A NEW LASER DOPPLER FLOWMETER PROTOTYPE

FOR MICROCIRCULATION SKIN DEPTH MONITORING

In Vitro Validation and In Vivo Preliminar Results

Edite Figueiras

, Rita Campos

, Ricardo Oliveira

, Luís F. Requicha Ferreira

and Anne Humeau-Heurtier

Instrumentation Center (GEI-CI), Physics Department, Faculty of Sciences and Technology of Coimbra University,

Rua Larga, 3004-516, Coimbra, Portugal

Laboratoire d'Ingénierie des Systèmes Automatisés (LISA), Université d'Angers,

62 Avenue Notre Dame du Lac, 49000, Angers, France

Keywords: Laser Doppler flowmetry, Measurement depth, Skin microcirculation.

Abstract: A new laser Doppler flowmeter with depth discrimination capabilities is being developed to monitor skin

microvascular perfusion. This new laser Doppler flowmeter is a multi-wavelength device with different

spaced detection optical fibres. In order to obtain an in vitro validation of this prototype, measurements in

two phantoms, one consisting of Teflon

microtubes and the other consisting of acrylic plates, are

performed. The prototype validatation in vivo is also presented. Results obtained for both validations are

compared with the ones obtained with a commercial laser Doppler flowmeter. The measurements show

quite good agreements between both flowmeters.

1 INTRODUCTION

Laser Doppler flowmetry (LDF) is a technique for

real-time and non-invasive monitoring of the

microcirculation blood flow based on the Doppler

Effect. In this technique, a monochromatic light

beam is carried from the laser by an emitting optical

fiber to the tissues under study. In the tissues, the

light can be reflected, scattered, absorbed or

transmitted. When photons hit moving red blood

cells (RBCs) a change in wavelength occurs

(Doppler shift), while photons that hit static objects

have an unchanged wavelength. The magnitude and

frequency distribution of these changes in

wavelength are directly related to the number and

velocity of the RBCs in the sampled volume. The

backscattered Doppler shifted and non-Doppler

shifted photons are detected and they will produce a

stochastic photocurrent in the photodetector. This

photocurrent contains information on velocity and

concentration of RBCs (Bonner and Nossal, 1981).

LDF can be used for skin microcirculation

monitoring. Skin microcirculation is present in the

dermis, and it is organized into two horizontal

plexuses: the most superficial is situated in the

papillary dermis at 0.4 - 0.5 mm below the skin

surface; the second plexus is located at the dermal

subcutaneous interface at 1.9 mm from the skin

surface where arteriovenous anastomoses can be

found (Brevarman, 2000).

Currently, LDF human skin measurements lack

in estimating the sampling depth. These difficulties

lead to ambiguities in the discrimination of the

fraction of light scattered from superficial and

deeper blood microcirculation skin layers (Oliveira

et al., 2011). Besides this, commercial available

flowmeters use different signal processing

algorithms and calibration procedures making

impossible the comparison of their results.

The most commonly used laser wavelength in

LDF monitors is 780 nm and the most used fibre

separation is 0.25 mm. Some studies proposed the

use of other wavelengths and fibre separations in

order to modify the sampling depth of the LDF

technique (see for example Larsson et al., 2002;

Murray et al., 2004). However, further work is

required to have better knowledge and control over

the mean sampling depth (and hence volume) in skin

tissue.

154

Figueiras E., Campos R., Oliveira R., F. Requicha Ferreira L. and Humeau-Heurtier A..

A NEW LASER DOPPLER FLOWMETER PROTOTYPE FOR MICROCIRCULATION SKIN DEPTH MONITORING - In Vitro Validation and In Vivo

Preliminar Results.

DOI: 10.5220/0003765401540159

In Proceedings of the International Conference on Biomedical Electronics and Devices (BIODEVICES-2012), pages 154-159

ISBN: 978-989-8425-91-1

 2012 SCITEPRESS (Science and Technology Publications, Lda.)

Besides that, only few papers reported the

influence of the fibre distance and wavelength in

vivo, and simultaneously (Gush et al., 1984;

Freccero et al., 2006). Gush et al. (1984) used

wavelengths in the visible spectrum where the light

penetration is very influenced by the tissue optical

properties. Very large fibre distances were used, and

were recorded separately as the probe used has only

one collecting fibre, that was placed at a certain

distance from the emitting fibre before each

acquisition. In Freccero et al. (2006) different

flowmeter apparatus are compared. When different

devices are compared, there are instrumental factors

whose effects cannot be precisely judged (Freccero

et al., 2006).

