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Quantitative measurement of blood flow dynamics in chorioallantoic
membrane of chicken embryo using laser Doppler anemometry
M.A. Borozdova, E. S. Stiukhina,
1 A. Yu. Sdobnov,1 I. V. Fedosov,1
D. E. Postnov,1 and V. V. Tuchin1,2,3
1Research-Educational Institute of Optics and Biophotonics, N.G. Chernyshevsky Saratov State
University, 83 Astrakhanskaya, Saratov, 410012 Russian Federation
2 Institute of Precision Mechanics and Control, Russian Academy of Sciences,
24 Rabochaya, Saratov, 410028 Russian Federation
3Interdisciplinary Laboratory of Biophotonics, National Research Tomsk State University, 36 Lenin
Ave., Tomsk, 634050 Russian Federation
ABSTRACT
We report the results on in ovo application of developed Laser Doppler Anemometer (LDA) device. The chorioallantoic
membrane (CAM) of 9-13 days chicken embryos was used as a biological model that allows an easy access to both
arterial and venous vessels of different size. The key point of our study was to find out how the periodic and aperiodic
pulsations of blood flow (which are inevitable in living organism) will affect the LDA functions and measuring
capability. Specifically, we (i) developed the technique to extract and refine the pulse rhythm from the signal received
from a vessel, and (ii) analyzed the changes in power spectra of LDA signal that are caused by heart beating and
considerably complicate the reliable measurement of Doppler shift. Our main conclusion is that the algorithms of LDA
data processing need to be improved, and this possibly can be done by counting the information on current phase of
cardiac cycle.
Keywords: laser Doppler anemometer, blood microcirculation, light scattering, digital signal processing, chorioallantoic
membrane
INTRODUCTION
Fast and reliable quantitative assessment of blood velocity is very demanded in different fields of physiology and
medicine. The availability of such measurements is of critical importance for early diagnostics of the visual nerve
atrophy in the cases of glaucoma and diabetic retinopathy,1-3 cerebral stroke4 or myocardium ischemia; atherosclerosis;
diabetes and other pathologies, including those associated with inflammatory states.5 The development and refinement of
tools for monitoring of blood flow at different parts of vascular system, from systemic vessels to microcirculation
appears to be the highly relevant trend in biophotonics.
Along with a variety of modern optical methods for study of microcirculation6-10 that to a great extent are focused on
blood vessel imaging, and estimation of blood volume relative changes, laser Doppler anemometers (LDAs)1-3,11,12 and
Doppler optical coherence tomographs (DOCTs)13-16 are designed to provide information on the movement of
backscattering particles and thus enable quantification of flow rate at medium-to-small size vessels.
LDA and DOCT operation principle is based on measuring of the magnitude of Doppler frequency shift (DFS) of light
backscattered by moving cells. DFS is directly proportional to velocity vector projection on light scattering direction
vector, which can be defined as the difference of incident and scattered light wave vectors.11,17,18 The absolute velocity
can be determined if the angle between velocity vector and scattering vector is known. In practice during in vivo
measurement, this angle often cannot be determined directly. In this case, the absolute blood flow velocity and direction
can be determined by measuring DFS of light scattered simultaneously in two or more different directions.
Saratov Fall Meeting 2015: Third Annual Symposium Optics and Biophotonics; Seventh Finnish-Russian Photonics
and Laser Symposium (PALS), edited by E. A. Genina, V. L. Derbov, D. E. Postnov, A. B. Pravdin, K. V. Larin, I. V. Meglinski,
V. V. Tuchin, Proc. of SPIE Vol. 9917, 99170W · © 2016 SPIE · CCC code: 1605-7422/16/$18 · doi: 10.1117/12.2229830
Proc. of SPIE Vol. 9917 99170W-1
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LDAs and DOCTs are mainly used for studying the vessels located not too deep and lying in parallel to the surface of a
strongly scattering tissue. The tissue is illuminated perpendicular to its surface and the backscattered radiation is
recorded. In this case, the scattering vector is practically perpendicular to the axis of the studied blood vessel; the DFS
strongly depends on the angle between the flow velocity vector and the scattering vector and turns into zero when this
angle is equal to 90°. On the other hand, if the angle between the velocity vector and the scattering vector is small, then
one can neglect the dependence of DFS on the angle.19
In our previous work, we proposed the novel technique to suppress low frequency components of LDA signal. Our
approach is based on elimination of spectral components caused by movement of the scattering particles outside the
measuring volume. This method does not require any devices for pre-shifting frequency of laser light.20 In the framework
of current research, we applied the method above in ovo, using the CAM of chicken embryos to access the arterial blood
flow.
