Assessment of pulpal vitality using laser speckle imaging.

Abstract

The pulpal chamber of each tooth contains the vasculature necessary to maintain a viable tooth. A critical need exists to develop an objective, repeatable method to assess pulpal viability. We hypothesized that the existence of blood perfusion within the pulp can be determined with analysis of laser speckle imaging (LSI) patterns generated by transillumination of the tooth.
We used nine extracted human cuspids and incisors. A Tygon tube was inserted into a channel created within each tooth and Intralipid pumped through the tube in a controlled manner with a syringe infusion pump. We evaluated the feasibility of LSI for flow assessment using both transillumination and epiillumination imaging configurations. With the transillumination geometry, we also assessed the effect of the angle of incidence of the probe laser light on the speckle flow index (SFI) values extracted from the collected speckle images.
Transillumination LSI, and not epiillumination LSI, enables differentiation between the absence and presence of perfusion in an in vitro tooth model. SFI values are insensitive to the relative angle of incidence of the laser light, over a wide range of angles.
Our preliminary in vitro data suggest that transillumination LSI is a promising method to identify the presence of blood flow in the pulpal chamber. Future in vivo evaluation is warranted.

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Lasers in Surgery and Medicine 43:833–837 (2011)
Assessment of Pulpal Vitality Using Laser Speckle Imaging
Charles Stoianovici, BS,1Petra Wilder-Smith, DDS, PhD,1and Bernard Choi, PhD1,2,3?
1Department of Surgery, Beckman Laser Institute and Medical Clinic, University of California, Irvine,
California 92612
2Department of Biomedical Engineering, University of California, Irvine, California 92697
3Edwards Lifesciences Center for Advanced Cardiovascular Technology, University of California, Irvine,
California 92697
Background and Objective: The pulpal chamber of
each tooth contains the vasculature necessary to maintain
a viable tooth. A critical need exists to develop an objec-
tive, repeatable method to assess pulpal viability. We hy-
pothesized that the existence of blood perfusion within
the pulp can be determined with analysis of laser speckle
imaging (LSI) patterns generated by transillumination of
the tooth.
Study Design/Materials and Methods: We used nine
extracted human cuspids and incisors. A Tygon tube
was inserted into a channel created within each tooth
and Intralipid pumped through the tube in a controlled
manner with a syringe infusion pump. We evaluated the
feasibility of LSI for flow assessment using both transillu-
mination and epiillumination imaging configurations.
With the transillumination geometry, we also assessed
the effect of the angle of incidence of the probe laser light
on the speckle flow index (SFI) values extracted from the
collected speckle images.
Results: Transillumination LSI, and not epiillumination
LSI, enables differentiation between the absence and
presence of perfusion in an in vitro tooth model. SFI val-
ues are insensitive to the relative angle of incidence of the
laser light, over a wide range of angles.
Conclusions: Our preliminary in vitro data suggest that
transillumination LSI is a promising method to identify
the presence of blood flow in the pulpal chamber.
Future in vivo evaluation is warranted.
Med. 43:833–837, 2011.
? 2011 Wiley-Liss, Inc.
Lasers Surg.
Key words: blood flow; dentistry; laser Doppler; laser
speckle contrast analysis; laser speckle contrast imaging;
perfusion
INTRODUCTION
The pulpal chamber of each tooth contains the vascula-
ture necessary to maintain a viable tooth. Trauma or in-
fection typically elicit an inflammatory response, which
could either resolve on its own or cause the pulpal tissue
to become necrotic, leading to gangrene and abscess for-
mation. The most common methods used to assess pulpal
health, are hot-and-cold thermal testing and electrical
stimulation of the Ad nerve fibers [1]. These methods
assess the degree of intact neural innervation in the
interrogated tooth. However, recent reports demonstrate
that blood flow is a far better indicator of pulpal health
[2]. The current inability to accurately diagnose and mon-
itor pulpal perfusion, and its response to noxious stimuli
or treatment, provides a strong incentive for clinicians to
avoid the risk of attempting measures to maintain pulpal
vitality by performing devitalization and root canal thera-
py as the treatment of choice. Disadvantages include
a long, costly and arduous procedure, medical contra-
indications, unsuitability of some teeth, as well as long-
term discoloration and risk of tooth fracture.