We present herein a new Doppler flowmeter

prototype that can bring depth discrimination

information to the skin blood perfusion

measurements. Validation tests are performed in

vitro and in vivo and the preliminary results are

compared with the ones obtained with a commercial

laser Doppler flowmeter (Periflux 5000, Perimed,

Sweden).

2 MATERIALS AND METHODS

2.1 Prototype

A new prototype with depth discrimination

capabilities is being built in order to discriminate

between different microcirculation skin layers (see

figure 1).

Figure 1: Laser Doppler flowmeter prototype.

The system has three constant power laser diodes

drivers to supply three laser diodes of 635, 785 and

830 nm wavelength. The probe used [from Perimed

(Sweden)] has a central emitting fibre and several

collecting fibres located at 0.14, 0.25 and 1.2 mm

from the emitting fibre. Three bi-cell photodetectors

(PD 1, PD 2 and PD 3) are used for backscattered

light detection (Oliveira et al., 2011). The prototype

has also a calibration system which provides the

light intensity for the photodetectors calibration.

This calibration system consists of three light

emitting diodes (LEDs), with the three wavelengths

used, with variable current sources (Oliveira et al.,

2011).

2.2 Measured Variables

The conventional parameters obtained from laser

Doppler technique are blood perfusion (Perf) and

concentration of moving red blood cells (CMBC).

Perf and CMBC can be estimated from the

Doppler power spectrum (Bonner and Nossal, 1981).

The CMBC is proportional to the zero order moment

of the power spectrum P(ω) of the AC component of

the light:

∫

∝ dwwPCMBC )( (1)

and Perf is calculated as the first order moment of

the power spectrum of the AC component of the

light:

∫

∝ )(wwPPerf d(ω) (2)

In our work, the voltage signal is sampled at

50 kHz and the digitalised signal is then processed to

give as an output a parameter proportional to the

average Perf.

2.3 Calibration and Normalization

The calibration is performed through two steps.

First, a LED illuminates the photodetector surface

with a number of selected DC intensity levels. The

intensity is regulated by software, where the DC

level of the photodetectors is used as feedback in

order to produce a stepwise, linearly increasing

intensity function (Oliveira et al., 2011). A block of

2048 points is acquired in each DC step and the Perf

is computed for each step. The blood perfusion

obtained during the DC steps is fitted to a first order

polynomial, called the detector noise curve:

bDCmPerf

noise

+×= (3)

where m is the slope of the curve, b is the y-axis

value and DC is the DC voltage value. This curve is

subtracted to the blood perfusion measurements.

For the second step, the perfusion in a motility

standard solution (Perimed, Sweden) is scaled with a

constant, M, in order to obtain 250 PU.

In order to make the perfusion independent of the

total light intensity at the detector surface, it is

Photodetectors

Laser Diodes

Probe tip

A NEW LASER DOPPLER FLOWMETER PROTOTYPE FOR MICROCIRCULATION SKIN DEPTH MONITORING

- In Vitro Validation and In Vivo Preliminar Results

155

normalized with the factor 1/DC

. The normalized

perfusion formula can then be expressed as:

()

noise

PerfPerf

Perfusion −=

(4)

2.4 In vitro Validation

In vitro validation was performed in order to

evaluate the performance of the prototype to

different scatterer concentrations and velocities.

For in vitro validation two phantoms have been

used. One possesses six layers of Teflon

microtubes with internal diameter of 0.30 mm and

an external diameter of 0.76 mm. The other phantom

consists of two glued acrylic plates with a 4×5 mm

excavated depression in one of them, used to study

the linearity of the prototype related to different

scatterer velocities and concentrations. Milk was

pumped through the microtube, in the Teflon

phantom, or through the depression in the acrylic

phantom. Commercial skimmed milk has been

chosen as a moving fluid, because it has various

components that act as scatterers (Waterworth et al.,

1995).

