Journal of Biomedical Optics 15共1兲, 011109 共January/February 2010兲

Laser speckle contrast imaging in biomedical optics

David A. Boas Abstract. First introduced in the 1980s, laser speckle contrast imaging
Harvard Medical School is a powerful tool for full-field imaging of blood flow. Recently laser
Massachusetts General Hospital speckle contrast imaging has gained increased attention, in part due to
Anthinoula A. Martinos Center for Biomedical Imaging
149 13th Street its rapid adoption for blood flow studies in the brain. We review the
Charlestown, Massachusetts 02129 underlying physics of speckle contrast imaging and discuss recent de-
velopments to improve the quantitative accuracy of blood flow mea-
sures. We also review applications of laser speckle contrast imaging in
Andrew K. Dunn neuroscience, dermatology and ophthalmology. © 2010 Society of Photo-
The University of Texas at Austin Optical Instrumentation Engineers. 关DOI: 10.1117/1.3285504兴
Department of Biomedical Engineering
1 University Station, C0800 Keywords: laser speckle contrast; blood flow; biomedical imaging.
Austin, Texas 78712
Paper 09230SSR received Jun. 8, 2009; revised manuscript received Jul. 20, 2009;
accepted for publication Jul. 29, 2009; published online Jan. 13, 2010.

1 Introduction An important calibration procedure for handling static scatter-

When a photon scatters from a moving particle, its carrier ing 共from the skull, for instance兲 that would otherwise con-
frequency is Doppler shifted. By analyzing the Doppler shift found the estimate of blood flow from spatial laser speckle
of the photon, or the distribution of Doppler shifts from a contrast was developed,13,14 while Li et al.15 showed that tem-
distribution of photons, it is possible to discern the dynamics poral laser speckle contrast imaging 共TLSCI兲 is intrinsically
of the particles scattering the light. Analogously, when coher- less sensitive to static scattering. Recently, Kirkpatrick et al.16
ent light scatters from a random medium, the scattered light and Duncan et al.,17 presented important theoretical efforts
produces a random interference pattern called speckle. Move- quantifying the impact of speckle size and sampling windows
ment of scattering particles within the random medium causes on the statistics of laser speckle, and others have demon-
phase shifts in the scattered light and thus changes the random strated more efficient algorithms for calculating speckle con-
interference pattern, producing temporal fluctuations in the trast to enable real-time visualization.18,19 Finally, LSCI is be-
speckle pattern that is analogous to the intensity fluctuations ing implemented in multimodal imaging schemes to obtain
that arise from Doppler shifts. The theoretical basis for ana- additional information about the tissue.20–23
lyzing speckle intensity fluctuations dates back to the late In the next sections, we review the theoretical background
1960s with the development of dynamic light scattering.1,2 of speckle contrast and the important considerations when us-
There was extensive activity in the 1970s rigorously relating ing spatial speckle contrast or temporal speckle contrast to
speckle temporal dynamics to various forms of particle dy- estimate blood flow. We then review several of the application
namics in dilute single-scattering suspensions.3–5 Extensions areas for LSCI that have been advancing over the last twenty
to highly scattering systems was made in the 1980s in various years, including imaging blood flow in the retina, skin, and
guises by Bonner and Nossal6 and Pine et al.7 While provid- the brain.
ing detailed information about system dynamics, these meth-
ods were slow at obtaining images of blood flow.8,9 A solution
was presented in the 1980s using a camera to obtain a quick 2 Theoretical Background
snapshot image of a time-integrated speckle pattern,10 from 2.1 Speckle Basics
which blood flow in the retina was estimated from the spatial
Speckle arises from the random interference of coherent light.
statistics of the speckle pattern. This method, laser speckle
Whenever coherent light interacts with a random scattering
contrast imaging 共LSCI兲, was advanced in the 1990s for im-
medium, a photodetector will receive light that has scattered
aging blood flow in the retina and skin with the availability of
from varying positions within the medium and will have thus
faster digital acquisition and processing technologies.
traveled a distribution of distances, resulting in constructive
In the last decade, there has been an accelerated adoption
and destructive interference that varies with the arrangement
of the technology as the neuroscience community has used it
of the scattering particles with respect to the photodetector.
to measure blood flow in the brain, starting with Ref. 11.
Thus, if this scattered light is imaged onto camera, we will see
During the last eight years, there have been more than 100
that the interference varies randomly in space, producing a
publications on the topic, significantly advancing the theory
randomly varying intensity pattern known as speckle. If scat-
and application of laser speckle imaging. Bandyopadhyay et
tering particles are moving, this will cause fluctuations in the
al.12 presented an important correction in the calculation of
interference, which will appear as intensity variations at the
laser speckle contrast and its relation to temporal fluctuations.
photodetector. The temporal and spatial statistics of this
speckle pattern provide information about the motion of the
Address all correspondence to: David A Boas, Harvard Medical School, Massa- scattering particles. The motion can be quantified by measur-
chusetts General Hospital, Anthinoula A. Martinos Center for Biomedical Imag-
ing, 149 13th Street, Charlestown, Massachusetts 02129. Tel: 617-724-0130;
Fax: 617-643-5136; E-mail: [email protected] 1083-3668/2010/15共1兲/011109/12/$25.00 © 2010 SPIE

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 Boas and Dunn: Laser speckle contrast imaging in biomedical optics

pattern.17,27 A spatial speckle contrast of 1 indicates that there

is no blurring of the speckle pattern and therefore, no motion,
while a speckle contrast of 0 means that the scatterers are
moving fast enough to blur all of the speckles. The speckle
contrast is a function of the exposure time T of the camera
and is related to the autocovariance of the intensity fluctua-
tions in a single speckle Ct共␶兲 by10

