Bio-speckle

application
blood flow

phenomena
and their
to the evaluation of

Y. AIZU, T. ASAKURA
The study of time-varying speckle phenomena observed in light-fields scattered
from living objects is reviewed. The laser speckles produced from living objects
may be called bio-speckles and fluctuate temporally due to various physiological
movements such as blood flow. The time-varying properties of the bio-speckles
are experimentally investigated from the analyses of the power spectrum and
the autocorrelation function. Based on the knowledge of dynamic bio-speckles,
some methods are introduced for evaluating blood flow in the skin surface,
internal organs, and ocular fundus. The experimental results show that the
degree of blood flow is reflected sensitively by the time-varying properties of the
bio-speckles and this can be utilized for monitoring the blood flow.
KEYWORDS:

speckles, blood flow, flow measurement, biological tissue

Introduction
When a diffuse object with an optically-rough surface
is illuminated by laser light, laser speckle phenomena
can be observed in the reflected or transmitted light.
If the diffuse object moves with a certain velocity,
the individual grains of the speckle pattern also
move and change their shape: the moving speckle
pattern thus contains information about the objects
motion. Therefore, the dynamic properties of the
speckles have been extensively studied and are being
applied to velocity measurements. However, most of
these studies have been carried out for inanimate
objects - such as a solid diffuse late with a simple
structure. Except for a few studies Y. 3, the dynamic
speckles produced from living objects have not been
sufficiently investigated. The speckles observed from
living objects generally fluctuate in a space-time
random fashion owing to the complicated structure
and inconsistent physiological activity of living
objects. Therefore, the dynamic behaviour of such
speckles is considerably different from that of
speckles produced from solid diffuse objects. From
this point of view, the speckles from living objects
may be called bio-speckles4. .
With the recent increase of laser applications

in the

YA is in the Department of Mechanical Systems Engineering,

Muroran Institute of Technology, Muroran, Hokkaido 050, Japan
TA is in the Research Institute of Applied Electricity, Hokkaido
University! Sapporo, Hokkaido 060, Japan. Received 20 March
1991.

0030-3992/9
Optics 8 Laser Technology
Vol23 No 4 1991

l/040205-

medical and physiological fields, bio-speckles are

receiving much attention from researchers. This is
because the temporal fluctuation of bio-speckles is
expected to provide much information about the
physiological activity of living objects, such as blood
flow and heartbeat. In addition, the dynamic behaviour of bio-speckles is an interesting subject in the
field of laser light scattering. This is due to the fact
that bio-speckles result from multiple scattering from
various complex media of living objects.
On the other hand, a number of studies6-4 have
been performed for the measurement of blood flow
using the laser Doppler effect. This Doppler method
has been reported to be useful because of its noninvasive and repeatable characteristics. However, it
is limited to cases where the blood vessel is exposed
to the laser beam or in which the vessel permits
insertion of an optical tibre probe. In other cases,
such as skin blood flow, some of the studies which
are described as laser Doppler velocimetry may, in
fact, be distinguished from the original principle of
laser Doppler velocimetry: red blood cells are illuminated through a surface tissue, which acts as a
diffuser, and the Doppler-shifted light is detected,
also through this diffuser. Therefore, the phase distribution of light fields to be detected is fully
randomized by the mass scattering of the tissues
and blood flows. These scattering processes seem to
be more suitably described as dynamic speckle
phenomena rather than the Doppler effect and, therefore, may be appropriately reinterpreted from the
viewpoint of speckle phenomena. On the basis of

15 0 1199 1 Butter-worth-Heinemann

Ltd
205

this consideration, some recent articles lsq7 have

discussed the relation between the bio-speckle fluctuation and blood flow.
In this paper, the time-varying properties of biospeckles are experimentally investigated on the
basis of the power spectral and autocorrelation
analyses. As one of the interesting applications of
bio-speckles, several techniques are introduced for
non-invasively evaluating the blood flow in the skin
surface, internal organs, and ocular fundus. Some
other applications of bio-speckle phenomena are
also described briefly.

g
E

I~
a

Bio-speckles from the skin surface

/[

Time {ms)

oO

We first discuss the dynamic properties of biospeckles observed from the human skin surface t5 as
shown in Fig. 1. A HeNe laser beam illuminates a
fingertip and is scattered back to an observation
plane Op, where a bio-speckle pattern is formed.
The photographs (a) and (b) show bio-speckle
patterns taken at the plane Op with different exposure
times of (a) 0.5 s and (b) 1 s. With increasing exposure time, it is found that the bio-speckle pattern is
blurred and that its contrast becomes lower, The
photograph (c) shows a high-contrast speckle pattern
obtained from a static styrene-form plate. These
observations imply that the bio-speckle pattern from
the skin surface fluctuates spatially and temporally
in a random fashion. This is due to dynamic scattering by the blood flow in dermal capillaries. Figure 2
shows the intensity fluctuations of bio-speckles detected by a photomultiplier, through a pinhole placed
at the plane Op. The signals (a) and (b) were recorded
for normal flow, and for reduced flow with an
inflated cuff on the upper arm, respectively. High
frequency components are clearly suppressed in
Fig. 2(b) and this seems to reflect the reduction of
blood flow. Thus, frequency analysis of the biospeckle fluctuations may give useful information on
the degree of skin blood flow.

laser

>I

.>

Observation of bio-speckles

HeNe

100

t//

|~

r.~

100
Time (msl

Fig. 2
Bio-speckle fluctuations (a) and (b) obtained from the
normal and reduced skin blood flows, respectively
It can be observed that the entire structure of the
low-contrast pattern in the photograph Fig. l(b) is
stable with a further increase of exposure time
unless the skin surface moves laterally. This means
that the bio-speckle pattern produced by the skin
tissue consists of two components: (1) the fast fluctuation due to the skin blood flow and (2) the slow
movement due to the delbrmation of the outer skin
tissue. The latter slowly-moving component causes
the low frequency noise in the output signals. Then,
the bio-speckles from the blood flow in the inner
skin tissue may be modulated by the movement of
the outer skin tissue before detection.