Research groups have studied the feasibility of various
methods to assess the viability of the pulpal chamber.
Gazelius et al. [3] demonstrated that laser Doppler flow-
metry (LDF) can monitor blood flow in the pulpal cavity of
intact enamel and dentin. Their data suggest that LDF
can differentiate between healthy and necrotic pulpal
tissue. However, problems with LDF include low signal-
to-noise ratio [3,4] and the strong dependence of the sig-
nal on the probe position [5]. Fried et al. [6] determined
that enamel and dentin are strongly forward scattering
and minimally absorb infrared light. This seminal obser-
vation led to subsequent investigation of various optical
methods such as pulse oximetry [7] and photoplethysmog-
raphy [8–10], to assess pulpal vitality. Due to the difficul-
ty in obtaining reproducible data, these methods are not
accepted in the dental clinic. Additionally, each method
requires the use of a dental splint, further limiting its
widespread use.
A critical need exists to develop an objective, repeatable
method to assess pulpal viability. For widespread clinical
acceptance, this method ideally involves rapid data collec-
tion and does not require use of a dental splint. Based
on our laboratory’s prior experience with laser speckle
Contract grant sponsor: Arnold and Mabel Beckman Founda-
tion; Contract grant sponsor: National Institutes of Health Laser
Microbeam and Medical Program; Contract grant number:
RR001192.
*Corresponding to: Bernard Choi, PhD, Beckman Laser Insti-
tute and Medical Clinic, University of California, Irvine, 1002
Health Sciences Road East, Irvine, CA 92612, USA.
E-mail: choib@uci.edu
Accepted 2 June 2011
Published online in Wiley Online Library
(wileyonlinelibrary.com).
DOI 10.1002/lsm.21090
? 2011 Wiley-Liss, Inc.

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imaging (LSI) to study blood-flow dynamics in preclinical
animal models [11,12] and human subjects [13,14], we in-
vestigated its efficacy in assessing fluid flow in an in vitro
tooth model. We hypothesized that the existence of
blood perfusion within the pulp can be determined with
analysis of laser speckle patterns generated by transillu-
mination of the tooth. This hypothesis is based on the
high scattering anisotropy of dental tissue reported
previously [6,9].
MATERIALS AND METHODS
Ex Vivo Tooth Preparation
We used nine extracted human cuspids and incisors.
Upper and lower cuspids and incisors were chosen as they
are easy to access in the clinical setting. Samples were
stored in water with thymol at a temperature of 0–48C.
We used a diamond-wafering blade (Buehler 10 ?
0.3 mm2) to detach the tooth crown from its root approxi-
mately 2–5 mm below the cement–enamel junction (CEJ).
The root canal was enlarged to a 1-mm diameter channel
from the apical end up to the CEJ using a Rite Dent V1
hand piece equipped with a White1HP4 carbide dental
bur. This work was performed in compliance with a
protocol approved by the Institutional Review Board
at University of California, Irvine, and with the docu-
mented, informed consent of the subjects.
LSI Instrument
The LSI instrument consisted of a laser source, a CCD
camera, and a macro lens. It is similar to the instrument
described in previous publications [13,14]. Briefly, a
12-bit thermoelectrically cooled CCD camera (1,600 ?
1,200 pixel resolution, Model 2000R, QImaging, Surrey,
Canada) was used to image the raw speckle image remit-
ted from the tooth. A 633-nm HeNe laser (30 mW power)
was used to transilluminate each tooth sample. By con-
trolling the magnification and aperture of the external
macro lens, we set the minimum resolvable speckle size to
be at least the width of two camera pixels [15].
Data Reduction
Each raw speckle image first was converted to a speckle
contrast image, then to a speckle flow index (SFI) image,
using the methodology described previously [16]. Each set
of SFI images was averaged to a single mean SFI image.
A region of interest was extracted from each mean SFI
image and a single mean SFI value calculated. The select-
ed region included pixels between the top of the root and
the CEJ.
