively long integration
times as the base-band filter in a homodyne demodulation
system or they may be used at sub-kilohertz rates to acquire
images for post-processing and averaging. These factors limit
the signal-to-noise ratio of detection and mean that wide-field
coherence-gated imaging systems generally can penetrate to
significantly lower depths than OCT. This is compounded by
the observation that, while OCT uses all of its radiation in a
single channel, whole-field detection necessarily divides the
available power between each pixel and so the dynamic range
is reduced accordingly—unless higher power sources are used
to provide an equivalent photon flux per pixel.
A further limitation of whole-field coherent detection
compared to OCT is inter-pixel cross-talk as discussed in
section 2.2. This can be particularly challenging for coherent
imaging because these inter-pixel scattered photons will have
an arbitrary phase and this leads to speckle noise on the
coherent image. When imaging through liquid scattering
media, however, the Brownian motion of the scatterers tends
to randomize the phase of the scattered photons and so they
merely contribute to the incoherent scattered light background.
It is much more of a challenge when imaging through static
turbid media for which stable speckle noise may be observed
superimposed on the ballistic light image. Most of the coherent
imaging experiments reported in the literature relate to imaging
through liquid scattering media and so one should be cautious
when evaluating their success. One way to eliminate inter-
pixel cross-talk in coherent imaging systems is to use spatially
incoherent light—thus ensuring that all transversely scattered
photons are uncorrelated with the ballistic light and result in
a uniform background. This additional d.c. signal will be
detected by the CCD camera, however, and reduce the effective
dynamic range. Typically CCD cameras with high bit depths
(dynamic) range are available but it is prohibitively expensive
to use systems with a bit depth greater than 16. Spatial
filtering can provide some rejection of scattered light in wide-
field systems, however, as discussed in section 2.3.2.1 and
imaging attenuation lengths of ∼16 MFP have been achieved
in practice, albeit at modest transverse resolution. Of course
averaging maybe used to increase the signal-to-noise ratio but
this will be at the expense of image acquisition time and this
makes video-rate wide-field coherence-gated imaging through

0 MFP scattering media practically impossible.
Chiang et al [86] demonstrated sub-milimetre depth-
resolved wide-field imaging through a liquid scattering
medium of 17 (roundtrip) scattering MFP using ‘c.w. broad-
band interferometry’ with a SLD of 8 nm bandwidth. The
image-processing algorithm involved subtracting the incoher-
ent background from each (coherent + incoherent) acquired
image acquired and then averaging the ‘coherent’ images. The
use of a spatially coherent SLD resulted in some speckle noise
on the acquired images. The speckle was somewhat reduced
by averaging over multiple frames and over adjacent pixels.
A more sophisticated wide-field coherence-gated imaging
system using optical heterodyne detection was reported by
Beaurepaire et al [87]. Figure 10 shows a generic schematic
of this approach, which involves recording four wide-field
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CCD
(modulated@Ω)
Reference
arm
Modulator
Broadband
Ω/4
radiation source
Imaging
optics
Scattering medium
Sample
Figure 10. Shows a generic schematic of whole-field optical
heterodyne imaging.
interferometric images with different phase delays (φ1 − φ2 )
between the object and reference beams and processing them
to recover both the coherent amplitude and phase images.
This technique was originally implemented using an LED
source and a photoelastic modulator in a Michelson/Linnik
interferometer set-up, in which the reference and object beams
are orthogonally polarized. It has recently been modified
to utilize a thermal broadband light source to provide sub-
micron depth resolution and a simple piezo-actuator at the
reference mirror to provide the required phase differences
between the two arms [88]. This simple but powerful technique
can use considerable averaging to achieve a sensitivity of
∼90 dB for an image acquisition time of 4 s. Images have
been acquired through both liquid and solid scattering media,
including biological samples, with the use of broadband light
sources of low spatial coherence eliminating the problem of
speckle noise. A particularly powerful feature is the ability
to calculate the phase of the coherent image, as well as
the amplitude [89]. This can provide depth information at
sub-wavelength precision (i.e. a few nanometres).