Despite the modulation, the bio-speckle signals in

Fig. 2 possibly show the dependence of bio-speckle
fluctuations on the skin blood flow. This observation
was also confirmed by a simulation study Is in which
the dynamic speckles were produced by two groundglass plates set in layers with a small separation
distance. Static-front ground-glass and the movingback ground-glass plates were used, respectively, for
modelling the static outer skin tissue and the blood
flow in the inner skin tissue. The illuminating laser
light was firstly transmitted through the static front
diffuser, secondly reflected by the moving back diffuser, and thirdly passed through the static front
diffuser. Thus, the triply-scattered speckle is detected
and its intensity fluctuation is investigated from the
autocorrelation function. In spite of the phase
modulation by the front ground-glass, the intensity
fluctuation of triply-scattered speckles was found to
depend linearly on the moving velocity of the back
ground-glass plate.

Evaluation of skin blood f l o w

Fig. 1 Bio-speckle patterns produced in (a) 0.5 s; and (b) 1 s;
from the skin surface of a fingertip and a usual speckle pattern
(c) obtained from a styrene-form plate

206

The time-varying properties of bio-speckles may be

applied to measurements of skin blood flow. Figure 3
Optics 8 Laser Technology
Vo123 No 4 1991

FI

HeNe laser

OL

Fig. 3

Schematicdiagram of a blood-flow monitoring system using bio-speckle phenomena

shows schematically 15 the measuring system using

an optical fibre probe. A HeNe laser beam is radiated
onto the skin surface via a multimode optical fibre
F1 with a core diameter of 80 p m. The scattered light
is picked up by a single-mode optical fibre F2 with a
core diameter of 10~m installed in parallel to fibre
Fl. This pair of multimode and single-mode fibres
is effective for achieving high output radiation from
the illuminating fibre, high signal-to-noise ratio, and
low modal noise from the vibration of the fibre. Biospeckle fluctuations are detected by a photomultiplier, PM, and are fed into a spectrum analyser
where the power spectral distribution is obtained.
Figure 4 shows the typical power spectral distributions obtained from three different regions of the
human skin surface. Two interesting features are
found in Fig. 4: (1) the power spectral distribution
plotted on a log scale decreases monotonically from
the low to the high frequency; and (2) the rate of
decrease is characterized by the region of human
skin being measured as the degree of blood flow
may be different. These features indicate that skin
blood flow can be characterized by the slope of the
power spectral distribution.
To evaluate quantitatively the slope of the power
spectral distribution, a simple signal processor was
constructed. This has two band-pass filters with
central frequencies of 640 Hz and 40 Hz. By
measuring the powers [ VH [ 2 and ] VL] 2 of the high
and the low frequency components obtained from
these filters, a blood flow parameter HLR can be
calculated by
H L R - ] VH] 2

I vLI 2

(l)

and used to evaluate the skin blood flow. Figure 5

shows the variation of the blood flow parameter
H L R according to the skin blood flow in the palm of
the hand. Region (a) shows normal flow, while
regions (b) and (c) show the flow stopped by an
inflated cuff on the upper arm and the enhanced
Optics ~ Laser Technology
Vol 23 No 4 1991

flow as the cuff is deflated, respectively. The value of

HLR is found to indicate sensitively the change of

skin blood flow. The separation of the two passbands determines the sensitivity of the measuring
system. It should be carefully noted that the biospeckle phenomena observed here reflect the overall
characteristics of the blood flows, but do not directly
give flow properties, such as velocity.
The depth of the probed volume is generally determined by the depth of the light penetration into the
skin. In Fig. 6, HeNe laser light with a wavelength of
632.8 nm penetrates into the dermis, while blue light
with a wavelength of nearly 450 nm is mainly
absorbed and scattered in the epidermis, under
which papillary loops exist. Selection of the wavelength of the illuminating light may provide the
discriminative measurements of the total dermal
flow and the superficial dermal flow in the papillary
loops. In the measuring system of Fig. 3, however,
the discrimination of skin-tissue layers to be probed
is made easier by adjusting the separation distance d
between the illuminating and detecting fibre ends as
shown in Fig. 6. Figure 7 shows the power spectral
distributions obtained from the palm of a hand by
means of the three fibre probes with different
separation distances d. With increasing distance d,
the low frequency components decrease and the
high frequency components increase. With small
distance d, the detecting fibre F2 mainly receives the
scattered light propagated in the short path in and
near the region of epidermis. This light is modulated
by the low degree of blood flow in papillary loops
and shows the low frequency fluctuation. With large
distance d, the light coming from the deeper dermal
region dominantly contributes to the detected biospeckles, and the high degree of blood flow in the
dermis results in the high frequency fluctuation. The
relation between the separation distance d and the
depth of the probed volume was quantitatively calibrated by the simulation study using a Teflon sheet
as a skin model. This study shows that the separation
distance of d = 1-2 mm is suitable for evaluating the

207

Fingertip

-~-- o

H=

~-i c l<-

-10

t.q

-20

>.
O_
u~

-30

25

o
-40
]

I
20

I
50

I
100

I
200

Frequency

I
500

I
1000

1
2000

\.