Experimental Design
Flow phantom. A Tygon tube was inserted into the
channel created within each tooth. The tube had an outer
diameter of 1 mm and inner diameter of 0.25 mm. One
end of the tube was connected to a 29-gauge insulin sy-
ringe. The syringe was mounted onto an infusion pump
(Harvard Instruments). The syringe contained 1 ml of a
Liposyn solution. The stock solution (20% intravenous fat
emulsion) diluted to a 1:20 Liposyn:water volume ratio.
The tooth was fixed in space with an optomechanical
mount. The buccal surface of the tooth was imaged on to
the camera sensor. We used the following average flow
speeds: 0, 0.34, 1.7, and 3.4 mm/second.
EpiilluminationVersus
evaluated two imaging configurations. In one set of
experiments, we used a transillumination configuration,
for which laser light was incident on the lingual side (i.e.,
the side facing the tongue) and resultant speckle pattern
imaged from the buccal side (i.e., the side facing the lips).
In a second set of experiments, we used an epiillumina-
tion configuration, for which laser light was incident on
the buccal side and the pattern imaged from the buccal
side. For each flow-speed setting and imaging configura-
tion, a sequence of 10 raw speckle images was collected.
The data were reduced as described above, to a single
mean SFI value. Three replicates were performed for each
flow-speed setting and imaging configuration. For transil-
lumination experiments, data were collected from nine
teeth. For epiillumination experiments, data were collect-
ed from three teeth. For each imaging configuration, a t-
test was used to test the null hypothesis that the SFI val-
ues collected at 0 mm/second were similar to those collect-
ed at 0.34 mm/second (i.e., the lowest nonzero blood-flow
speed used) (Fig. 1).
Relative angle of incidence of laser light. For
in vivo application of LSI, we postulated that the relative
angle of incidence of the laser light on the tooth surface,
may differ for each tooth. To determine the effect of a
varying angle of incidence, we performed experiments
with the transillumination configuration described above.
We used four angles of incidence, relative to normal inci-
dence: 08, 158, 308, and 458. A 3608 manual rotational
mount was used to control precisely the angle. For each
flow speed setting and relative angle of incidence, a
sequence of 10 raw speckle images was collected. The
data were reduced as described above, to a single mean
SFI value. Three replicates were performed for each flow-
speed setting and angle of incidence. For each flow speed,
a single-factor analysis of variance test was used to test
the null hypothesis that the SFI values collected at each
angle of incidence were similar.
transillumination. We
RESULTS
Transillumination LSI, and not epiillumination LSI,
enables differentiation between the absence and presence
of perfusion in an in vitro tooth model (Fig. 2). With trans-
illumination, an ?70% increase in SFI was observed
(Fig. 2A). In general, the mean SFI values were at least
three times higher for transillumination LSI than for
epiillumination LSI, for all flow speeds. For transillumi-
nation LSI experiments, the mean SFI value was signifi-
cantly lower at 0 mm/second than at 0.34 mm/second
(P < 0.05). For epiillumination LSI experiments, the
mean SFI value was insensitive to flow speed (P > 0.30).
SFI values are insensitive to the relative angle of inci-
dence of the laser light, over a wide range of angles
834STOIANOVICI ET AL.

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(Fig. 3). Based on a single-factor analysis of variance test,
SFI values for a given flow speed, are identical over a
large range of incidence angle settings (P > 0.62).
DISCUSSION
Our preliminary in vitro data support our hypothesis
that use of the transillumination LSI method enables de-
termination of the presence of pulpal perfusion. A signifi-
cant increase in SFI values was observed with a change
from stagnant flow to an average flow speed of 0.34 mm/
second (Fig. 2). In contrast, epiillumination LSI was in-
sensitive to flow speeds ranging between 0 and 3.4 mm/
second (Fig. 2).
We propose that transillumination LSI has several
advantages over other diagnostic methods which have
been described in the peer-reviewed literature. It has the
capability to serve as an objective method to study blood
flow in the pulpal chamber, as opposed to thermal testing.