Like all wide-field coherence-gated imaging techniques
based on direct detection with a CCD camera, this approach is
limited by the scattered light that reaches the detector (CCD
camera) and reduces the dynamic range. This is discussed
further in section 2.3.4.4. A further potential disadvantage
for biological imaging is that this approach requires the
sample to be interferometrically stable during the acquisition
of the four phase-stepped interferometric images. Although
the system can acquire images at 100 Hz using a high-
speed CCD camera, the interferometric stability requirement
still implies that the sample is effectively stationary on
a millisecond timescale. An approach to overcome this
limitation has recently been demonstrated that acquires all
four phase-stepped interferometric images in a single-shot
[90]. This is done by using appropriate polarizing optics
and beamsplitters to project the four phase-stepped images
simultaneously on a single CCD camera and has been used to
acquire depth-resolved images of moving objects at video rate.
2.3.4.3. Holographic imaging. This section deals with
techniques that preferentially select unscattered light based on
its coherence with a reference beam and exploit heterodyne
detection in the spatial domain.

Holography may be considered as a wide-field two-
dimensional coherent recording technique, making it an
obvious choice for a fast coherent gating method to image
through turbid media. It was with holography that Stetson
first showed the principle of coherence gating for imaging
through a fog-like medium [55]. In 1989, light-in-flight (LIF)
holography was demonstrated to facilitate depth-resolved
imaging of three-dimensional objects [91], including through
optical diffusers. However, photographic film was used
as the holographic recording medium and so each image
acquisition had to be chemically developed before being
reconstructed. Subsequent research has concentrated on
using real-time methods to perform the holographic recording
and reconstruction, focusing mainly on two techniques:
‘electronic’ or ‘digital’ holography and photorefractive
holography.
A schematic of holography applied to imaging through
turbid media is shown in figure 11. Holography can
be considered as a spatial heterodyne technique, with the image
information being modulated onto a spatial carrier (i.e. a fringe
pattern). There is ‘heterodyne gain’ in the process since a
powerful reference beam can interfere with a weak signal beam
to overcome any dark noise in the recording medium. This may
be a conventional photographic film or a CCD camera, as in
the case of ‘electronic’ or ‘digital’ holography, or it may be
a photorefractive medium. In principle, if there is no inter-
pixel cross-talk, only the ballistic image will be coherent with
the reference beam and contribute to the hologram—although
the diffuse scattered light may saturate the recording medium.
In the case of the film or the photorefractive medium, there
is an additional mechanism for gain when the (potentially
weak) hologram may be reconstructed using a powerful read-
out beam for acquisition by a CCD camera. Furthermore,
the read-out beam may be a different wavelength from the
writing beams and so all light scattered by the object can
be excluded by a filter. For the case of digital holography,
in which the reconstruction is done numerically, the CCD
camera will record any incident scattered light along with the
hologram and this will degrade the dynamic range of detection
and compromise the ability to reconstruct the ballistic light
image.
(a)
Image beam
Reference beam
Scattered light
(b)
Read out beam
Reconstructed
image beam
Figure 11. Holographic imaging through turbid media:
(a) recording and (b) reconstructing the hologram.
Topical Review
As a whole-field coherent imaging technique, holography
suffers from many of the same disadvantages as wide-
field heterodyne detection, particularly inter-pixel cross-talk
and saturation of the detector by the scattered light background.
The first of these issues may be addressed by the use of
incoherent light—in fact Leith has shown that holography with
incoherent light is formally equivalent to confocal imaging
[92]. Note that this is not necessary if imaging through a
dynamic (e.g. liquid) turbid medium. The second issue is
less tractable and provides the ultimate limitation to many
holographic imaging systems—but it should be noted that
photorefractive holography is insensitive to a diffuse light
background, as discussed later.
Unlike wide-field heterodyne detection, off-axis holog-
raphy is intrinsically a single-shot technique and so may be
applied to high-speed two- and three-dimensional imaging
of dynamic objects. This significant advantage is somewhat
offset by issues associated with the necessarily noncollinear
interference geometry. These include optical aberrations aris-
ing from the off-axis image propagation through the optical
system, the issue of beam walk-off, encountered when inter-
fering beams whose coherence length is comparable or less
than the beam diameter, and the issue of spatial resolution.
