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8.7 Theory 153
where σ
2
s
is the spatial variance of the intensity in the speckle pattern, T is the
integration time, and
˜
g
2
(τ ) is the autocovariance of the temporal fluctuations in
the intensity of a single speckle. The autocovariance is a normalized version of the
autocorrelation function and is defined as follows:
˜
g
2
(τ ) =
˜
G
2
(τ )
˜
G
2
(0)
(8.3)
where
˜
G
2
(τ ) =
I(t) −
I
t
I(t + τ) −
I
t
t
Equation (8.3) defines the relationship between LASCA and those techniques
that use the intensity fluctuations in laser light scattered from moving objects or
particles. LASCA measures the quantity on the left-hand side of Eq. (8.3); photon
correlation spectroscopy, laser Doppler, and time-varying speckle techniques mea-
sure the quantity on the right-hand side. (It is also worth noting that LASCA uses
image speckle, whereas most of the temporal techniques use far-field speckle. How-
ever, this does not detract from the fundamental equivalence of the two approaches
expressed in Eq. (8.3)).
Provided the assumptions made in establishing Eq. (8.3) are valid, LASCA is now
on an equal footing with all the temporal techniques (photon correlation, Doppler,
time-varying speckle) so far as linking the measurements to actual velocities is
concerned. All the techniques now allow the correlation time τ
c
to be determined.
In the case of photon correlation, this parameter is measured directly. In the case of
LASCA, some further assumptions must be made in order to link the measurement
of speckle contrast (defined as σ
s
/
I
) with τ
c
.
Various models can be used, depending on the type of motion being monitored.
For the case of a Lorentzian velocity distribution, for example, the equation
becomes [14]
σ
s
I
=
τ
c
2T
1 − exp
−2T
τ
c
1
2
(8.4)
Equation (8.4) (and similar equations for other types of scatterer motion) relates
the speckle contrast, defined as the ratio of the standard deviation of the speckle
pattern intensity to its mean intensity, to the ratio of correlation time τ
c
to the
integration time T. The relationship is plotted in Figure 8.2.
Figure 8.2 shows that the speckle contrast increases from near zero to near
its maximum value of 1 over about 3 orders of magnitude of τ
c
(and hence of
velocity). (For a single exposure, of course, T is a constant.) For flow velocities
corresponding to values of τ
c
less than about 0.04T, the speckle contrast is very
low, that is, the speckles are completely blurred out by the motion. For velocities
corresponding to values of τ
c
greater than about 4T, the speckle pattern has
high contrast. Between these limits, flow velocity will be mapped as variations
in speckle contrast. If a different velocity distribution is assumed, for example, a
Gaussian or a uniform distribution, the curve is slightly different, but still shows
this characteristic S-shape. The dynamic range – the range of velocities that can be
covered – remains similar.
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154 8 Laser Speckle Contrast Analysis (LASCA) for Measuring Blood Flow
1.0
0.8
0.6
0.4
0.2
0.0
0.001 0.01 0.1 1 10 t
c
/
T
s/<I >
Figure 8.2 Variation of speckle contrast with the ratio
decorrelation time to integration time for the Lorentzian
model of LASCA.
8.8
Practical Considerations
From this point on, LASCA experiences the same problem as all the temporal fre-
quency measurement techniques – photon correlation spectroscopy, laser Doppler,
and time-varying speckle. This is the problem of relating the correlation time τ
c
to
the velocity distribution of the scatterers. It is not straightforward. Problems include
the effects of multiple scattering, the size of the scattering particles (blood cells in
the present case), the shape of the scatterers, non-Newtonian flow, non-Gaussian
statistics resulting from a low number of scatterers in the resolution cell, spin
of the scatterers, and so on. Much work is being done on these effects, and the
question is far from settled. Because of the uncertainties caused by these factors, it
is common in all of these techniques to rely mainly on ratiometric rather than on
absolute measurements.