Fig. 1 共a兲 Raw speckle image from the thin skull of a rat, showing a
grainy pattern in which it is possible to discern some spatial variation
in the speckle contrast, and 共b兲 when the spatial speckle contrast is
K2 =
␴2s 共T兲
具I典2
=
1
T具I典2
冕 0
T
C t共 ␶ 兲 d ␶ , 共2兲
estimated from a 7 ⫻ 7 window of pixels, the blood vessels on the
surface of the brain become apparent with high spatial resolution. although it has recently been indicated that the proper calcu-
lation of a second moment is12

ing and analyzing either the temporal variations4 or the spatial

variations.10 Using the latter approach, 2-D maps of blood
␴2s 共T兲
具I典 2 = 2
1
T 具I典2
冕冕 T

0
T

0
C t共 ␶ − ␶ ⬘兲 d ␶ d ␶ ⬘

冕冉 冊
flow can be obtained with very high spatial and temporal res-
olution by imaging the speckle pattern onto a CCD camera 2 T
␶
= 1− Ct 共␶兲d␶ . 共3兲
and quantifying the spatial blurring of the speckle pattern that T具I典2 0 T
results from blood flow. In areas of increased blood flow, the
intensity fluctuations of the speckle pattern are more rapid, While Eq. 共3兲 is the correct formulation and should be used in
and when integrated over the CCD camera exposure time data analysis, it appears to produce insignificant differences in
共typically 1 to 10 ms兲, the speckle pattern becomes blurred in biomedical applications when considering relative changes in
these areas. By acquiring an image of the speckle pattern and blood flow and the integration time is sufficiently long.14,28,29
quantifying the blurring of the speckles in the image by mea- The autocovariance is defined as
suring the spatial contrast of the intensity variations, spatial
maps of relative blood flow can be obtained.24 Alternatively, Ct共␶兲 = 具关I共t兲 − 具I典t兴关I共t + ␶兲 − 具I典t兴典t , 共4兲
instead of measuring the spatial contrast of the speckle pat-
where 具 ¯ 典t indicates a time-averaged quantity. It is conve-
tern, one can measure the temporal contrast.15,25,26 Each has
nient to consider that the intensity temporal autocorrelation
its advantages and disadvantages, as we detail further in the
function g2共␶兲 and its relation to the autocovariance since
following. In short, spatial contrast offers superior temporal
g1共␶兲 has been calculated5 explicitly for a wide range of dy-
resolution at the expense of spatial resolution and vice versa
namically varying media in the dynamic light scattering com-
for temporal contrast, while the advantages of both can be
munity for the last 30 yr.5 The temporal autocovariance func-
obtained with spatiotemporal algorithms.17,26 tion is related to the autocovariance by
To quantify the blurring of the speckles, the speckle con-
trast, defined as the ratio of the standard deviation to the mean
Ct 共␶兲
intensity, is computed as,24 g 2共 ␶ 兲 = 1 + , 共5兲
具I典t2
␴s
Ks = . 共1兲
具I典 g2共␶兲 = 1 + ␤兩g1共␶兲兩2 , 共6兲
Note that we use ␴s to refer to the spatial standard deviation where g1共␶兲 is the electric field temporal autocorrelation func-
of the speckle intensity and ␴t to refer to the temporal stan- tion given by
dard deviation. Similarly for the spatial speckle contrast Ks
and the temporal speckle contrast Kt. A typical example of a g1共␶兲 = 具E共t兲E*共t + ␶兲典/具E共t兲E*共t兲典 . 共7兲
raw speckle image of the rat cortex, taken through a thinned
skull, and the computed spatial speckle contrast are shown in Note that the average in Eq. 共7兲 is an ensemble average that is
Fig. 1 under normal conditions. The raw speckle image illus- only equivalent to a temporal average for ergodic systems
trates the grainy appearance of the speckle pattern. The 共i.e., systems that evolve through all ensembles or arrange-
speckle contrast image, computed directly from the raw ments over time兲. Equation 共6兲 is widely known as the Siegert
speckle image using Eq. 共1兲, represents a 2-D map of blood relation, where ␤ accounts for loss of correlation related to the
flow. Areas of higher baseline flow, such as large vessels, have ratio of the detector size to the speckle size and polarization.
lower speckle contrast values and appear darker in the speckle If the source is polarized and the detector is not, then
contrast images. ␤ = 0.5. A detailed discussion and derivation of the value of ␤
Theoretically, the speckle contrast has values between 0 can be found in Ref. 30. Note that the coherence length of the
and 1, provided that the speckle pattern is fully evolved. A light must be longer than the width of the distribution of de-
speckle pattern is considered fully evolved provided that the tected photon path lengths through the scattering medium
phases of the interfering electromagnetic fields are uniformly such that all detected photons will interfere coherently. If the
distributed, as can be verified by confirming a negative expo- coherence length is comparable to or less than this distribu-
nential probability distribution of the speckle intensity tion width, then the relation in Eq. 共6兲 is not valid and the