For electronic holography, aberrations in the imaging
system may be corrected at the image-processing stage but
for optical holography, including photorefractive holography,
they can only be minimized by using appropriate optical
systems. The problem of beam walk-off, which arises when
using low coherence light to provide depth resolution and/or
rejection of scattered light is illustrated in figure 12. This
presents a significant problem for angles of incidence much
greater than a few degrees with broadband radiation of 10 nm
[93]. By adjusting the group delay with respect to the
phase delay (wavefront) as a function of lateral position, e.g.
using prisms, it is possible of overcome this walk-off and
recover the whole field of view [94]. The issue of transverse
spatial resolution is a function of the number of resolution
elements in the holographic recording medium. For electronic
holography the pixel size of the CCD camera is generally of
the order of 5 µm (or greater for CCD sensors with large well
capacity—high dynamic range). To ensure adequate sampling
of the holographic interference pattern, the fringe period
must be at least twice this and the corresponding spatial
θ θ
Image Reference
beam beam
Figure 12. Walk-off of low coherence beams during holography.
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frequency must be at least three times the highest spatial
frequency in the image to avoid aliasing in the holographic
reconstruction. In practice, micron resolution is achievable
in systems with optical magnification but this consideration
does place constraints on the optical system that are not
present for collinear wide-field optical heterodyne imaging.
For photorefractive holography, the diffraction efficiency will
depend on the fringe period according to the photorefractive
medium used. For electro-optic crystals, the highest diffraction
efficiencies are obtained for fringe periods around 1 µm, which
is fine for spatial resolution but a problem with respect to beam
walk-off. For photorefractive MQW devices, the fringe period
should be greater than ∼20 µm and this presents similar spatial
resolution considerations as large CCD pixels do for electronic
holography.
2.3.4.3.1. Electronic holography. Electronic holography was
demonstrated as a means to coherence-gate the early-arriving
light when imaging through a scattering medium by Chen et al
[95] in 1991. In this technique, the interference pattern is
incident directly upon a CCD camera and recorded onto a
computer. With knowledge of the reference beam intensity
distribution the recorded hologram can be processed computa-
tionally to produce the original coherent portion of the trans-
mitted beam. By spatially filtering the transmitted beam before
it was incident on the CCD camera and using a low coherence
source a resolution of <1 mm has been achieved through a scat-
tering solution equivalent to ∼4 mm of tissue [96]. As with
wide-field coherent heterodyne detection, the chief limitation
of this technique is the scattered light background, from which
the coherent holographic image must be subtracted.
The principal advantage of electronic or digital
holography is that, because the holographic reconstruction is
done in software, there is great flexibility in the processing
that may be carried out. For instance, it is possible to
use a tunable narrowband laser and compute the time-gated
hologram that would have been obtained using a broadband
source from a series of holograms recorded using the narrow
band source tuned over the equivalent spectral range. This has
been described as Fourier synthesis holography [97]. Digital
holography also has the advantage that post-processing may
also be applied to compensate for aberrations in the imaging
system, as long as they are characterized [98], and one can
filter out zero order and twin images [99]. In some situations,
aberrations may be reduced by locating the recording (CCD)
plane away from an image plane. With today’s computers, the
computation required to reconstruct the holograms takes only
a few milisecond and real-time imaging is practical. As with
coherent heterodyne detection, it is possible to obtain both the
amplitude and the phase of the coherent image.
2.3.4.3.2. Photorefractive holography. In 1993, Mamaev et al
[100] showed that a photorefractive crystal called strontium
barium niobate (SBN) could be used to image through a
suspension of milk in real-time with no chemical developing.
This demonstrated the efficacy of photorefractive holography
although narrowband c.w. radiation in the green was used and
the imaging configuration was in transmission. In 1995, Hyde
et al [101] demonstrated, for the first time, real-time two- and
three-dimensional imaging through a scattering medium using
both c.w. and ultrashort pulse NIR radiation with rhodium-
doped barium titanate (Rh-Ba : TiO3 ) as the recording medium.
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This work highlighted the observation that photorefractive
holography is insensitive to a uniform background of, e.g.
multiply-scattered diffuse light. This reflects the unique
properties of photorefractive media that make them sensitive,
not to the incident intensity but to the spatial derivative of
the incident intensity distribution—in contrast to all other
wide-field imaging detectors (apart from arrays of smart
pixels discussed in section 2.3.4.2.3) for which the uniform
scattered photons are detected and can saturate the detector,
compromising its dynamic range. Figure 13 shows a typical
set-up for low coherence photorefractive holography for three-
dimensional imaging through turbid media. The set-up is
essentially the same as for electronic holography except that
the CCD in the holographic recording plane is replaced by a
photorefractive medium, from which the reconstructed image
is read out using a laser that may be at a different wavelength
from the broadband source. Thus, all scattered photons may
be blocked at the CCD camera using a colour filter.