Another problem that occurs with LASCA is the difficulty in achieving the
full range of contrasts that should theoretically be available. A stationary object
should give a speckle contrast of one (σ =
I
, in accordance with well-established
speckle statistics theory and experiment). A fully blurred speckle pattern produced
by rapidly moving scatterers should have zero contrast. The Lorentzian model of
Eq. (8.4), for example, predicts the relationship between contrast (σ /
I
) and the
ratio τ
c
/T presented in Figure 8.2. This suggests that, for a given integration time
T, the dynamic range of the technique corresponding to contrasts between 0.1
and 0.9 should be about 3 orders of magnitude in τ
c
(and hence in velocity). In
practice, maximum contrasts of only 0.6 were being measured, even for stationary
random diffusers [19]. One possible cause of this problem is the CCD camera.
Most CCD cameras are designed to produce a DC offset from zero called the
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8.9 Applications and Examples 155
pedestal. This is to avoid cutting off the negative peaks produced during any
processing. It is formed because of the dark current and other anomalies in the
video circuitry of the camera. By carrying out some preprocessing on the data,
it is possible to remove the effect of the dark current. When this was done [37],
the measured speckle contrast on the image of a stationary diffusing surface rose
to 0.95, very close to the theoretical value of 1.0 for a fully developed speckle
pattern. Similarly, the measured contrast in the speckle pattern recorded from
a hand increased from 0.33 to 0.68. This lower contrast (compared with the
stationary diffuser) is due to the scatterers in the hand (the red blood cells) being
in motion.
Other problems with the statistics occur as a result of effects such as the Gaussian
profile of the laser beam and the nonlinearity of the CCD camera [37]. However,
many of these problems also affect the laser Doppler, photon correlation, and other
time-varying speckle techniques.
Other parameters can be varied. The effect of using different exposure times
(integration times) has been investigated, and times as short as 1 ms have been
used. Also, wavelengths other than 633nm could be used. There is a need in some
clinical situations, for example, eczema and other dermatological problems, to
measure the superficial dermal blood flow [38]. This has prompted several groups
to use other wavelengths with the laser Doppler technique [38 –40]. Penetration of
tissue is highly wavelength dependent, with infrared light penetrating the furthest
(several millimeters) and green and blue hardly penetrating (typically 150 µmfor
green light).
8.9
Applications and Examples
The original LASCA technique was developed specifically for monitoring capillary
blood flow in the skin. Figure 8.3 shows an example from the mid-1990s. It shows
part of a forearm with a superficial hot-water burn. The increased perfusion around
the burn is clearly visible.
Some researchers have continued this theme of using LASCA and related
techniques to measure skin and other microcirculation blood flow [41–45]. Others
have used it to characterize atherosclerotic plaques [46]. There has also been
a return to the area that first prompted the development of single-exposure
speckle photography in the 1980s – ophthalmology [7, 15, 47–49]. However, the
most important application of the LASCA technique is currently in the area of
neurological research, for monitoring cerebral blood flow [21, 31, 50–60]. A specific
investigation was into the causes of migraine [61]. (It is stressed that these examples
are just a selection from the large amount of work that is going on – the list is
not meant to be exhaustive. We are aware of more than 40 groups in at least 18
countries who are working on or with the LASCA technique.)
The intensive activity involving the LASCA technique in recent years has,
of course, been accompanied by improvements in the images produced. Two
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156 8 Laser Speckle Contrast Analysis (LASCA) for Measuring Blood Flow
Figure 8.3 LASCA image of part of a forearm, showing
increased perfusion around the site of a hot-water burn [24].
(a) (b)
Figure 8.4 Raw image of part of a rat cortex (a), and its
processed version (b). (Source: D. Boas, Harvard Medical
School.)
examples are reproduced here. Figure 8.4 is from David Boas’ group at the Harvard
Medical School, and Figure 8.5 is from Andrew Dunn at the University of Texas.
In both cases, the improvement in the quality of the images is clear.
8.10
Recent Developments
In general, researchers using LASCA have developed their own version of
the technique. This has naturally led to some significant improvements.
Developments include optimization of the exposure time [22], noise reduction
[62], and some significant contributions to the theory [23, 63]. Optimal integration
time varies from 1 ms for faster flow to 20 ms where the flow in smallest vessels
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8.10 Recent Developments 157
0 20406080100
Figure 8.5 Blood flow changes during stroke: relative cere-
bral blood flow 10 min after occlusion of the middle cerebral
artery in a rat, demonstrating the spatial gradient in the
blood flow deficit due to the ischemia. (Source: A. K. Dunn,
University of Texas.)