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 Boas and Dunn: Laser speckle contrast imaging in biomedical optics

calculation of the speckle contrast becomes extremely

complex.31
Starting with the first paper on laser speckle flowmetry,10 it
has been common to assume that the field temporal autocor-
relation function has the form

g1共␶兲 = exp −冉 冊 ␶
␶c
. 共8兲

This gives a speckle contrast

再 冋 冉 冊 册冎
Fig. 2 共a兲 Speckle contrast sensitivity to particle dynamics as given by
␶c ␶2c 2T 0.5
camera integration time divided by decorrelation time and 共b兲 the
K=␤ 0.5
+ 2 exp − −1 , 共9兲 normalized contrast-to-noise ratio of changes in speckle contrast ver-
T 2T ␶c sus camera integration time due to changes in rodent cerebral blood
flow. Reproduced from Ref. 33 with permission.
where the correlation time ␶c is assumed to be inversely pro-
portional to a measure of the speed or flow of the scattering
particles.24 As in the case of laser Doppler measurements, it is
theoretically possible to relate the correlation times ␶c to the 2.2 Spatial Speckle Contrast
absolute speed of the red blood cells, but this is difficult to do 2.2.1 Effect of speckle size and window size
in practice since the number of moving particles that the light
Accurate estimation of the speckle contrast from the spatial
interacts with and their orientations are unknown.6,24 How-
statistics of the speckle pattern generally assumes that the
ever, relative spatial and temporal measures of speed can be
speckle intensity distribution follows a negative exponential
easily obtained from the ratios of the correlation times. Note
probability distribution function.17,27,34 We always mentioned
the reminder from Ref. 12 that the form in Eq. 共8兲 is related
that one requirement for this is that the speckle pattern be
only to speed in the highly scattering regime where photons
fully evolved, i.e., that the received light have an effectively
have each experienced multiple scattering events from mov-
uniform phase distribution. Additionally, care must be taken
ing particles. In the single dynamic scattering regime, when
in spatial sampling of the speckle pattern. Specifically, the
photons scatter no more than once from moving particles,
speckle size relative to the camera pixel size must be consid-
then the argument of the exponent in Eq. 共8兲 should be
ered as well as the number of pixels that are used to estimate
squared to represent blood speed. In the single dynamic scat-
the speckle contrast.
tering regime, Eq. 共8兲 is correct if the moving particles are
Typically, the speckle pattern is imaged onto a camera in
undergoing Brownian motion.12 Further, the relation between
which case the minimum speckle size will be given by
␶c and the speed of the scattering particles depends on the
speed distribution of the scattering particles sampled by the
␳speckle = 2.44␭共1 + M 兲 f/#, 共10兲
detected light, a topic that is receiving increased attention in
the speckle contrast community.28,29,32 Finally, the speckle where ␭ is the wavelength of light, M is the magnification of
contrast literature tends to use speed and flow interchangeably the imaging system, and f / # is the f number of the system. As
when the speed is a length per unit time, while flow is a shown by Kirkpatrick et al.,16 the Nyquist sampling criteria
volume per unit time. While the theoretical derivation sug- must be satisfied by having the minimum speckle size be two
gests that speckle contrast is measuring speed, it is possible times larger than the camera pixel size, i.e., ␳speckle 艌 2␳pixel,
that sensitivity to the number of moving particles would ren- to obtain a negative exponential distribution. It has been com-
der speckle contrast a measure of flow, as has been rigorously monly stated that the speckle size should match the pixel size,
shown for laser Doppler blood flowmetry6 and suggested by but as indicated by Ref. 16, this results in a slight distortion of
some LSCI studies.11 A rigorous analysis of this question is the intensity distribution function with the result that the
needed. speckle contrast is underestimated by approximately 20% 共see
As indicated by Eq. 共9兲, the speckle contrast is a function Fig. 3, reproduced from Ref. 16 with permission兲. Underesti-
of the camera exposure time T and the time scale of the mating the speckle contrast, if not calibrated, will result in a
sample dynamics ␶c. As the exposure time goes to zero, the systematic error in the estimated correlation time ␶c and the
spatial speckle contrast approaches a value of 1. As the expo- associated sample dynamics. This reduction in speckle con-
sure time becomes long compared to ␶c, the speckle contrast trast is absorbed by ␤ in Eq. 共9兲 and can be easily estimated
approaches a value of 0. At both extremes, the speckle con- empirically for calibrating the speckle contrast
trast will not be sensitive to spatial or temporal variations in measurement.13 However, any reduction in ␤, despite calibra-
the speckle contrast. As detailed in Yuan et al.,33 the sensitiv- tion, does reduce sensitivity to spatial and temporal variations
ity to and contrast to noise ratio for variations in the speckle in the speckle contrast. Additionally, the distortion in the in-
contrast is optimized for T ⬇ ␶c, falling sharply for shorter T tensity distribution function likely produces systematic errors
and gradually for longer T 共see Fig. 2, reproduced from Ref. in the estimated sample dynamics, but the magnitude of these
33 with permission兲. As discussed by Duncan and errors remains to be estimated.
Kirkpatrick,28 we are assuming that the ␶c related to blood Speckle contrast is estimated from the mean and standard
flow is significantly shorter than the correlation time of any deviation of the speckle intensity, which are generally deter-
other dynamic processes within the sample, such as Brownian 1/2
mined from a square region of Npixels 共i.e., Npixels ⫻ Npixels
1/2
兲.
motion. Clearly a larger value of Npixels will give a more accurate

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 Boas and Dunn: Laser speckle contrast imaging in biomedical optics

+<< +=:
!" +=+ #"
+<E+
+=<

&53#!5 J3-/(!./
<=O
+<E:
<=@
G A"

+<E; <=P

<=?