\
%,

5000

(Hz)

'

1()

1'1

1'2

Time (min)
Fig. 5
Variation of the blood flow parameter HLR for the
different degrees of skin blood flow in the palm of a hand

0
Neck

-I0

Laser
input

"(3

Laser
detection

_ =

-20

F1

o_

F2

"Eg
Lu
f

-30
c_-

-=-z---T

z-=-~-~-=

-40

Z
20

50

200

500

1000

2000

5000

Frequency
lHz)

100

_IOoL

Leg

20

30

a)

g~

4o
I

0
C

20

50

100

200

Frequency

500

1000

E -

2000

5000

(Hz)

Fig. 4
Power spectral distributions obtained from three different
skin surfaces

blood flow in the dermis, although the simulation is

not directly applied to the actual skin tissue.
Two-dimensional blood flow map
The technique of monitoring blood flow using biospeckles can be extended to the visualization of a
two-dimensional microcirculation map 3 m. Figure 8

Papillary loops
Subpapillary
plexus
( a r t e r i o l e s and
venules)

Fig. 6
Schematic description of the optical fibre probe and the
vascular structure of skin

(see Ref. 19) schematically shows an optical system

for visualizing a two-dimensional blood-flow map
using a C C D linear image sensor. A skin surface is
illuminated, via mirror M, with a HeNe laser spot
expanded into a line by cylindrical rod lens Ll. The
scattered light is reflected by the mirror M and
collected by lens L2 to form an image of the line
spot on a C C D linear image sensor (with an array
of 256 elements) where a bio-speckle pattern appears.
When the intensity pattern of the bio-speckle image
is scanned successively by the C C D linear sensor,
the profile of the output signals fluctuates temporally
due to the blood flow in the dermal tissue layer. The
rate of this fluctuation depends on the blood-flow
degree.
Figure 9(a) shows two successive scanning outputs o f
the C C D image sensor. The left and right-hand
sides of the scanned position correspond to the areas
where the skin blood flow is artificially controlled to
be slow and fast, respectively. Considerable difference
is found between the two successively scanned signals
in the right-hand side. This is due to the rapid
Optics ~t Laser Technology

208

V o 1 2 3 No 4 1 9 9 1

~.~

d = 0.125mm

Slow

~ I-

-I

-10,

Fast

~ I

-I

k.

-20.

O_
o3

taJ'

tn
c

-30

o
C

~.

-40
4

40

400

4000

F r e q u e n c y (Hz)

. . . .

256
0

2
E

d = 1.27 mm

Position

-10'

~ -20'
~

-3e

-40

!
40

l
400
Frequency

(Hz)

-a
E

4000

d = 2.91 mm

256

-10
b

Fig. 9 (a) Two successive scanning outputs of a CCD linear

image sensor and (b) a one-dimensional blood flow map

~ -3O ~
-40 4

410

4~0

4000

Frequency (Hz)

Position

owing to the slow blood flow. The difference between

a pair of output signals for successive scans, defined

as
N

Fig. 7 Power spectral distributions obtained by three fibre

probes with the different separation distance d

kl ~ ~ H e N e

laser

ou, face

Fig. 8 Schematic diagram of a system for visualizing the twodimensional blood flow map

fluctuation of bio-speckles resulting from the fast

blood flow. The two successive signals in the lefthand side are almost the same because the slow biospeckles hardly fluctuate in one scanning interval
Optics B Laser Technology
Vol 23 No 4 1991

r~

--

[Ik(n)

-- I k + l ( n ) [

,-,,

is measured and integrated for each pixel. In (2),

Ik(n) is the amplitude value of the output signal for
the nth pixel point of the kth scan in the CCD
linear sensor. This process was repeated for more
than 100 scans to average the difference data. Figure
9(b) shows the integrated value D(n) of the average
difference which provides a one-dimensional bloodflow map. The average difference becomes high for
the area where the blood-flow degree is high and
vice versa. Figure 10 shows a one-dimensional flow
map on the back of a hand where a small scratch
scar is left. There was a slightly itchy region in the
scar which is represented by a circle in Fig. 10(a).
The blood-flow map was measured and plotted
along the solid line across the scar. The result,
Fig. 10(b), clearly shows that the wound healing
process induces a high degree of blood circulation
around the scar.
A two-dimensional map of the microcirculation is
obtained by scanning the illuminating line spot in a
direction perpendicular to the line spot. In Fig. 8,
this is done by tilting the mirror M step by step and
repeating the same data analysis as in the onedimensional measurement. The two-dimensional
spatial variation of the flow degree is visualized by

209

I~

50 mm

~1

co

u
,m
co
>
~3
C~

125

250

Position

Fig. 10 (a) A scanned line across the scar on the back of a

hand and (b) the corresponding one-dimensional blood flow map

graphically displaying in colours the integrated

values of the average difference. Figure II shows
black and white photographs of a coiour CRT display
showing the microcirculation map of h u m a n skin
under a tuberculin test. The values of the average
differences are colour-coded in 32 levels. The results
(a) and (b) were obtained 6 h and 24 h after injection
of the drug into the skin. As expected, the spot of
higher blood-flow degree is small in (a) and is
enlarged due to the reaction in (b). Using a twodimensional area sensor, instead of tilting the mirror
mechanically, is more suitable for a practical
instrument. However, the scanning rate of commercially available area sensors is not sufficiently
high enough to follow the high frequency fluctuation
of bio-speckles. Thus, the use of an area sensor is
promising for the skin blood flow, but needs to have
an improved scanning rate if the more active blood
flow in large vessels is to be visualized.