The milk was pumped with a motorized syringe

at 1.56, 3.12, 4.68, 6.25, 7.78 and 9.35 mm/s in the

Teflon® phantom and at 0, 0.5, 1.0, 1.5, 2.0, 2.5,

3.0, 3.5, 4.0, 4.5, 5.0, 5.5 and 6.0 mm/s in the acrylic

phantom. Measurements were taken with milk, and

with two different aqueous milk solutions (50% and

25%) during ten minutes with Perimed probe

positioned perpendicular to the surface of the

phantom. The mean signal for each velocity and

concentration was computed for three minutes of

blood perfusion signal and a linear regression study

was performed.

The same protocol was executed with the

commercial Periflux 5000 flowmeter, from Perimed,

and with the prototype in order to compare the two

flowmeters.

2.5 In vivo Validation

The prototype was also tested in vivo in healthy non-

smoking subjects. The subjects were asked to refrain

from drinking coffee during the measurements day.

Perfusion has been recorded during thirty three

minutes in the forearm with the subjects in the

supine position: baseline blood flux was recorded for

20 min. Then, an arterial occlusion test was

performed with a pressure cuff placed around the

upper limb, inflated for 3 min at 200 mmHg. The

cuff was then released to obtain a post-occlusive

hyperemia and the signal was recorded during

10 min after the release of the occlusion.

For each subject, the protocol was repeated using

the three laser diodes existing in the prototype: 635,

785 and 830 nm, and also for the commercial

flowmeter, Periflux 5000 from Perimed, with the

probe positioned in the same position during the four

measurements.

The Ethics Committees of the Centro Cirúrgico

de Coimbra (CCC) in Portugal, approved this study.

Informed consent was obtained from the subjects

before the recordings were made. The measurements

were made in 20 subjects and the preliminary results

are presented.

3 RESULTS AND DISCUSSION

3.1 Calibration

The steps obtained with our prototype during the

calibration with the 830 nm LED are shown in figure

2 where a pair of signals was obtained for each

photodetector (as the detection system is made by

bi-cell photodetetors). It can be seen that the

intensity level pairs diverge when the voltage

increases. This is due to the lack of alignment

between the photodetectors and the LEDs.

0 0.5 1 1.5 2 2.5 3 3.5

x 10

-8

-7

-6

-5

-4

-3

-2

-1

number of points

Voltage steps (V)

bi-cell PD 1 A1

bi-cell PD 1 A2

bi-cell PD 2 A1

bi-cell PD 2 A2

bi-cell PD 3 A1

bi-cell PD 3 A2

Figure 2: Photocurrent generated by the three bi-cell

photodetectors during calibration: PD 1 A1 and A2 are the

PD 1 signals, PD 2 A1 and A2 are the PD 2 signals, PD 3

A1 and A2 are the PD 3 signals.

The detector noise perfusion curve is presented

in figure 3. The slope of the curves are (-6.86, -6.67

and -6.73)x10

-5

for PD 1, PD 2 and PD 3,

respectively and the y-axes value are (0.157, 0.165

and 0.162)x10

-3

with a coefficent of determination,

, close to one.

BIODEVICES 2012 - International Conference on Biomedical Electronics and Devices

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-7 -6 -5 -4 -3 -2 -1 0

x 10

-4

DC Voltage (V)

Perfusion (PU)

PD 1

PD 2

PD 3

Figure 3: Detector noise perfusion curves obtained for

each photodetector: PD 1, PD 2 and PD 3.

0 50 100 150 200 250 300 350

230

235

240

245

250

255

260

265

270

number of points

Perfusion (UP)

0.14 mm

0.25 mm

1.2 mm

Figure 4: Perfusion obtained in the motility standard with

the prototype for a 785 nm laser light for the three fibre

distances.

The perfusion obtained in the motility standard

solution after the calibration for the 785 nm laser

light is presented in figure 4 with a mean value of

250.6± 6.8, 250.5± 6.1 and 250.4± 6.0 for 0.14, 0.25

and 1.2 mm fibre distances, respectively. These

values are in the range of the ones obtained with the

Perimed flowmeter which can be 250±15.

3.2 In vitro Validation

3.2.1 Periflux 5000 - Teflon

Phantom

In the Teflon

phantom, results obtained with

Periflux 5000 show that the perfusion increases with

the velocity and concentration of milk and with the

emitting-receiving fibre distance. Non-linearities

were found for 0.14 and 0.25 mm fibre separations.