+<E> <=F

<=>
+<EF
<=;
<=:
+<E? < + : ; > F ? P @ O
< <=: <=> <=? <=@ +=< +=: +=> +=? +=@ :=<
A B CAD H%I)5. H)( .H)JK5)L 2 2M: %. 21N'%./"

Fig. 3 共a兲 Speckle intensity probability distribution function considering different numbers of pixels per speckle N and the distribution is an
exponential for N = 2. 共b兲 The speckle contrast for a static medium equals its expected value of 1 when N = 2. Reproduced from Ref. 16 with
permission.

estimate of the speckle contrast, but at the expense of spatial E共t兲 = E f 共t兲exp共− i␻t兲 + Es exp共− i␻t兲 , 共12兲
resolution. If the window size is too small, then the large
variation in the estimate of the speckle contrast will reduce where E f 共t兲 is the fluctuating electric field scattered from
sensitivity to vascular variations. A survey of the literature moving particles, Es is the static electric field scattered from
indicates that the community has empirically settled on a win- nonmoving particles, and ␻ = 2␲v / ␭ is the angular frequency
dow size of 7 ⫻ 7 pixels as a reasonable trade-off between of the electric field related to the wavelength ␭ and speed v of
spatial resolution and uncertainty in the estimated speckle light in the sample. Substituting into Eq. 共6兲 and Eq. 共7兲 gives
contrast, but this all depends on camera resolution, speckle an intensity temporal autocorrelation function of
size, and desired contrast resolution.
Duncan et al.17 recently presented an important analysis g2共␶兲 = 1 + ␤关␳2兩g1,f 共␶兲兩2 + 2␳共1 − ␳兲兩g1,f 共␶兲兩 + 共1 − ␳兲2兴
that formulated the dependence of the mean speckle contrast
and variation in the estimated speckle contrast as a function of
2
+ Cnoise , 共13兲
the window size. Assuming that the Nyquist sampling crite- where ␳ = I f / 共I f + Is兲, I f = 具E f E*f 典 is the time-averaged intensity
rion is met, they determined that the variation of the estimated
of the fluctuating dynamically scattered light, Is = EsEs* is the
speckle contrast could be expressed by a dimensionless width
intensity of the statically scattered light, and g1,f 共␶兲
parameter ␴g as
= 具E f 共t兲E*f 共t + ␶兲典 / 具E f 共t兲E*f 共t兲典. Cnoise is a term added to ac-
count for contrast that arises from measurement noise such as
␴g = 1 + 0.454p0.672Npixels
−0.373
, 共11兲
shot noise or camera readout noise.13,14 We can now obtain a
where p = ␳speckle / ␳pixel is the number of pixels per speckle. new formulation for the speckle contrast by substituting Eq.
Their formulation reveals that the variation in the speckle 共13兲 into Eq. 共3兲 and assuming an exponential decay for g1共␶兲
contrast dramatically decreases as the window size is in- 关Eq. 共8兲兴,
creased to 7 ⫻ 7, but then diminishing returns are obtained for
larger window sizes, i.e., a 7 ⫻ 7 window sits near the bend in
the curve 共see Fig. 9 in Ref. 17兲. Note that the variation in the K = ␤0.5 ␳2冋 exp共− 2x兲 − 1 + 2x
2x2

册
estimated speckle contrast is a strong function of the number
of pixels per speckle. Essentially, increasing the speckle size exp共− x兲 − 1 + x 0.5
+ 4␳共1 − ␳兲 + 共1 − ␳兲2 + Cnoise ,
means that a given window of Npixels is estimating the speckle x2
contrast from fewer speckles. Finally, smaller window sizes
共14兲
result in an underestimation of the speckle contrast 共see Fig. 8
in Ref. 17兲. where x = T / ␶c.
The implications of Eq. 共14兲 are straightforward. If the
2.2.2 Effect of static scattering relative contributions of the fluctuating signal I f and the static
It is important to consider the effect of static scattering on the signal Is expressed by ␳ are unknown, then it is not possible to
estimate of the sample dynamics from the speckle contrast. A estimate the dynamics of the sample ␶c. A simple multiexpo-
purely static component to the scattered electric field will pro- sure calibration scheme was proposed by Refs. 13 and 14.
duce a speckle contrast that remains constant as the camera Briefly, performing measurements on a static sample with the
integration time is increased. If this effect is not considered, same light source and imaging optics provides an estimate of
then it results in an underestimation of the spatial and tempo- ␤. Similarly, ␤ can be estimated on the sample of interest by
ral variations in the sample dynamics.13–15,35 Consider a scat- obtaining a measurement with T Ⰶ ␶c. Measurements on the
tered electric field reaching the detector of the form sample with T Ⰷ ␶c drive the first two terms of Eq. 共14兲 to

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 Boas and Dunn: Laser speckle contrast imaging in biomedical optics