Bio-speckles from internal organs

We next discuss the bio-speckle p h e n o m e n a observed
from internal organs. Figure 12 shows the experimental set-up of the optical fibre probe shown in
Fig. 3, which was used for observing the bio-speckle
fluctuation from the gastric mucous m e m b r a n e of an
anaesthetized albino rabbit. The optical fibre probe
was held by a flexible arm to avoid the effect of
body movements. Figure 13 shows the power spectral
210

Fig. 11 CRT display of the blood flow map at the skin surface
under tuberculin test: (a) 6 h and (b) 24 h after the injection

Optical fibre /

probo

Gastricn

<"~,~~

.:i. :-~

Fig. 12 Schematic illustration of the experimental set-up for

evaluating the blood flow in the gastric mucous membrane using
bio-speckles

distributions obtained under three diflerent

conditions of blood flow in the gastric mucous
membrane. The results (I) and (II) were obtained
when norepinephrine and prostaglandine E I were
applied to the mucous m e m b r a n e as a vasoconOptics 8- Laser Technology
Vol 23 No 4 1991

"O

lk

Normal

-1o

-I0

Normal

L
+a

-20

-20,

fc

O-

fc

-30
g_

-40

-30

.o4

0.4

O
[3.

4O

0.4

Frequency (kHz)
0

-40
0.04

40

Frequency (kHz)
0

'%,1

73

-10

Otn
L
03

vasoconstrictor 1

-20

-20,

fc

Applied
vasodilator 1
h ~ ~ , ~

-30

-30
O
el

-40
0.4

0.04

Frequency

-40

4O

0.04

0
__

I
4

"~.
40

Frequency (kHz)

0
,~-u

I
0.4

(kHz)

73

"(3

-10

fc

A ~pplied
soconstrictor 2

E
L

-20
{3in

O
r~

O-

O
el

-10

-10

ldTIdtor 2

-20

-30

-40

o..

0.04

--30

0.4

40

Frequency (kHz)

([)

-40

0.04

I
g

0.4

I
40

Frequency (kHz)

(ll)

Fig. 13 Powerspectral distributions obtained for (I) the normal and the reduced blood flows and (11)the normal and the enhanced
blood flows, in a gastric mucous membrane

strictor and a vasodilator, respectively. In addition,

the power spectrum (a) is measured for the normal
flow, while the power spectra (b) and (c) are measured
for single and continuous applications of these drugs,
respectively. The blood flow was expected to decrease
with the vasoconstrictive effect of norepinephrine
and to increase with the vasodilative effect of prostaglandine El. The power spectral distribution is
nearly flat from the low frequency to a certain
frequencyfc which may be regarded as a cut-off
frequency and, then, begins to decrease in the higher
frequency region. With the application of norepinephrine and prostaglandine El, the cut-off frequency
fc is seen to become lower in (I) and higher in (II),
respectively, from (a) to (c). This implies a relative
increase of lower and higher frequency components
in the power spectral distribution corresponding to the
reduced and the enhanced blood flows, respectively.
It should be noticed that the form of power spectral
distributions shown in Fig. 13 is clearly different
from the result for the skin, as shown in Fig. 4. A
difference is also found in the variation of the power
Optics Et Laser Technology
Vol 23 No4 1991

spectral distribution according to the change of

blood-flow degree. Figure 14 schematically illustrates
such a difference in the form of power spectral
distributions obtained from the skin surface and the
gastric mucous membrane. This difference is probably
due to the structure of tissues and vessels. The blood
vessels and capillaries in the gastric mucous membrane are directly exposed to the laser light for
illumination and detection, and the photomultiplier
dominantly receives the high-frequency fluctuation
scattered by the blood flow. In the skin, the lowfrequency fluctuation due to involuntary movements
of the outer skin surface primarily contributes to the
photodetector signals. This latter effect results in the
relative increase of low frequency components as
shown in Fig. 14(a).
The blood flow parameter HLR in (1), which is used
for evaluating the skin blood flow, is not suitable for
analysing the power spectral distributions obtained
from the gastric mucous membrane, because of their
different decreasing forms. To evaluate appropriately
the variation of the power spectral distribution

21 1

Skin tissue

VC: Vasoconstrictor
VD: Vasodilator

1.5

Apply VC
N
"12

Apply VC

/'

"

/~
1.0
q~

0J
O-

& 0.5

Apply VD

\\ Apply saline

'

1 +0

2+0

'

3'0

'

4'0

Time (min)
Variation of the mean frequency <f) for the application
Fig. 15
of vasoconstrictor (VC) and vasodilator (VD)

Frequency

Gastric mucous membrane

E
L

arteriole and venule. According to this operation, the

power spectral distribution shows a substantial decrease in higher frequency components, and the
usefulness of the method is also verified for the
blood flow in the intestinal mucous membrane. One
of the promising potentials in the clinical application
of this method may be the installation of the present
measuring system into a fibre endoscope.