The results obtained for all velocities and the three

emitting-receiving fibre distances at 25% milk

concentration are presented in figure 5. Perfusion

saturates for 6.25 mm/s in milk using the 1.2 mm

fibre separation. A good fitness between the linear

model and the obtained results was obtained in the

linear regression analysis: R

was always close to

one and the p-values were always smaller than 0.01,

except for results collected with 1.2 mm fibre

distance in milk. In this case it was only considered

three samples for the regression analysis (due to the

saturation mentioned above).

The saturation of the perfusion can be explained

by the fact that the Periflux 5000 flowmeter is for

perfusion measurements in living tissues and the

phantom does not perfectly mimic them.

Positioning the probe in the top of the microtube

Teflon

-based phantom was difficult due to the

microtube curvature. This, together with the small

milk volume in the microtube, when compared with

the tube volume, lead to the sub-estimation and

uncertainties of the perfusion measurements. These

factors could be the reason for the non-linearity

obtained. That is why an acrylic phantom was

developed 1) with a plain surface easing the contact

probe/phantom and 2) with a higher volume of milk.

0 2 4 6 8 10

100

200

300

400

500

600

700

Velocity (mm/s)

Perfusion

0.14 mm

0.25 mm

1.2 mm

Figure 5: Perfusion obtained in the Teflon

phantom with

the Periflux 5000 flowmeter (Perimed, Sweden) for all

velocities at 25% aqueous milk solution.

3.2.2 Prototype - Teflon

Phantom

Some drawbacks in the prototype results obtained in

the Teflon phantom were found, resulting from the

positioning difficulties of the probe in the top of the

microtube. These drawbacks are difficult to

overcome as real time signal processing is not yet

implemented in the prototype.

A NEW LASER DOPPLER FLOWMETER PROTOTYPE FOR MICROCIRCULATION SKIN DEPTH MONITORING

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3.2.3 Periflux 5000 - Acrylic Phantom

For the acrylic phantom, perfusion increases with

the velocity and with the concentration of the

moving fluid for each emitting-receiving fibre

distance. Moreover, we also noted that increasing

the emitting-receiving fibre distance leads to a larger

perfusion value. This is due to the larger volume

measured with larger emitting-receiving fibre

separations. For the 1.2 mm emitting-collecting fibre

distance, perfusion saturates for the higher

velocities. The statistical analysis showed good

correlation between the fitted model and the results

since R

was close to one and the p-value was lower

than 0.01. Therefore, we can conclude that the

relation between perfusion and velocity is linear, as

expected.

The perfusions obtained with the Periflux 5000

flowmeter in the acrylic phantom with 1.2 mm

emitting-fibre distance are shown in figure 6. It can

be seen that the perfusion saturates at 3.5, 4 and

4.5 mm/s for milk, 50 and 25% aqueous milk

solution, respectively.

0 1 2 3 4 5 6 7

100

200

300

400

500

600

700

800

900

1000

Velocity ( mm/s)

Perfusion

Milk

50% aqueous milk solution

25% aqueous milk solution

Figure 6: Perfusion obtained in the acrylic phantom with

the Periflux 5000 flowmeter (Perimed, Sweden) for all

velocities with the 1.2 mm emitting-receiving fibre

distance.

3.2.4 Prototype - Acrylic Phantom

In the acrylic phantom, results obtained with the

prototype show an increase of the perfusion with

milk velocity for all wavelengths of incoming light,

as it was expected. Moreover, perfusion also

increases with the emitting-receiving fibre distance.

Concerning the milk concentration, for the three

laser light wavelengths, the perfusion increases with

milk concentrations for each fibre distance and

velocity. To investigate if the relationship between

perfusion and milk velocity was linear, a linear

fitting was performed. R

values obtained were

higher than 0.94 and the p-values were always lower

than 0.01. Therefore, we can conclude that our

perfusion measurements are linearly proportional to

the velocity of the moving fluid.

Perfusion obtained in the acrylic phantom with

785 nm laser light for all velocities with the 1.2 mm

emitting-receiving fibre distance are shown in

figure 7.

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7

200

400

600

800

1000

1200

Velocity (mm/s)

Perfusion

Milk

50% aqueous milk solution

25% aqueous milk solution

Figure 7: Perfusion obtained in the acrylic phantom with

the prototype for a 785 nm laser light for all velocities and

with the 1.2 mm emitting-receiving fibre distance.