zero, providing an estimate of 共1 − ␳兲. Care is taken in these the relative changes since 具I典t cancels in the ratio as long as it
two measurements to estimate Cnoise or to ensure that it is remains constant. From the relative changes in Kt we can
negligible. Finally, measurements can be made with multiple estimate the relative changes in ␶c and subsequently the rela-
intermediate T’s or the optimal T ⬇ ␶c to then estimate the tive changes in flow.
spatial and temporal variations of ␶c within the sample. From
the relative variations in ␶c we then obtain relative measures
of flow.
2.4 Spatial versus Temporal Speckle Contrast
Speckle contrast can be estimated from either the spatial sta-
2.3 Temporal Speckle Contrast
tistics or the temporal statistics and related to blood flow. The
Laser Doppler blood flowmetry is based on measuring the spatial statistics offers better temporal resolution but at the
temporal fluctuations of speckle with sufficient temporal res- expense of spatial resolution because of the requirement to
olution to resolve the power spectrum of the intensity fluctua- use a sufficiently large window of image pixels in the calcu-
tions, which forms a Fourier transform pair with the intensity lation. If the pixel size is too small relative to the speckle size,
temporal autocorrelation function1,6 g1共␶兲. This requires a then speckle contrast will be underestimated and sensitivity to
temporal sampling resolution of more than 20 kHz for super- spatial and temporal variations in speckle contrast will de-
ficial measurements and 10 MHz for deeper tissues, as mea- grade. Ideally, the speckle size is twice as large as a pixel and
sured with diffuse correlation spectroscopy. The high data the contrast is estimated from at least 7 ⫻ 7 = 49 pixels. The
bandwidth limits measurements to a few points in space at a temporal statistics offer better spatial resolution at the expense
given time precluding rapid imaging.8,9 TLSCI, on the other of temporal resolution. If temporal samples are statistically
hand, images the time-integrated speckle and subsamples its independent, i.e., the time between samples is greater than the
temporal variation at 10 Hz or faster.15,25,26 In this way, it correlation time, then a minimum of only 15 samples is re-
images all pixels in parallel but does not resolve the high- quired to accurately estimate the speckle contrast and have
frequency temporal variation. Instead, it obtains the temporal good sensitivity to spatial and temporal variations in speckle
speckle contrast Kt from the temporal standard deviation ␴t of contrast. For typical brain applications, the correlation time is
the speckle intensity divided by the mean intensity. This tem- approximately 5 ms. Thus, given an image sample every
poral speckle contrast can then be related to the sample dy- 10 ms, temporal speckle contrast could be estimated every
namics via Eq. 共9兲. By using temporal sampling to estimate 150 ms with pixel spatial resolution. Compare this with spa-
the speckle contrast, rather than spatial sampling, one main- tial speckle contrast, which can be estimated at the camera
tains a higher spatial resolution but at the expense of temporal frame rate with a resolution of 7 ⫻ 7 pixels. Note that while
resolution. this spatial resolution difference is important on the surface of
A rigorous analysis of the effect of speckle size and num- the sample, it is quickly blurred by photon scattering beneath
ber of samples on Kt has not been published. Cheng et al. the surface of the tissue. The difference in spatial resolution
showed in Fig. 3 of Ref. 25 that Kt correlated well with the between spatial and temporal statistics likely makes no differ-
true speed when estimated from 15 or more independent ence when imaging more than 100 ␮m in highly scattering
samples of the speckle intensity. This is significantly fewer tissue.
than the minimum of 49 that is required in a 7 ⫻ 7 window to Spatial and temporal statistics do have important differ-
estimate the spatial speckle contrast with minimal ences when it comes to the treatment of static scattering.
uncertainty.17 Although a rigorous analysis comparing similar Static scattering will produce an additive offset to the spatial
statistical quantities between temporal and spatial contrast is speckle contrast. If this is not properly calibrated, then one
still needed, the temporal analysis is likely to perform better would overestimate the absolute speckle contrast and under-
with fewer samples, provided that temporal samples are more estimate relative changes in the speckle contrast, with the re-
statistically independent than the spatial samples. The spatial sult that both absolute blood flow and relative changes in
samples will be correlated because one typically imposes that blood flow would be underestimated. Static scattering does
the speckle size be 2 times greater than the pixel size to sat- not produce an additive offset in the temporal speckle con-
isfy the Nyquist theorem. Temporal samples, on the other trast, but instead scales the speckle contrast by a factor related
hand, will be uncorrelated provided that the sampling time to the relative contribution of statically and dynamically scat-
Ts ⬎ ␶c. In the exposed rodent brain, ␶c ⬃ 5 ms, enabling more tered photons. While this confounds estimates of the absolute
than 100 statistically independent samples per second. speckle contrast, the scale factor generally cancels when esti-
Temporal speckle contrast appears to be inherently less mating relative changes in speckle contrast.
sensitive to the contribution of static scattering than spatial Computational efficiency has plagued the calculation of
speckle contrast.15,32 When estimating speckle contrast from speckle contrast, usually requiring much more computational
spatial sampling, static scattering has an additive effect in time than data acquisition time, thus preventing real-time vi-
increasing the speckle contrast 关see Eq. 共14兲兴. Static scattering sualization. While temporal speckle contrast is generally less
does not induce temporal variation in the speckle intensity computationally expensive than spatial speckle contrast,36
and therefore will not contribute to the temporal standard de- Tom et al.18 recently developed improved algorithms that en-
viation ␴t. However, it does contribute to the mean intensity able speckle contrast calculation at rates that exceed image
具I典t = I f + Is and thus reduces the estimate of the temporal acquisition rates so that the acquisition time is now the limit-
speckle contrast since Kt = ␴ / 具I典t. While this impacts the abil- ing factor in the temporal resolution. As a result, true real-
ity to quantify an absolute measure of speed from an absolute time calculations of speckle contrast and blood flow changes
measure of speckle contrast, it does not prevent estimation of are now possible.