0
tn

Bio-speckles from ocular fundus

Evaluation of fundus blood flow
Bio-speckles are also observed from the ocular

Frequency

Fig. 14
Schematic comparison of the power spectral distributions obtained from (a) the skin tissue and (b) the gastric
mucous membrane

according to the change of the blood-flow degree,

the mean frequency (f) is introduced here and is
given by
ZfiPtf,-)
I

Oc}- ZP{f)

(3)

where P(f,-) is the signal power at frequency f,.

Figure 15 shows the variation of the mean frequency
Q") obtained for the application of norepinephrine
as the vasoconstrictor (VC) and prostaglandine E1 as
the vasodilator (VD). As expected, the mean frequency
Q') decreases with the VC-application and then
increases again with the VD-application. Therefore,
the mean frequency (,f) is found to be another
useful indicator for the evaluation of blood-flow
degree as well as for the blood-flow parameter H L R
The experiment was also carried out for the intestinal
mucous membrane of an anaesthetized albino rabbit.
The blood flow was reduced by the clamping of

212

fundus tissue in which retinal vessels are dire_ctly~

exposed to the laser light. Fercher and Briers 3 20-_;
showed a method for visualization of the retinal
blood-flow map. In their method, the bio-speckle
fluctuation from the ocular fundus is recorded on a
photographic film as the decorrelation effect and is
used for visualizing a blood-flow map using optical
filtering or image processing. In this section, we
discuss the dynamic properties of bio-speckles
obtained from the ocular fundus (including retinal
and choroidal tissue layers) and introduce a new
method24.25 for evaluating the ocular-fundus blood
flow. Figure 16 shows two basic optical systems for
detecting the bio-speckle fluctuations from the ocular
fundus (I) at the diffraction plane and (II) at the
image plane. In Fig. 16 (I), laser light illuminates a
certain area of the retina with an extended spot and
is scattered by ocular fundus tissues and blood
flows. The scattered light produces, via lenses Li, L2,
and L 3, a dynamic bio-speckle pattern at the diffraction plane where a detecting aperture DA is
placed. Through the detecting aperture DA, the biospeckle fluctuation is detected by a photomultiplier,
PM. Owing to the extended illuminating spot and
the setting of the detecting aperture at the diffraction
plane, this aperture DA receives the superposition of
whole light fields scattered from every point in the
spot. Therefore, this optical system enables us to
measure the overall activity of the various blood
flows observed in the illuminated spot area, rather
than the absolute flow velocity at a certain point on
Optics 8 Laser Technology
Vol 23 N o 4 1991

Object

Image plane
LI

L2

~ ~ 1-........
_. ~ ~~-

.-

Diffraction
plane
L 3 DA % . L 4

.......

PM

(I) Diffraction plane detection

Object

Image plane
L1

~fl

L2

~ -

fl

,r,

~' ~Y~DA

f2 ~ f 2

a Vessel
(,l) Image plane detection

b Tissue

Fig. 16 Two basic optical systems for detecting bio-speckles

from the ocular fundus (I) at the diffraction plane and (11) at the
image plane

a blood vessel. In addition, a consistent phase relation

between those scattered light fields may not be
retained at the detecting point but may be fully
randomized. This is due to the rapid fluctuation of
the positions of the red blood cells. It is, therefore,
noticeable that the bio-speckle fluctuation observed
here may be distinguished from the usual Doppler
effect.
Figure 17 (see Ref. 25) shows a schematic diagram of
the apparatus for detecting the bio-speckle fluctuation
from the ocular fundus and for evaluating the ocularfundus blood flow. A HeNe laser beam with 50100 p W power illuminates the ocular fundus with an

Ocular fundus

//y._~ ~

area

\M 2

approximately 1 mm diameter extended spot using

the illuminating optical system of a fundus camera.
This exposure follows the security conditions for the
human retina presented by the WHO 26. The light
scattered from the ocular fundus is received and
detected by a photomultiplier PM via a 80 p m diameter detecting aperture, DA, placed at the Fraunhofer diffraction plane. The probe area is easily
positioned by a target fixation. The probe volume
depth cannot be strictly defined in the present
method, but we employ a HeNe laser of 632.8 nm
wavelength so that the light penetrates into the
choroidal tissue layers Then, the method may probe
both the retinal and choroidal layers.
Figure 18 (see Ref. 24) shows typical power spectral
distributions of the bio-speckle fluctuation obtained
from the ocular fundus of an anaesthetized albino
rabbit. Result (a) was measured when the rabbit was
breathing in room air. Results (b) and (c) were
measured when the rabbit's mouth was covered with
a vinyl bag filled with a gas mixture of CO2 and 02
for 2 min and 3 min, respectively. This application
was expected to increase the blood flow artificially.
With the gas application, the low frequency components of the power spectral distribution are clearly
reduced and higher frequency components increase
relatively. This variation indicates the substantial
increase of the ocular-fundus blood flow. It is
interesting to note that the form of the power spectral
distribution in Fig. 18 is very similar to the result for
the gastric mucous membrane in Fig. 13 but is
different from the result for the skin surface in
Fig. 4. This is probably due to the fact that the
structure of the ocular fundus tissue is closer to that
of the gastric mucous membrane than that of the
skin tissue. This observation implies that the
frequency components of bio-speckle fluctuations
are significantly influenced by the structure of the
tissues and vessels. Figure 19 shows the temporal
variations of the mean frequency (f~ as the two
kinds of gas mixture (a) and (b) were applied to two
anaesthetized rabbits. The mean frequency ~) increases with the gas mixture (a) after time A and
then decreases with room air after time B, but it
remains unchanged with gas mixture (b). This
difference arises from the effect of CO2 density on
the blood flow. The mean frequency (f~ reflects
favourably the change of the ocular fundus blood
flow according to the gas application.