3.3 In vivo Validation – Preliminary

Results

The preliminary in vivo results are in accordance

with the literature and with the commercial

prototype results. The results obtained for one

subject using the 635 nm laser light of the prototype

and the commercial flowmeter are shown in figures

8 and 9. The baseline blood flux was recorded for 20

min (T1). During the occlusion, perfusion decreases

for any fibre distance (T2). After the occlusion

release the reactive hyperaemia peak, P, occurs and

then the signal returns to the baseline value (T3).

0 5 10 15 20 25 30 3434

100

150

200

Time (mi n)

Perfusion

0.14 mm

0.25 mm

1.2 mm

T2 T3

Figure 8: Signal collected in the human forearm with the

non-invasive prototype with a 635 nm laser diode. The

results for the three fiber distances are shown.

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5 10 15 20 25 30

100

120

Time (mi n)

Perfusion

0.14 mm

0.25 mm

1.2 mm

Figure 9: Signal collected in the human forearm with the

Periflux 5000 flowmeter (Perimed, Sweden). The results

for the three fiber distances are shown.

4 CONCLUSIONS

Calibration was performed with success despite the

lack of alignment between the LEDs and the PDs. In

general, there are good agreement between the in

vitro results of the Periflux 5000 flowmeter and our

prototype taken on the acrylic phantom. In both

systems Perf increases with the increasing of the

parameters under evaluation, i.e, velocity, milk

concentration and emitting-receiving fibre distance.

There is only a mismatch with the theoretical

expectations for the 635 nm laser diode when blood

perfusion of different milk solutions are compared

for the signals collected in the Teflon

phantom.

Furthermore, it was statistically proved that Perf is

linearly proportional to the velocity of the moving

fluid, as theoretical principles indicate. Moreover, in

vivo preliminary results obtained with the non-

invasive prototype are in accordance with the

literature and with the commercial flowmeter used.

ACKNOWLEDGEMENTS

The authors thank the “Instituto de Investigação

Interdisciplinar (III)” of the University of Coimbra,

“Acções Universitárias Integradas Luso–Francesas”

(PAUILF) programme and “Fundação para a

Ciência e a Tecnologia (FCT), Lisbon”, for

supporting this work.

REFERENCES

Bonner, R. F., Nossal, R. (1981). Model for laser Doppler

measurements of blood ﬂow in tissue. Appl Opt; 20,

2097–2107.

Braverman, I. M.(2000). The Cutaneous Microcirculation.

J Invest Dermatol Symp Proc; 5, 3-9.

Freccero C., Wollmer P., Sundkvist G. and Svensson H.

(2006). The influence of wavelength and probe

configuration on findings of a skin vasoconstriction

test when using laser Doppler perfusion devices.

Microvascular Research; 71,64-67.

Fredriksson I., Larsson M. and Stromberg T. (2009).

Measurement depth and volume in laser Doppler

flowmetry. Microvascular Research; 78, 4-13.

Gush, R. J., King, T. A. and M. I. V. Jayson. (1984)

Aspects of laser light scattering from skin tissue with

application to laser Doppler blood flow measurement.

Phys.Med. Biol. 29;1463-1476.

Larsson M., Steenbergen W. and Strömberg T. (2002).

Influence of optical properties and fiber separations on

laser doppler flowmetry. J. Biomed.Opt; 7,236-243.

Murray A. K., Gorodkin R. E., Moore T. L., Gush R. J.,

Herrick A. L. and King T. A. (2004). Comparison of

red and green laser Doppler imaging of bloof flow.

Lasers in surgery and medicine; 35,191-200.

Oliveira, R., Semedo, S., Figueiras, E., Requicha Ferreira,

L. F., Humeau, A. (2011). Laser Doppler Flowmeters

for microcirculation measurements, 1st Portuguese

Meeting in Bioengineering - Bioengineering and

Medical Sciences - The Challenge of the XXI century,

Portuguese chapter of IEEE EMBS; Technical

University of (Portugal).

Waterworth, M. D., Tarte, B. J., Joblin, A. J., van Doorn,

T. and Niesler, H. E. (1995). Optical transmission

properties of homogenized milk used as a phantom

material in visible wavelength imaging. Australasian

Physical and Engineering Sciences in Medicine;18,

39-44.

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