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 Boas and Dunn: Laser speckle contrast imaging in biomedical optics

3 Applications
The need for high-resolution blood flow imaging spans many
applications, tissue types, and diseases. LSCI and related
techniques have been used for a large number of blood flow
imaging applications in tissues such as the retina, skin, and
brain. These tissues are particularly well suited for LSCI since
the microvasculature of interest is generally superficial. Be-
cause of its measurement geometry LSCI is unable to sense
blood flow in deep tissues. One of the earliest uses of speckle
imaging was in the retina, where the vasculature and blood
flow of interest is accessible.37,38 More recently, LSCI has
become one of the most widely used methods for in vivo
imaging of blood flow in the brain, particularly in small ani-
mal models of both normal and diseased brain.11 Imaging of Fig. 4 Illustration of LSCI for monitoring PWS treatment. Left: Photo-
graph of patient with PWS in the area indicated by the rectangle. LSCI
cerebral blood flow with LSCI typically requires thinning or images were acquired immediately before 共upper兲 and 15 minutes
removal of a portion of skull to access the cortex. Prior to its after 共lower兲 laser therapy. Figure graciously provided by Bernard
application to cerebral blood flow studies, the standard Choi.
method for in vivo blood flow determination in animal models
was laser Doppler flowmetry, which typically consists of a
fiber optic probe that provides relative blood flow measure-
ments at a single spatial location. Although scanning laser
Doppler instruments exist,8,9 their temporal resolution is lim- 3.2 Retinal Blood Flow
ited by the requirement to scan the beam. LSCI instruments Another of the earliest applications of LSCI and related tech-
on the other hand, enable noncontact full-field imaging of niques was in visualization and quantification of blood flow in
blood flow without requirement any scanning. In addition, the the retina in both animals and humans.37,38,42 Since the retina
relatively simple instrumentation makes LSCI instruments is accessible and blood flow is an important indicator in a
easy to construct and use. In this section, several applications variety of ophthalmology conditions, considerable effort has
of LSCI are illustrated. been invested in developing blood flow imaging methods for
the eye. Speckle-based imaging of retinal blood flow has been
performed with diode lasers as well as argon ion lasers that
are coupled into fundus cameras. The group of Tamaki et al.38
3.1 Skin Perfusion
have developed a measure called the normalized blur 共NB兲,
Full-field monitoring of skin perfusion was one of the earliest which is used as an alternative to the speckle contrast, but is
uses24 of LSCI. Initially, skin was a convenient in vivo test still computed from the time-integrated speckle intensity fluc-
sample. However, images of skin perfusion are complicated tuations. Applications in the retina have included analysis of
by the fact that the majority of the vasculature is contained the effects of a wide range of pharmacological agents on
beneath a layer of tissue that has few blood vessels. There- blood flow43,44 as well as analysis of flow around the optic
fore, it is usually difficult to monitor flow in single vessels in nerve head.45 Despite the large number of reports of the use of
the skin, although LSCI is able to quantify overall perfusion speckle techniques for measuring retinal blood flow, however,
in the capillary bed.26 Recently Choi et al. have taken advan- very few images of retinal blood flow in humans have been
tage of this and demonstrated that LSCI can be used in con- reported. The majority of studies have reported average flow
junction with laser therapy for feedback during treatment of values that are calculated from images without showing any
port wine stain39,40 共PWS兲. In laser therapy of PWS birth- spatial maps of blood flow. The study by Isono et al.46 is one
marks, pulsed visible laser light is used to treat vascular le- of the few publications to show spatial flow maps in the hu-
sions. Although laser therapy is now the standard treatment man retina, and these maps were generated by stitching to-
for PWS, some areas of residual perfusion can exist following gether images from adjacent areas to create a composite im-
treatment. LSCI has the potential to provide real-time feed- age covering a 3-mm2 area of retina. One reason that many of
back during PWS treatment by indicating the spatial distribu- the earlier human retinal studies neglected to show spatial
tion of perfusion in the skin before and after laser therapy, as maps could be that most instruments had limited spatial res-
illustrated41 in Fig. 4. olution due to small camera sensors47 共100⫻ 100 pixels兲.
LSCI is also highly effective in quantifying microvascular
blood flow in the rodent dorsal skinfold model in which a
section of skin is resected and a window is inserted. This
preparation enables chronic studies of microvascular structure 3.3 Brain Applications
and function within the tissue under the window. Studies of For most applications in the brain, a portion of the skull is
dermatalogical laser treatments in skin have utilized this either thinned or removed and saline or mineral oil is placed
preparation as well as a large number of tumor biology studies on the surface to improve image quality by minimizing the
in which tumors can be implanted in the tissue beneath the effects of static scattering elements. Most applications in the
window. LSCI has enabled quantification of blood flow brain involve quantifying the changes in speckle contrast at
changes in individual vessels to be monitored in the dorsal each pixel as an indication of the relative cerebral blood flow.
skinfold model.39 Next we summarize applications of this approach to the brain.

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 Boas and Dunn: Laser speckle contrast imaging in biomedical optics

Fig. 5 Imaging of stimulus induced changes in blood flow in the brain. 共a兲 Sequence of images showing the percent changes in blood flow in
response to 10 s of forepaw stimulation and 共b兲 graph illustrating the percent change in blood flow over a 1.75⫻ 1.75-mm region of interest
centered on the activation 关see 共a兲兴. Reproduced from Ref. 54 with permission.