I D;~a~t....L~9 ,ip-

Llo

i]i

i i i I

I I

(a)

-10
"D

-20
O

-30

(cl Gas m i x t u r e , 3 rain

-40

I I I I

0.1

"~-:..__
I I I

l~l.~.~J

10

Frequency (kHz)

Fig.17 Schematicdiagramoftheapparatusforevaluating the

blood-flow at the ocular fundus

Optics 8- Laser Technology
Vol 23 N o 4 1991

Fig. 1 8 Power spectral distributions obtained from the ocular

fundus of a rabbit for the three different conditions of breathing

213

A:CO 2 and

0 2

B:Room

air

Rabbit

14
Z
I
1

;-_

7
~

{a)

--o

[b}
0

o--32,9% CO 2 and 24.3% 02

--e-----o--9.7%
I

CO 2 and 22.4% 02

Time,

t {1 rain d i v - i /

Fig. 1 9
Variation o f the m e a n f r e q u e n c y ( f ) as t h e t w o kinds
o f t h e gas m i x t u r e (a) and (b) are applied to t h e t w o anaesthetized
rabbits

Because of the weak-intensity speckles from the

ocular fundus, the photon correlation technique is
used for analysing effectively output signals. In
Fig. 17, photoelectron pulses obtained from the photomultiplier PM are fed into a 256 channel digital
correlator, CORR, with which the autocorrelation
function of the bio-speckle fluctuation is obtained.
Figure 20 shows the fundus photograph of a human
ta)

volunteer including an illuminated spot, along with

typical correlation functions of the bio-speckle
fluctuation obtained from the probe positions (a)
and (b) indicated in the photograph. The correlation
function attenuates rapidly in (a) and slowly in (b).
The probe area (a) contains two major retinal vessels
(vein and artery), but the area (b) includes no visible
vessel. Thus, the blood flow is considered more
active in (a) than in (b). The more and less active
blood flows in (a) and (b) increase higher and lower
frequency components and result in the rapid and
slow decays of the correlation functions, respectively.
The attenuation of the correlation function is
evaluated by analysing the time-correlation length r~
which gives half the maximum correlation value of
the correlation function. Figure 21 shows the distribution maps of the time-correlation length r,, obtained
from each ocular fundus of three normal volunteers
(a), (b) and (c). From the rough estimation of Fig. 21,
the smallest or largest values of v~. are obtained from
those probe areas where retinal vessels cross or do
not, respectively. In these results, the absolute wdues
of c~, cannot be directly compared among the three
volunteers, because of their individuality with respect
to the inhomogeneous structure of tissue and vessels.
But the similar dependence of r~ on the existence or
non-existence of blood vessels was observed tor the
three volunteers. Thus, these results are helpful for
investigating the blood circulation at the human
ocular fundus. In the present method, the bio-speckle
fluctuation mainly reflects blood flows in retinal
vessels and capillaries, although the light from capillaries has weak intensity. The light scattered from
the underlying choroidal tissue also contributes to
the bio-speckle fluctuation, but this is modulated
and indirect since the detected light experiences
twice the phase modulation due the retinal blood
flows and tissues. Therefore, the only possible and
useful measurement in the method is to evaluate the
overall degree of blood-flow activity in the probed
area including both the retinal and choroidal
vasculatures.

beam
(~1 mmO)

Retinal blood-flow velocity

1.0

0.5
~,

- L

-'. . .~. ' . . .

-...

"-

'.v<:

'.-.~[:...=..,.......

..

..":.i "~
-' ...

'.:'
.

.
..

-.'""<,.:-.-::
(a)
"
- - " .i..,
"' " : . ' .
0

i
0

I
200

I
qO0

.
,

.-

i: ;. L . . . !::
i

"

1
600

Fig. 2 0
F u n d u s p h o t o g r a p h of a h u m a n v o l u n t e e r and
correlation f u n c t i o n s o b t a i n e d f r o m t h e t w o p r o b e points s h o w n
in t h e p h o t o g r a p h

21 4

As well as the overall degree of blood flow in a

finite fundus area, the blood-flow velocity at a point
on a retinal vessel also has important information
for many diagnostic purposes. Several researchers ~ s
have extensively studied the laser Doppler method,
and recently the usefulness of the method has been
reported for the non-invasive assessment of some
eye diseases. We shall study a method for measuring
the retinal blood-flow velocity using bio-speckle
phenomena. In Fig. 16(II), the intensity fluctuation
of bio-speckles scattered by red blood cells in a
retinal vessel is detected by the aperture DA at the
magnified image plane. By positioning the centre of
the detecting aperture DA to a measuring point of
interest, this optical system enables us to measure
the velocity of retinal blood flow at the selected
point. Arrangements (a) and (b) in Fig. 16(II) show
schematic examples of the measuring point on a
vessel and surrounding tissue, respectively. Output
pulses are analysed to obtain the autocorrelation
Optics 8 Laser T e c h n o l o g y
Vol 2 3 No 4 1 9 9 1

60

120

180

240

Time, t (las)