Furthermore, SFI values obtained with transillumination
LSI, were insensitive to the relative angle of incidence of
the laser light (Fig. 3). Multiple optical scattering of the
light is expected to occur, resulting in a homogenization of
both the spatial intensity distribution and the degree to
which the speckle pattern is modulated by the moving
scatterers in the tube. This result suggests that precise
positioning of an eventual probe design in the mouth,
is unnecessary to enable accurate interrogation of the
pulpal chamber for the presence of blood flow. Published
studies using LDF probes, required the use of stents
to affix the probe in place, and the measurements were
easily corrupted by relative differences in probe place-
ment or even angulation [5].
To the best of our knowledge, in vivo measurements of
blood flow in the pulp, remain unknown. Arterioles, ven-
ules, and capillaries are present in the pulp. If we assume
that the blood flow in these vessels is similar to that found
in other arterioles, venules, and capillaries in other
organs, then the maximum blood flow speed is several
Fig. 2. A: With a transillumination LSI configuration, SFI
images enabled visualization of a small increase in flow
speed. The mean SFI value increased by 70% as the flow
speed increased from 0 to 0.34 mm/second. B: With transillu-
mination LSI (triangles), an increase in SFI values was
observed as the fluid flow changed from Brownian fluid
motion (0 mm/second) to forced fluid flow (?0.34 mm/second)
within the tooth. In contrast, epiillumination LSI (squares)
was insensitive to any change in flow speed. For (A), the error
bars represent the standard deviation of the mean. For
(B), the error bars are not visible due to the low coefficient of
variation (<3.2%) in measured SFI values.
Fig. 1. Schematic of experimental setups used to evaluate (left) epiillumination and (right)
transillumination LSI. The buccal and lingual sides of the tooth are those that face the
cheek and tongue, respectively.
ASSESSMENT OF PULPAL VITALITY835

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mm/second [17]. Hence, the range of flow speeds used
in our experimental design, is appropriate to represent
in vivo pulpal blood flow.
Our data in Figure 2B suggest that transillumination
LSI potentially enables identification of the presence or
absence of viable perfusion in the interrogated tooth, but
it has limited utility to enable reliable quantitation of the
actual perfusion value over potential blood-flow speeds in
the pulpal vasculature. A possible explanation for this re-
sult is the presence of static optical scatterers in the hard
dental tissue. Static scattering is known to modulate the
measured speckle pattern and diminish the sensitivity of
LSI to blood flow [18]. Additional studies are planned to
determine the effects of tooth thickness on measured SFI
values and the dynamic range and flow-speed resolution
of LSI.
Our current experimental setup is bulky and hence not
well suited for imaging of teeth other than the incisors.
With the rapid advances being made in both laser diode
and digital-camera technology, we plan to reduce consid-
erably the footprint of the LSI instrument, to enable
development of a probe that could be used with other
teeth.
A potential issue is that the intra- and interpatient dif-
ference in SFI values in healthy teeth may be greater
than the difference observed within our data set. The
thickness of the tooth is expected to affect the SFI meas-
urements. To address this issue, various methods can be
used to assess tooth thickness, such as the use of calipers.
A more difficult issue to address, is that the ratio of dentin
to enamel thicknesses is expected to differ for each type
of tooth.
One approach that we plan to explore in vivo, is to de-
termine the degree of similarity between SFI values asso-
ciated with contralateral teeth (i.e., the left and right
central incisors, the left and right lateral incisors, etc.)
from each subject’s mouth. If matched teeth are associat-
ed with similar SFI values, then transillumination LSI
may be used to identify relative differences in blood flow
between matched teeth, which may be used as an objec-
tive indicator that pulpal blood flow is compromised.
In conclusion, our preliminary in vitro data suggest
that transillumination LSI is a promising method to iden-
tify the presence of blood flow in the pulpal chamber.
Future work involves development of a robust in vivo
transillumination LSI instrument and pilot clinical data
collection to assess intra- and interpatient variability in
SFI values.
ACKNOWLEDGMENTS
We thank Joe Youssef and Elaine Nguyen for their as-
sistance on this project. This study was funded in part by
the Arnold and Mabel Beckman Foundation and the Na-
tional Institutes of Health Laser Microbeam and Medical
Program (LAMMP, a P41 Technology Research Resource,
award RR001192).
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