While it is widely assumed that blood flow pulsations caused by the heart beating should vanish at the capillary level, it
is not true in many cases. Increased blood pressure during systole causes the elastic stretching of the vessel walls and, in
turn, increase of cross-section and volume of vessel segment. The emerged pulse wave propagates at a much greater
velocity than the velocity of blood flow.21
Periodic pressure fluctuations not only significantly affect the processes trans-capillary sharing, but also provide the
variation of shear stress that in turn activates the vessel wall response. Thus, the resulted modulation of LDA signal
might be far from "just harmonic". Our specific goal was to test what will happen under the conditions of real living
organism, to diagnose problems and to find possible ways to escape it.
In the next section, we discuss the essential elements of our LDA device, including the developed beam modulator, and
describe the object under study. In the section 3, we illustrate and discuss the obtained results related to changes in power
spectra of LDA signal and extracted pulse rhythm. Our findings are summarized in section 4.
2. MATERIALS AND METHODS
2.1 Laser Doppler Anemometer
LDA scheme is presented in Fig. 1. As a light source, we use the diode laser module ML-09 (Skat-R, Russia) with CW
radiation output of 15 mW at wavelength of 650 nm. Laser beam was divided into two parallel beams with prism. Then
these beams were focused on the blood flow in a vessel by 100 mm-focal length lens and 30 mm-clear aperture diameter
and Mirror 1.
When red blood cells (RBC) in the flow cross the interference fringes within the probe volume they cause the light
scattering toward Mirror 2 and then to detector (photodiode PD-256). Thus, each time a RBC passes through the probe
volume, backscattered light passes through the objective lens 1 toward lens 2. Lens 2 produces an image of the
measuring volume on a 50 µm-pinhole, placed in front of a photodetector that registers pulsed modulation, which
frequency is proportional to particle velocity. In order to keep the point of beams intersection on the target of
measurements, we precisely adjusted the position of blood vessel under study by means of 3D-movable stage.
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Miror 1 Lens 1 Miror 2 Reference
photodetector
:
Object --411'
Pinhole Mod
Lens 2
Photodetector
eamspliter
prism
Laser
Figure 1. Experimental setup.
The optical modulator presented in Fig. 1 consists of (i) modulator disk with a single blade attached to a DC motor, and
(ii) position encoder, which contains a LED and a reference photodetector with an aperture between them. The
experimental setup also includes an analog amplifier and a computer for signal processing that are not shown in Fig. 1.
Photodetector’s signal was passed to the audio adapter of a personal computer with a frequency 44100 kHz and 16-bit
resolution. In order to determine the Doppler frequency shift we use the Fast Fourier Transform (FFT) of recorded time
series. Specifically, both FFT and other stages of signal processing were performed with the LabVIEW (National
Instruments, USA). The typical observation time was 10 sec, it was splitted into periodograms with length of 512 points
each. This delivered us about 861 periodograms per measurement. Since a high-frequency component of the power
spectrum which corresponds to the modulation of the scattered light is hidden by the low-frequency component (which is
often has higher intensity and broader frequency band), we performed the averaging of non-overlapping modified
periodograms using the Hanning data window.22
Generally, the power spectrum of LDA-signal has two components,17,18 the first one is concentrated at a low frequency20
being the contribution from light scattering by particles that move outside the probe volume, and the second one at the
higher frequency near the DFS being the contribution of moving particles inside the probe volume. Because of
interference between waves scattered by different cells, LDA-signal spectrum has components with frequencies in the
range from 0 to a maximal frequency, so-called cutoff frequency.20 The cutoff frequency is equal to the difference
between frequency of light scattered by the cells with the lowest possible velocity (fixed) and the frequency of light
scattered by a particle moving as fast as possible (on flow axis). These components can be observed if the vessel is
illuminated by only one of two beams.
Note, in our setup there is the additional data channel, see Fig.2. While channel 1 (the main one) is for a signal received
from the flow, the channel 2 carries information from the Position encoder, via which we can identify its current position.