Using Rh-Ba : TiO3 as the photorefractive medium, the
detection of a weak coherent image in the presence of a 106 ×
greater incoherent background was demonstrated using a low-
cost video camera with a bit depth of ∼6 [102]. Three-
dimensional imaging through scattering media of up to 16 MFP
(roundtrip) thickness was demonstrated for the first time with
sub-100 µm depth and transverse spatial resolution [103],
albeit with a long (∼300 s) integration time owing to the
relatively slow response of Rh-Ba : TiO3 as a photorefractive
recording medium for such weak intensities. Rh-Ba : TiO3 was
used for this application because it offers the highest diffraction
efficiency of the electro-optic photorefractive crystals in the
optical ‘window’ of transmission of biological tissue around
800 nm. It’s slow response, however, makes it impractical
for most real-world applications. To obtain both high
speed and NIR sensitivity, the most promising candidates
are semiconductor media. To date, the highest sensitivities
and fastest responses have been obtained from semi-insulating
photorefractive MQW devices exploiting the transverse Franz
Keldysh effect [104].
CC
D
Photorefractive
medium
Read-out
laser
Broadband
radiation source
Imaging
optics
Scattering medium
Sample
Figure 13. Low coherence photorefractive holography.

The application of photorefractive MQW (PRQW)
devices to rapid three-dimensional imaging through turbid
media with NIR radiation was demonstrated to permit depth-
resolved image acquisition with integration shorter than 0.4 ms
[105]. Later three-dimensional imaging was demonstrated
with image acquisition direct to a video-cassette recorder,
without recourse to a digital frame-grabber [106, 107].
Recently, the technique has been adapted to the use of
spatially incoherent light from LEDs, providing sub-10 µm
depth resolution [108] and demonstrating speckle free images
through static turbid media including sandstone and chicken
tissue [109]. Using a high-speed CCD camera, this approach
has realized depth-resolved imaging at 476 frames s−1 [110],
The chief drawback of this approach is that, while
photorefractive holography does have the potential to enhance
the ability to detect a weak coherent image against a
uniform background of scattered light and so increase the
effective dynamic range compared to direct detection with a
CCD camera, the scattering of the read-out beam from any
inhomogeneities in the photorefractive medium introduces a
source of additional noise that can degrade the reconstructed
holographic image. This is a significant issue for current
PRQW devices (and for bulk semiconductor crystals)—as
discussed in the following section.
2.3.4.4. Dynamic range of wide-field coherence-gated
imaging techniques. Wide-field coherence-gated imaging
through scattering media involves the detection of a (weak)
interferometric signal against a (usually larger) diffuse light
background. When attempting to measure such small
variations in intensity directly using a CCD camera, it is
primarily the full-well capacity that determines the smallest
change in incident intensity that can be detected. If the total
signal is sufficiently large, the CCD camera should optimally
be operated near to the full-well capacity of the pixels and the
detection is then a reasonable approximation to being shot-
noise limited as the number of electrons stored in the well
is significantly larger than the typical readout, digitization
and other noise sources. It is therefore the shot noise that
determines the smallest detectable change in signal and this is
proportional to the square root of the number of electrons stored
in the pixel-well. Typically, large pixel CCD cameras can have
full-well capacities of ∼105 or greater. This size of pixel-well
√ information in an interference or diffraction pattern.
could therefore resolve a change in illumination of ∼1/ 105 .
If spatial averaging (pixel binning) or temporal averaging is
employed then the effective number of electrons in the pixel-
well is increased by a factor equal to the number of pixels and/or
frames averaged. Temporal averaging is a powerful method
for increasing the dynamic range, although the apparatus
and object must be stationary to within a wavelength for
the duration of the acquisition of an interferogram and this
may imply some practical limitations—particularly for living
tissue. The ultimate ability of a CCD based coherence-
gated imaging technique to resolve images in the presence
of a background of scattered light will always depend on the
precise experimental configuration but generally will vary as
the square root of the effective CCD full-well capacity.