30

63 194

75

9
II0

39

/43

74
80

176

~
205

47

Fig. 21

3q

,,~

Distribution maps of r c obtained from each ocular fundus of the three normal volunteers

for 22.8X magnification and an X - Y micro stage for

positioning a measuring point. Nearly 200 p m diameter grains of bio-speckles were detected by a
400 p m diameter detecting aperture, DA, at the mag-

function. The blood-flow velocity is measured in a

relative value from the reciprocal of the timecorrelation length, l/re. Figure 22 shows the
measuring apparatus modified with a microscope

Reticule
pot

Ocular

fundus

~_ ~ / . ~ i~~:;~: ~PCU

Fig. 2 2

Vessel image

Schematic diagram of the measuring apparatus modified with a microscope and an X - Y micro stage

Optics 8" Laser Technology

Vol 2 3 No 4 1991

215

al

Diameter d (pro)
250 - ~

280

- - * 200

200 - ~---..u150
c--~125

N
2~

150

. . . . 75

a2

b2

a3

b3

aq

bq

//

/.,~ ..,."

.."
// // ..."

/__

bl

~,/

.~//~/"""//

o
t/3

2
I00

/
50
a

V(mm s 1)

100

/
/

100 300 1000

b

d (urn)

Fig. 2 3
(a) Relation between the blood-flow velocity in capillaries
and the reciprocal of time-correlation length; (b) Relation between
the slope of lines and the capillary diameter obtained from (a)

nified image plane. With this condition, the probe's

cross-sectional area was defined to be nearly 20pm.
Thus, more than 50pm diameter of the vessel is
available for blood-flow measurements in this
arrangement. By using the reticle of the eyepiece, the
position of the detecting aperture can be adjusted to
the measuring point of interest.
Before applying the method to human subjects, we
performed a preliminary experiment 27"2s by using
blood flow in glass capillaries of various diameters.
Figure 23(a) shows the relation between the velocity
of the blood flow and the reciprocal of timecorrelation length, 1~re. Good linearity was obtained
for each glass capillary, but the slope of the lines
increased with increasing capillary diameters. This
means that a certain constant flow velocity was
evaluated to be different values of l/v~ according to
the capillary diameter. We found, however, that the
slope of the lines is linearly proportional to the square
root of the diameter. Figure 23(b) shows this linearity
obtained from the results of Fig. 23(a). Therefore, the
diameter dependence of the bio-speckle fluctuations
may be compensated for by this experiment, and the
velocity of the retinal blood flow can be measured
by the present method.
On the basis of the knowledge from the preliminary
experiment, we applied the method to human volunteers. Figure 24 shows the fundus photograph of a
human volunteer, in which al-a4 indicate the
measuring points on the vessel while bl-b4 represent
the measuring points on the surrounding tissue.
Figure 25 shows typical correlation functions obtained
from the points a3 and b 3 of Fig. 24 and co-histograms obtained by repeating the measurements
several times at all points of Fig. 24. Each correlation
function was measured in 2 s. The correlation
function (a) demonstrates an extremely early decay
owing to the high blood-flow velocity in the vessel, but
the function (b) shows a slow decay owing to the
low degree of capillary blood flow and indirect
choroidal blood flow. In the histograms, the v~values of (a) and (b) are distributed in shorter and
longer time regions, respectively. Therefore, the biospeckle fluctuation well reflects the retinal bloodflow velocity.
Figures 26 and 27 show the temporal variations of
the correlation functions and their l/v~ values when

216

Fig. 2 4
Fundus p h o t o g r a p h of a human volunteer and s o m e
measuring points

3
" " E ' : : l
0

200

q00

"~

" ~l

1f

"'ii'

600

/
T

200

(psi

-~
q00

600

(ps)

,o[_i]"'""

101

1 bl bu

i,.iiJ
0

100

200

I00

200

Fig. 2 5
Typical correlation functions and rc-histograms obtained
from the measuring points; (a) a 1 -a 4 on retinal vessels; and
(b) b l - b 4 on s u r r o u n d i n g tissue areas s h o w n in the p h o t o g r a p h
of Fig. 2 4

the measuring point was moving out from (a) the

centre of a vessel to (b) the surrounding tissue. We
stored the photon count data lbr about 6 s and then
analysed the correlation functions every 52.5 ms. In
Fig. 26, the decay of the correlation curve is tound to
become slower after 4 s because of displacement of
the measuring point. This variation is clearly shown
by the 1/re-value in Fig. 27. This means that the
blood flow velocity can be favourably evaluated by
the value of l/v~. Figure 28 shows the variations of
1/re-values and the electrocardiogram (ECG) obtained
every 65 ms from the retinal artery (a) and vein (b).
In (a), the value of 1/vc clearly changes over the
same period with the ECG, and the pulsating blood
flow is successfully measured. In (b), however, the
pulsation of the blood flow is not recognized as
expected from the elementary medical knowledge.
These experimental results show the usefulness of
the present method for measuring the blood-flow
Optics t Laser Technology
Vol 2 3 No 4 1 9 9 1

t (s)
0

3.150

3.675
X

~. o5o

:~~'::~~'''!:;~i~!i::'::'i!~'i!i::'l

(a) Vessel

4.200

I .575

4. 725

~(':;'":" 7

2.100

5,250

(b) Tissue

2.625

5.775

P
3. ~50

;q~:~:::",:.t~;~:~;.~:(.:~i~.!
;.. ::,

~:';:":!~.".':"':t'~?~:..:.::..-.::::~.%:.:::.
256
-

6.291

{us)

256
v{ps)