In Fig. 2, the time interval between points 1 and 2 corresponds to the encoder state when both beams are opened,
between points 2-3 ‒ only beam 1 is opened, 3-1 – only beam 2 is opened.23 Thus we can easily sort out signal
components from the channel 1. The modulation frequency normally was 600 rotations per minute.
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Chanel 1 - Chanel 2
-maxi - t - max 2
12:
I.dih 111 T-1 T 1 ri T-1 T-1
fil
Ill. I. I. I. 1
_--r 71- _--
os 1Illlpll1llll'IIIIIIP 111,)II11IIII1IIP1NI1;1111IIII,
I1.iS U.'J
Figure 2. Timing during measurements: pulses and current position of modulator.
2.2 Object under study
The experiments were performed for the chorioallantoic membrane (CAM) of 13-days-old chicken embryos. Fertilized
red chicken (Gallus domesticus) eggs were incubated for 9 days at 37.8 °C at humid atmosphere being turned two times
per day. Embryos that showed no bleeding or development abnormalities were selected for the study. After opening of
egg shell above air cell, outer shell membrane around was gently removed and the entire egg was positioned within the
experimental setup in order to reach the proper location of beams intersection on the vessel segment selected for the
measurement. The later means that vessel segment was placed perpendicular to the direction of the laser beams and the
position providing better signal was determined empirically. We selected the vessels with the diameter in the range from
111 to 229 µm lying at a depth from 70 to 200 µm, see Table 1. Diameters and depths were obtained by using the optical
coherence tomography system (Ganymede 930 nm OCT System). Figure 3 illustrates all the 4 sample vessels that are
characterized below.
2.3 Processing of pulsating flow
Being heart-pumped, the blood flow in CAM considerably fluctuates during the cardiac cycle, even in relatively small
arterial vessels. According to the LDA measuring scheme this causes more or less pronounced broadening of the spectral
peak at Doppler frequency, that in turn makes the measurements challenging, especially if it is made with arbitrary
chosen recording options. For example, with observation time of 10 sec, there are only about 10-20 cardiac cycles
included. Thus, depending on specific value of heart rate and phase of cardiac cycle at the moments of the beginning or
the end of recording, the calculated absolute value of velocity can vary.
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i
*4
a) b)
c) d)
Figure 3. Vessels for study: diameter of 220 µm at depth 100 µm (a), diameter 229 µm at depth 150 µm (b), diameter 111 µm at depth 200 µm (c), and
diameter172 µm at depth 70 µm (d).
Table 1 Diameter and depth of the studied vessels
Vessel Diameter Depth
Vessel 1 220 µm 100 µm
Vessel 2 229 µm 150 µm
Vessel 3 111 µm 200 µm
Vessel 4 172 µm 70 µm
3. RESULTS AND DISCUSSION
The first problem to solve was to select an easiest way to obtain the timing of heart beating from already existing data
channels. To solve this problem we extracted the pulse wave signal in order to synchronize our measurements with its
phase. We did this by the appropriate filtering of the channel 1 signal by means of Chebyshev bandpass filter of order 5
with a bandwidth of 2500-2700 Hz. This specific frequency band was chosen empirically, but can be justified in terms of
frequency modulation of Doppler signal. Then, we used the direct method of linear convolution with data window 10000
and got a smooth curve that represents the pulse wave (see Fig. 4). In fact, we performed some digital implementation of
frequency modulation detector.
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2
1II I IIIIMI NIYI N
_
-
-2
0123456789
Time, s
.lig. .II ... .I,
-0,1
-0,2
0123456 7 89
Time, s
-.1nnn01nAnn
dIVVVVVVVV1
0
0123456 7 89
Time, s
a) b)
c)
Figure 4. Signal processing: raw data (a), data filtered with Chebyshev filter (b), and smoothed pulse wave (c).
With pulse signal extracted, we can use it for timing, since we know that each maximum of the pulse wave corresponds
to the highest frequency shift, and therefore to highest velocity, and minimum corresponds to the lowest velocity,
respectively.