For photorefractive holographic imaging, the most
significant source of noise is introduced by optical
imperfections in the photorefractive medium itself, as
Topical Review
discussed above. Small imperfections are often introduced
during the manufacture of the photorefractive medium and
may eventually be eliminated but they are an issue for the
devices currently available. When reconstructing a hologram
in a photorefractive medium, the desired first-order diffracted
light containing the image information is angularly separated
from the undiffracted light. Typical diffraction efficiencies for
photorefractive media may be of the order of 1–0.1% and so the
diffracted signal is generally at least two orders of magnitude
smaller than the undiffracted beam. Consequently, any small
optical imperfections can scatter a significant amount of the
undiffracted beam into the direction of the diffracted signal
and hence introduce an unwanted background of scattered
read-out light at the CCD camera. The dynamic range of
the photorefractive detection process thus depends on two
parameters: the dynamic range of the CCD and the optical
quality of the photorefractive device. These effects have been
quantified and the achievable dynamic range for PRQW based
photorefractive detection has been estimated as a function of
the optical quality of the PRQW device [111]. In practice, the
discrimination against a scattered light background achieved
with photorefractive holography using current PRQW devices
is comparable to that achieved with direct detection using a
12-bit CCD camera. Further development of, e.g. PRQW
growth and fabrication technologies, however, could result in
significant enhancements in optical quality and therefore in
effective dynamic range.
2.3.4.5. Depth-resolved imaging with no mechanical
scanning. A major advantage of whole-field coherent
detection techniques compared to confocal scanning systems
is that they acquire multiple channels in parallel, providing
faster image acquisition—albeit at the expense of reduced
spatial filtering of scattered light, reduced power per pixel
and the lack of synchronous detection using high frequency
lock-in amplifiers. A whole-field detector can clearly
simultaneously sample two dimensions of a three-dimensional
object volume and both XZ-scans (traversal) and XY-scans
(en face) are possible. For OCT, particular attention has been
paid to achieve simultaneous acquisition of all the pixels of a
XZ-scan, for which it is necessary to encode depth information
in some manner and map it onto one dimension of the CCD
detector. Most of these techniques involve encoding depth
One proposal is to use acousto-optic modulators (AOM)
to laterally scan a single pixel laser beam and to provide depth
resolution by recording the interference pattern between light
reflected from the object and from the sample [112]. The
modulators also provide a heterodyne signal. This system,
which was designed to be a rapidly scanned OCT system, is
ingenious but relatively complex with a depth range limited
to ∼400 µm by the spatial resolution of the optics used and
a depth resolution given by the spectral width of the source.
An alternative technique uses a diffraction grating in the OCT
interferometer to arrange that different wavelengths take paths
of different lengths [113]. This approach effectively encodes
different depth information in different spectral interferograms
and achieved a sample depth scan of 3 mm with 15 µm
resolution. A similar approach has been described as ‘spectral
radar’ [114]. This technique has been applied to imaging in
the eye [115].
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An alternative analysis of a similar approach is that
of ‘spectral holography’, given by Leith’s group [116, 117].
Their implementation combined the idea of Fourier synthesis
holography, sweeping a tunable dye laser over 15 nm, with
spectral interferometry. It had the very significant practical
advantage that it is not necessary to match the arm lengths
of the interferometer to acquire the depth-resolved image
information. This is because each spectral component has a
long coherence length and the short coherence information can
be extracted when a series of spectrally resolved interferograms
are combined. A similar spectral interferometry technique
utilising a broadband SLD source was shown to achieve 38 µm
depth resolution with a depth range of 7.5 mm in air [118].
Note that all of these techniques suffer from the usual
wide-field imaging issues when applied through scattering
media. It remains to be seen whether high-speed OCT will be
most widely deployed using rapid confocal scanning or wide-
field detection.