Figure 8.6 This image, produced by the
Moor Instruments laser perfusion imager
(FLPI), shows the mesenteric vessels in a
frog. The area shown is approximately 1 cm
2
,
and the gut runs around the right periphery
of the image. In this investigation, the user
is assessing angiogenic growth in mesenteric
vessels caused by a virus. This image was
taken before injection of the virus. (Source:
Moor Instruments Ltd: www.moor.co.uk.)
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158 8 Laser Speckle Contrast Analysis (LASCA) for Measuring Blood Flow
(with slower flow) is being studied [22, 35]. Issues such as the need to know the flow
range a priori and the fact that the theoretical basis is not as rigorous have meant
that laser speckle has a much slower uptake than LDPI. A proposal to use a multiple
exposure version of LASCA has recently been made [64]. The authors show that
this approach leads to a power spectral measurement that is exactly equivalent
to the Doppler spectrum and therefore provides a more robust theoretical basis
for the technique. This means that laser speckle contrast can be used to measure
perfusion with the same assumptions that are required for Doppler perfusion
measurements, but with the advantage of generating full-field images at video rates.
Some workers [25] have identified errors in the original theoretical formulations
(such as Eq. (8.4)). Some recent work, however, shows that the model still forms a
reliable basis on which to quantify velocities from the contrast measurements [59].
In late 2006, a major development took place with the announcement by Moor
Instruments Ltd, a UK company, of a commercial instrument that uses the LASCA
technique. Moreover, it has a real-time video capability. For the first time, this
allows the researcher or clinician to follow changes of blood flow in real time. Of
course, some resolution is sacrificed in order to achieve the high-speed processing
required, but the instrument also has a still facility for those cases where
resolution must be maintained. Typical images from the instrument are shown in
Figure 8.7 A composite image produced
by the Moor Instruments laser perfusion
imager (FLPI). The series of images of the
hand shows the baseline blood flow, the
changes during a partial occlusion using a
pressure cuff, and the flow following pres-
sure release. The images were taken at 3 s
intervals. The single image window is from
a video sequence taken at 25 frames per
second with a time constant of 0.3 s. The
single point measurement is taken from a
finger tip. (Source: Moor Instruments Ltd:
www.moor.co.uk.)
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References 159
Figures 8.6 and 8.7. A second company, Perimed of Sweden, has recently launched
their own version of LASCA.
8.11
Conclusions
Although in existence for more than two decades, very little has changed in LASCA
technology other than the acronyms. LASCA (whether known as laser speckle contrast
imaging or laser speckle imaging) offers a full-field, real-time, noninvasive, and
noncontact method of mapping flow fields such as capillary blood flow. Sensitive
to both the speed and morphological changes of the scattering particles, it is
particularly suitable for in vivo blood flow cytometry as well as investigation into the
evolutions of cell parameters. In its basic form, it uses readily available off-the-shelf
equipment. Laser Doppler, photon correlation spectroscopy, and time-varying
speckle are related techniques,but these work byanalyzing the intensity fluctuations
in scattered laser light. As they are essentially methods that operate at a single point
in the flow field, some form of scanning must be used if a high resolution full-field
velocity map of the flow area is required. Typical scanning laser Doppler systems
take some minutes to complete this scan. LASCA achieves this goal in a single shot
by utilizing the spatial statistics of time-integrated speckle. The technique produces
a false-color map of blood flow in a fraction of a second, without the need to scan.
Thus, LASCA is a truly real-time technique. In addition, the LASCA system is much
more cost effective and very simple to operate. The main disadvantage of LASCA
compared with the other techniques is the loss of resolution caused by the need to
average over a block of pixels in order to produce the spatial statistics used in the
analysis. In principle, at least, the time-domain techniques can operate on a single
pixel. However, in practice, even this disadvantage is not always present, as many
of the temporal methods use sampling in order to speed up the processing. In any
case, the advantage of real-time operation without scanning probably outweighs
the problem of loss of resolution, as demonstrated by the current global interest in
the technique. Finally, a more complete account of the history and development of
LASCA can be found in recent review papers [35, 65].
Acknowledgments
The authors are very grateful to David Boas of the Harvard Medical School, Andrew
Dunn of the University of Texas, and Moor Instruments Ltd, for their cooperation
and permission to use some of their images.
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