3.1 LDA signal analysis
At different points of the vessel, RBCs can move with different velocities. As a result of the interference of the waves,
scattered by different RBCs, in the LDA signal spectrum the components appear with the frequencies distributed within
the interval from zero to a certain maximal frequency (referred to as a cutoff frequency).23 The frequency components
corresponding to scattering of light by particles illuminated simultaneously by two beams can be identified by
subtracting the spectrum of the low-frequency component to obtain the difference spectrum (curve 4). The difference
spectrum has a maximum at the same frequency as signal detected at flow illuminated by two beams (raw data).
Figure 5 (a) shows the details of data processing for the 3-rd vessel: the spectrum of the signal obtained when flow is
illuminated with two beams (curve 1), spectra obtained at illumination by beam 2 only (curve 2) or beam 1 only (curve
3). The frequency components corresponding to scattering of light by particles illuminated simultaneously by two beams
can be identified by subtracting the spectrum of the low-frequency component to obtain the difference spectrum (curve
4). For reference, in Fig. 5 (b) we illustrate the spectra obtained during measurement of the flow velocity in strongly
scattering kaolin suspension flowing in the channel with the cross section of about 1 square millimeter. The LDA probing
volume was located at the flow axis in the channel at the distance 0.5 mm from its front window. Figure 5(b) shows flow
illuminated with two beams (curve 1), and the difference spectrum with a pronounced peak (curve 2). In contrast, in ovo
measurements provide the variety of spectral envelopes. Figure 5(c) shows difference power spectra of vessels 1-4. The
average velocity was calculated as 0.2 mm/s, 0.6 mm/s, 0.3 mm/s, and 0.2 mm/s, respectively. The inspection of curves
in the figure clearly shows that in all cases multiple peaks can be found. In all cases, in spite of successful removal of
low-frequency interference, there are still questions how to determine the actual Doppler shift.
Proc. of SPIE Vol. 9917 99170W-6
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oo1
I400
200-
11 ' _A ,,.
2.0
t. dlì
V0246810
f kHz
-2
300
i
4
iu .,... nn_.._
2
f, kHz
One can note, however, that peaks are located nearby the same central frequency of about 0.5 kHz. Thus, one can
hypothesize that this is the result of frequency modulation during the cardiac cycles. The later means that simple
procedure of velocity recovery from the LDA data using FFT should be somehow adopted to the case of non-stationary
flow. As a first trial, we use the knowledge of current phase of cardiac cycle to separate the periodograms according the
velocities.
(a) (b)
(c)
Figure 5. Power spectra of LDA signal: the Doppler signal spectrum
from vessel 3 when flow is illuminated by two light beams (1), by
beam 2 only (2) and beam 1 only (3): 1 – raw data, 2, 3 – low
frequency components, 4 – difference spectrum with a well-defined
frequency shift (a); power spectrum from strongly scattering kaolin
suspension in the channel with the cross section 1 ´ 2 mm shows flow
illuminated with two beams (curve 1), difference spectrum (curve 2)
(b); difference power spectrum of vessels 1-4 (c).
CONCLUSION
We have illustrated the application of the developed modification of LDA device to the in vivo measurement under the
conditions of pronounced modulation of the blood flow. Our results show that normal lifetime pulsations of blood flow
can considerably complicate the operation of LDA. Specifically, since the measurements are based on determination of
peak position in power spectra, its broadening and splitting makes the use of laser Doppler anemometer in vivo rather
challenging. The possible ways to escape this limitation may include the development of pulsation-specific algorithm of
spectra averaging, based on strobing provided by the current phase of cardiac cycle (PCC), which should be determined
separately, either by different method (EGG, photoplethysmography), or extracted from the same data, like we proposed
above. Then, the averaging of power spectra should be performed over the periodograms obtained at the same interval of
PCC in order to obtain the well-pronounced spectral peak. If the measurement is targeted on average blood flow velocity
measurement, then at the final stage the averaging over the set of PCC- dependent velocity values should be performed.
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ACKNOWLEDGMENTS
The experimental setup used in this work has been developed in the framework of research and development RF
governmental contract №2014/203, Research №1490 "Development of optical methods and instrumentation for
measurements and control of structure and dynamics of biological media".
The application of CAM-based model for study of vascular responses end experimental protocol were supported by the
Russian Ministry of Education and Science, project number 3.1340.2014/K ”Development of methods for diagnosis of
functional state of microvasculature cell layers on the basis of optical imaging techniques”.
VVT is thankful for support by Tomsk State University Foundation named after D.I. Mendeleev.
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