2.4. Imaging with scattered light
2.4.1. Introduction. When imaging through scattering
media after which no ballistic photons are detectable within
a reasonable integration time, or through which the ballistic
signal is below the shot-noise limit, then images must be
formed with light has been scattered. In such a case,
the inter-pixel cross-talk is usually so dominant that whole-
field imaging becomes practically impossible. Essentially,
the approach is to build up an image, pixel-by-pixel, with
the light that has scattered least (snake light), if it can be
detected, or to use average pathlength-resolved measurements
(in transmission or reflection) of the average optical properties
of thick scattering samples (such as ∼1 cm of biological tissue)
using diffuse light. As is illustrated in figure 14, the earlier
arriving light corresponds to photons that have taken the better-
defined path through a scattering sample, thereby probing the
optical properties of a smaller volume and ultimately providing
superior spatial resolution. In the reflection trajectory, time-
resolved measurements of scattered photons probe the volumes
illustrated, sometimes described as a ‘banana’ trajectories for
obvious reasons. If an array of sources and detectors is
employed, as illustrated in figure 15, it is possible to tackle
the inverse scattering problem and build up an image using
(a)
Tissue
Optical
fibre
Source Detector
(b) Optical fibre Optical fibre
Source
Tissue
Figure 14. Schematic illustrating the uncertainty in the volume of tissue interrogated for two common source-detector geometry’s for
time-resolved measurements in turbid media.
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CAT, usually based on the diffusion approximation, which is
essentially a statistical treatment of the propagation of photons
through a scattering medium.
2.4.2. Imaging with the earliest arriving light. Clearly if one
can form images with the earliest arriving, least-scattered light,
then one will achieve a higher spatial resolution. This is usually
done in the time domain using time-gated photon counting
or a streak camera (e.g. [36]), although the weak signal level
often requires a prohibitively long integration time for practical
applications and there is still much debate (e.g. [119]) about
whether it is possible to practically improve upon the 0.2L
diffuse light resolution limit for optical mammography and
brain imaging, for which the tissue thickness is >5 cm or
500 scattering MFP. One particularly intriguing technique
is to record the whole of the TPSF and, using a fit to the
diffusion approximation, extrapolate the TPSF to early times to
calculate what would have been the early-arriving light signal,
had it been possible to detect it [37]. This achieved ∼5 mm
spatial resolution through a phantom simulating a human breast
(∼500 MFP thickness).
For scattering media of <100 MFP thickness, it is possible
to use rather simpler technology to obtain enhanced images.
Holography has been applied to thick scattering media by Leith
et al who argue that holography is still applicable with scattered
light, albeit with a much degraded point spread function. Using
a technique called ensemble-averaged imaging combined with
holography using the first arriving light, enhanced images of
0.5 mm wide slits were obtained through a phantom of 60
scattering MFP thickness [120].
An even simpler technique to implement is polarization
gating, which can provide a limited but effective means
to discriminate against scattered light. In the process of
being scattered, photons lose their initial sate of polarization
and, after scattering a sufficient amount of times, the light
becomes effectively depolarized. Hence by only detecting
those photons with the same polarization state as that of the
input, some of the scattered noise can be rejected. Demos
et al [121] have demonstrated a technique whereby they collect
the TPSF of the transmitted light parallel and perpendicular
to the input polarization. The parallel polarization TPSF
contains information about the unscattered and least scattered
photons as well as noise due to the totally randomized photons
Earliest
light
Optical
fibre Detector
t
Detector Earliest light
Most scattered light
Detector
t

S
D
D
S Source
S S
D Detector
D
D
S
Figure 15. Illustration of source-detector geometry for optical
tomography using diffuse light and inverse scattering calculations.
appearing later in the TPSF. The orthogonal polarization
TPSF only contains information about those photons that
have scattered sufficiently enough to totally lose their initial
polarization state. Subtracting the TPSF of the perpendicular
from the parallel polarization component allows the light that
has scattered least or not at all to be preferentially selected as
it would have been by a ∼100 ps time gate. The birefringence
properties of different materials can also be exploited to image
them through scattering media. If a polarization analyser
is placed after the scattering media, Tyo et al [122] have
shown that a judicious choice of orthogonal polarization state
measurements allows objects that predominantly reflect a
single polarization to be imaged through scattering media.