3.3.1 Functional brain activation models.53 However, laser Doppler measurements of the blood
Over the past two decades functional magnetic resonance im- flow response are limited to single spatial locations due to the
aging 共fMRI兲 has become the standard technique for investi- relatively fast temporal dynamics of the blood flow response
gating how the brain responds to various types of stimuli.48 共typically a few seconds兲. Since LSCI does not require any
fMRI studies have been widely used in humans as well as scanning, full-field imaging is possible with high temporal
animals for both clinical applications and basic research stud- resolution that is sufficient to resolve the blood flow dynamics
ies. The majority of fMRI studies use BOLD 共blood oxygen due to functional activation in the normal brain.20,54–56
level dependent兲 fMRI, whereby the changes in blood oxy- An illustration of the ability of LSCI to image the spa-
genation, in particular deoxyhemoglobin, are detected. There- tiotemporal changes in cerebral blood flow 共CBF兲 following
fore, BOLD fMRI measurements sense the hemodynamic re- functional activation is given in Fig. 5. A 5 ⫻ 5-mm area of
sponse to brain activation. cortex was imaged in a rat through a thinned skull as the
Optical techniques are widely used to image the hemody- forepaw of the rat was stimulated with electrical pulses. The
namic response to various stimuli. In particular, optical imag- stimulus was applied for 10 s 共0.5 mA兲 and 20 trials were
ing of intrinsic signals is commonly used. This method has repeated. The speckle contrast images at each time, relative to
provided numerous insights into the functional organization of the stimulus were averaged. Speckle contrast values were
the cortex49–52 by mapping the changes in cortical reflectance converted to speckle correlation decay times 共␶c兲 at each pixel
arising from the hemodynamic changes that accompany func- in all images, and ratios of the inverse of the decay times were
tional stimulation. The majority of these studies have been used as a measure of the relative changes in CBF.
based on qualitative mapping at a single wavelength, and Figure 5 highlights the strength of LSCI in revealing both
while they have provided valuable insight into many aspects the spatial and the temporal blood flow dynamics. A series of
of cortical function, the techniques used in these studies have images are shown at 0.5-s intervals, and illustrate a localized
been unable to reveal quantitative spatial information about increase in CBF beginning approximately 1 s after stimula-
the individual hemodynamic 共hemoglobin oxygenation and tion onset. The relative changes in CBF are shown superim-
volume and blood flow兲 and metabolic components that un- posed on the vasculature 共derived from the speckle contrast
derlie the measured signals. image兲 and the changes in CBF are displayed for all pixels
The blood flow changes that accompany brain activation with an increase in CBF greater than 5%. The time course of
are particularly important in estimating the oxygen consump- the CBF changes averaged over a region of interest centered
tion of the brain. Laser Doppler flowmetry has been used on the activated area illustrates a peak increase in CBF of
extensively to quantify the blood flow changes in the soma- approximately 12% occurring 3 s after stimulus onset. The
tosensory cortex in response to stimulation in animal initial peak in CBF then decreases to approximately half of its

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 Boas and Dunn: Laser speckle contrast imaging in biomedical optics

maximum amplitude to a value of ⬃6%, where it remains

until the end of the stimulus, in a manner consistent with !"
previous laser Doppler measurements of CBF during ex-
tended forepaw stimulation.57 This time course also reveals
the very high SNR of LSCI for relative CBF measurements.
Since the CBF response to functional activation is only one
component of the overall hemodynamic response, a number
of groups have combined LSCI with other optical techniques
to simultaneously measure CBF and other hemodynamic and
metabolic parameters. Because the wavelength used for LSCI
can lie anywhere in the red or near-IR regions, LSCI can be
performed simultaneously with other techniques such as mul-
tispectral reflectance imaging 共MSRI兲 of hemoglobin oxygen-
#"
ation and blood volume,20,54,58 fluorescence imaging of
NADH and flavoproteins,55 and phosphorescence quenching
measurements of the partial pressure of oxygen.21 For ex-
ample, by adding a second camera and light source to the
LSCI setup, LSCI and MSRI can be performed
simultaneously.59 For MSRI, incoherent light of different
wavelengths 共typically 550 to 650 nm兲 sequentially illumi-
nates the cortex, while longer wavelength laser light
共700 to 800 nm兲 is used for speckle imaging. The reflected ;<<
light from each source is spectrally separated and two cam- J"

(Q$ R 3S #!.)5%-)"
eras are used to record the speckle and multispectral signals.
For MSRI, each set of spectral images can then be converted
+F< 4%775) 4)-%-&)!5 !(/)(1
to changes in oxyhemoglobin 共HbO兲 and deoxyhemoglobin
共HbR兲 at each pixel to yield maps of hemoglobin changes,
while LSCI images are processed as already described.
+<<
J3(/)I
3.3.2 Cortical spreading depression and migraine # J 7
headache < +< :< ;< >< F<
/%4) 4%-"
Cortical spreading depression 关or spreading depolarization
共SD兲兴 is a wave of negative potential shift that slowly propa- Fig. 6 Blood flow images following cortical spreading depression: 共a兲
gates across the cortex at a rate of 2 to 5-mm/ min, and was the speckle contrast image reveals vasculature in the cortex and dura,
first identified in rabbits in 1944 by Leao.60 SD is associated the middle meningeal artery is indicated by the arrow; 共b兲 relative
blood flow 20 min after induction of cortical SD reveals elevated
with large ionic shifts, increased metabolism, and hemody- blood flow in the middle meningeal artery; and 共c兲 time course of the
namic changes. SD has been studied extensively for more changes in blood flow in the cortex and the middle meningeal artery
than 50 yr and its role in the pathophysiology of stroke, mi- demonstrating the long-lasting blood flow increase that is restricted to
graine, and subarachnoid hemorrhage is well established.61,62 the dural vessel. Reproduced from Ref. 67 with permission.
Recent clinical studies have further emphasized the impor-
tance of SD in human stroke and brain injury.63–66 Despite
this, the physiologic changes that occur during SD are not middle meningeal artery 共see Fig. 6兲, Bolay et al. were able to
fully understood. In particular, the role of cerebral microcir- map the nerve pathway linking the triggering event of a head-
culation on the impact of SD on brain tissue is poorly under- ache 共spreading depression兲 to the subsequent headache.67
stood. In animal models, SD can be induced in normal brain These types of studies were not possible with methods such as
through topical application of KCl or through localized injury laser Doppler or MRI-based techniques that lack sufficient
such as pinprick. Once induced, SD propagates across the spatial resolution to resolve blood flow in single vessels.
cortex and is associated with large transient changes in dc LSCI has also been used to investigate other aspects of SD
potential. In response to the increased metabolism required such as the difference in hemodynamic response between rats
for cells to repolarize, SD is typically accompanied by a large and mice.68 These studies also demonstrated that LSCI can be
transient increase in blood flow 共hyperemia兲. performed through an intact skull in mice since the skulls of
SD was one of the first applications of LSCI in the mice are relatively thin. As in functional activation studies,
brain.11,20,67 Laser Doppler flowmetry had been used to quan- LSCI has been combined with other imaging methods for
tify the temporal changes in CBF for many years, and laser multiparameter measurements of the hemodynamic and meta-
Doppler was suitable for quantifying the magnitude and tim- bolic responses to SD. Sakadzic et al.21 investigated the blood
ing of the transient hyperemia associated with SD. However, flow and oxygenation changes during cortical spreading de-
when LSCI was used to visualize the spatial extent of the pression 共CSD兲 using combined LSCI and phosphorescence-
blood flow changes during SD, a transient but delayed blood quenching measurements, while another recent study23 com-
flow increase was discovered in the dural vessels overlying bined LSCI with voltage-sensitive dyes to simultaneously
the cortex.67 By imaging the blood flow response within the measure the hemodynamic 共blood flow兲 and neuronal re-