Fig. 2 6

Variation of correlation functions when the measuring point was shifting from a vessel to a tissue
100 "

~oo
N
32
0

50

\
0

~lt

t {s)

t(s}

Artery

v 200

t(s)

t Is)
Fig, 2 7
Variation of 1/Vc-value corresponding to the correlation
functions of Fig. 26
Lu

velocity in retinal vessels. The method detects the

temporal fluctuation of bio-speckles but not the
Doppler broadening components. Therefore, the
present technique is free from the severe scattering
geometry conditions.
Other examples

of

bio-speckles

A few studies 2,29. 30 have been made on bio-speckles

obtained from fruits and eggs. These studies are not
directly related to the evaluation of blood flow, but
are interesting in other applications of bio-speckle
phenomena. Briers 2 investigated the bio-speclde
fluctuation obtained from red and green tomatoes,
and proposed a method for evaluating the movement
Optics Et Laser
Vol23

No4

t is)

t(s)

t(s)

Vein

Fig. 2 8
Variations of I / r c and ECG obtained simultaneously
from (a) retinal artery and (b) vein

of plastids in the cells with relation to the wavelength of light used. The bio-speckle phenomena
observed from some fruits were also studied by
Oulamara et a129 and were applied to the quantitative analysis of the biological activity of the living
state of the cells. Another example30 of bio-speckle
was studied for measuring the heart rate of avian
embryos in an egg. Figure 29 presents a block diagram
of the experiment for measuring minute ballistic
movements of the egg using the bio-speckle

Technology

1991
217

Ballistic movement
ffCG

Position change
{non-contact)

Velocity

(contact)

Needle

Audio
cartridge

electrodes

b
II

l
amplifier

Fibre probe

Z ~J

C
Photo
multiplier

AC
amplifier

0.75

1.5

2.25

~.0

linic l<->l
Fig, 30
(a) ECO; {b) audiocartridge signals; and (c) bio-speckle
signals simultaneously measured from an embryo 1 9 days old

i 10 200 Hz
bandpass
filter

0.1 200 Hz
bandpass
filter

-200 Hz
low-pass
filter

FM tape
recorder

] l

and/or
recorder

Amplifiers
and A/D
converter

I Computer
and
xy plotter

Fig. 29
Block diagram of the experiment for measuring simultaneously minute ballistic movements of the egg using the ECG,
audiocartridge, and b/o-speckle technique

phenomena. Two contact methods with the electrocardiogram (ECG) and audiocartridge were also
used for comparison purposes. By using the optical
fibre probe shown in Fig. 3, a HeNe laser beam
illuminates an egg in a non-contact manner and the
scattered light is picked up and propagated to the
photomultiplier. The intensity fluctuation of b/ospeckles was measured via a bandpass filter and an
amplifier. The b/o-speckle signals are compared to
the results obtained by the two contact methods.
Figure 30 shows typical examples of (a) an electrocardiogram, (b) the output signals of an audiocartridge, and (c) the bio-speckle signals
simultaneously measured from an embryo 19 days
old. The periodic variation synchronized with the
electrocardiogram was clearly obtained in the biospeckle signals (c) with the merit of a non-contact
and non-disturbing operation. In addition to heartrate measurements, the bio-speckles from the egg are
expected to provide information about other physiological functions and responses of avian embryos
within their eggs to altered environments.

Conclusion
This paper reviews the time-varying properties of
dynamic b/o-speckles obtained from living objects

218

and introduces some methods lor measuring the

blood flow in the living tissue. The bio-speckle
fluctuation has various frequency components
according to the probing area of the living objects
being measured, but it has now been found to reflecl
sensitively blood-flow activity such as velocity. This
is quite useful tor evaluating non-invasively the
blood flow. The dynamic behaviour of b/o-speckles
depends on various factors within the living objects,
such as the spatial structures of tissues and vessel
network, and their absorption and diffusion characteristics. The theoretical analysis of itle rehttion
between these factors and the bio-speckle fluctuation
seems to be an interesting but a complicated subjecl
because of the complexity of living objects. However,
detailed obselwation and experimental analysis of
b/o-speckles are effective for the development of
their potential applicability. We hope that dynamic
bio-speckle phenomena will be further investigated
lor various kinds of living objects and will be applied
to the measurement of these objects" physiological
behaviour.

References
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application to velocity measurements ot" the diffuse object'.
Appl Ph).'~ 25 (1981) 179-194
2 Briers, J.D. "Wavelength dependence of intensity fluctuations
in laser speckle patterns lronl biological specimens'. Opt
('on,,mun 13 (19751 324-326
3 Fercher, A.F., Briers, J.D. "Flow visualization by means ot
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4 A s a k u r a , T. 1)ynamic properties of bio-speckles and their
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5 Aizu, Y., Asakura, T. "B/o-speckle phenomena and thcir
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6 T a n a k a , T., Riva, C., Ben-Sira, 1. "Blood velocity measurcmerits in h u m a n retinal vessels', &'fence 186 (19741 830-831
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9 Koyama, T., Horimoto, M., Mishina, H., Asakura, T.
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I0 S t e m , M.D. "In vivo evaluation of microcirculation by
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Optics 8 Laser Technology

Vol 2 3 N o 4 1991

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21

tinuous measurement of tissue blood flow by laser Doppler

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Watkins, D., Holloway, Jr,, G.A. "An instrument to
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Optics & Laser

Technology

Optics ~ Laser Technology

Vol 23 No 4 1991

219