2.4.3. Imaging with diffuse light. When imaging through
thick scattering media for which all the detected photons
are diffuse, images must be formed using highly scattered
photons. It is usually necessary to resort to some model of
photon transport in order to extract information concerning
objects embedded in such a scattering medium. Although light
propagation in random media may be accurately simulated
using Monte Carlo techniques, the prohibitive computing
resource requirements make it impractical to accommodate
the numbers of photons encountered in any real optical
measurement or experiment. To analyse experimental data
it is, instead, necessary to use mathematical models based
on the radiative transfer equation [123]. Unfortunately,
no general solution to this equation has yet been obtained
and so an approximation must be used. The simplest is
probably the diffusion approximation, which is valid for
photon distributions far from boundaries of the scattering
medium and photon sources. The diffusion approximation also
assumes that photon propagation is dominated by scattering,
i.e. µa  (1−g) µs . The time-independent diffusion equation
[124] is widely employed in near infrared spectroscopy
(NIRS) to calculate the approximate absorption and scattering
coefficients along an ‘average’ path through biological tissue,
as discussed above. These may be more precisely determined
using the time-dependent diffusion equation [125, 126], which
can also be employed to tackle the inverse scattering problem
that must be solved to achieve tomographic imaging with
diffuse light in thick tissue samples.
When making the line-of-sight or ‘banana’ measurements
of the average absorption (µa ) and transport coefficients (µs )
along a trajectory in a thick scattering medium, it is necessary
to acquire information concerning the corresponding average
optical path length. This is done in the time domain by
Topical Review
(a) Mean time-
of-flight
0 t 0 t
(b) Phase shift ∆θ
Attenuation
~e-µeffL
µeff = [3µa(µa + µ’s)]1/2
Figure 16. Illustration of (a) time domain and (b) frequency domain
techniques for line-of-sight measurements of attenuation and
average path length of optical signals in turbid media.
measuring the average time-of-flight of the detected photons or
in the frequency domain by measuring the average phase delay
of a sinusoidally modulated signal, as represented in figure 16.
Time-domain detection tends to lead to expensive
instrumentation, requiring ultrashort light pulse sources,
(typically mode-locked lasers) and fast detectors, but to date
that it has yielded the best resolution images through thick
scattering media. A notable current state-of-the-art instrument
for thick tissue imaging is MONSTIR, being developed at
University College, London, which boasts 32 time-gated
detector channels with 20 ps resolution and 8 tunable ultrafast
laser sources [127]. Recent advances in technology for TCSPC
technology, however, offer the promise to build comparable
instruments that are considerably less expensive and more user-
friendly.
The frequency domain technique requires a c.w. source
which is typically modulated at a frequency in the range
100 MHz–10 GHz. The resulting radiation may be considered
as a photon density wave. The detector is required to
measure the amplitude and the phase shift of the transmitted
photon density wave with respect to the source. The frequency
domain technique is considered to be simpler than the time
domain approach because it does not require mode-locked
lasers or ultrafast detectors. Such a measurement at a single
frequency, however, provides only the mean time-of-flight of
the photons through the medium and not the full TPSF, which
can be exploited to provide more information for the inverse
scattering algorithm. In principle, one can make a set of such
frequency domain measurements over an appropriate range of
frequencies and obtain the Fourier transform of the TPSF but
this is not usually technologically straightforward because of
the requirement to modulate and detect signals at very high
frequencies.
3. Conclusions
In conclusion, this article has aimed to review the range of
techniques being developed to image through scattering media
such as biological tissue. It is impossible to describe all the
relevant physics and technology in the space available but
the authors hope that they have presented a useful overview
or introduction to the subject and provided an indication of
suitable further reading. Optical imaging through biological
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tissue remains a tremendously exciting, challenging and
vigorous research area that is likely to deliver advances of
significant practical benefit. While it is likely that many of
the techniques discussed here will not progress to clinical
application, they have all contributed to the understanding that
will produce biomedical advances and most of them will be
employed in diverse areas of science and technology. One
topic that was not discussed here is fluorescence imaging.
Currently this has not been widely applied to scattering tissue
samples but its excellent specificity and the opportunities to
use exogenous ‘molecular beacons’, including genetically-
expressed fluorescent labels, are drawing increasing interest
from the biomedical optics community. The challenges
associated with extracting functional fluorescence-based
information from highly scattering tissue samples will
certainly build on the pioneering work on optical tissue
imaging.
Acknowledgments
C Dunsby gratefully acknowledges a QUOTA PhD studentship
from the UK Engineering and Physical Sciences Research
Council (EPSRC) and a CASE award from Holoscan UK Ltd.
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