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 Boas and Dunn: Laser speckle contrast imaging in biomedical optics

Fig. 7 Application of LSCI to cerebral ischemia: 共a兲 the spatial blood flow gradient following occlusion of an artery can be visualized using LSCI,
where the middle cerebral artery was occluded just outside the top region of the image and the color map shows the relative blood flow, expressed
as a percentage of preischemic flow; 共b兲 LSCI and MSRI can be performed simultaneously to image multiple hemodynamic parameters; and 共c兲 time
courses of changes in oxyhemoglobin 共HbO兲, deoxyhemoglobin 共HbR兲, total hemoglobin 共HbT兲, blood flow 共CBF兲, oxygen consumption
共CMRO2兲, and scattering during a stroke. The three graphs demonstrate the changes in each of these parameters in three spatial regions 共ischemic
core, penumbra, and nonischemic cortex兲. 共b兲 and 共c兲 reproduced from Ref. 59 with permission.

sponse to CSD. This combination could be very significant in Blood flow in the area closest to the affected region 共ischemic
future studies of neurovascular coupling. core兲 is typically reduced to less than 20% of its baseline
value, and as a result neurons depolarize and die rapidly. In
3.3.3 Stroke the region between the ischemic core and the normal tissue
Animal models of stroke are widely used to investigate the 共penumbra兲, neurons preserve the ability to maintain ion ho-
basic pathophysiology of stroke and to evaluate new stroke meostasis but are considered to be electrically silent since
therapies. A critical aspect of these studies is monitoring of evoked potentials and spontaneous electrical activity cease.72
blood flow dynamics in the brain. Laser Doppler flowmetry The reduction in blood flow in the penumbra is not as severe
has been used for many years in such studies and is widely as in the core since collateral blood supply to the area is
considered to be the gold standard for quantifying blood flow maintained. This reduction in flow in the penumbra can lead
changes in the brain in animal models of stroke. Although to secondary effects such as peri-infarct spreading depolariza-
MRI and positron emission tomography have also been used tions, inflammation, and ultimately cell death.73 The penum-
in animal models of stroke, the spatial and temporal resolution bra has been a primary target of treatment strategies since
of these techniques is usually not sufficient to enable detailed membrane function is preserved in cells in the penumbra.
studies of blood flow dynamics. LSCI has emerged as a pow- Restoration of blood flow to the penumbra, therefore, may
erful technique for quantifying both the spatial and temporal provide a means of salvaging tissue without loss of function.
blood flow changes during stroke due to its high spatial and Since the ischemic penumbra varies both spatially and
temporal resolution.11,63,69–71 temporally, LSCI is one of the few techniques that can pro-
During ischemic stroke, blood flow is reduced in a local- vide a dynamic view of the penumbra, as well as the ischemic
ized region of the brain leading to a cascade of cellular and core and nonischemic areas. LSCI has been used to visualize
molecular events that ultimately results in tissue damage. the CBF changes throughout the ischemic territory in stroke

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 Boas and Dunn: Laser speckle contrast imaging in biomedical optics

models in mice, rats, and cats.11,64,69,74 Figure 7共a兲 shows an Acknowledgments

example of the spatial gradient in blood flow following occlu- The authors acknowledge Sean Kirkpatrick, Donald Duncan,
sion of the middle cerebral artery in a rat. Closest to the and Shuai Yuan for insightful discussions that improved the
occlusion site, blood flow is reduced to less than 20% of quality of this manuscript, thank Bernard Choi for providing
preischemic values and the blood flow deficit gradually im- Fig. 4, and acknowledge support from the National Institutes
proves with distance away from the site of occlusion due to of Health 共NIH兲 Grants P50-NS010828, P01-NS055104, R01-
collateral blood flow. NS057476, the American Heart Association 共Grant No.
One of the mechanisms that may lead to cell death in the 0735136N兲, and the National Science Foundation 共Grant No.
penumbra is the presence of periinfarct depolarizations CBET/0737731兲.
共PIDs兲, which resemble the spreading depressions of Leao.60
PIDs are spontaneously generated following an ischemic in-
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