Noninvasive assessment of burn wound severity using optical technology: a review of current and future modalities.

Abstract

Clinical examination alone is not always sufficient to determine which burn wounds will heal spontaneously and which will require surgical intervention for optimal outcome. We present a review of optical modalities currently in clinical use and under development to assist burn surgeons in assessing burn wound severity, including conventional histology/light microscopy, laser Doppler imaging, indocyanine green videoangiography, near-infrared spectroscopy and spectral imaging, in vivo capillary microscopy, orthogonal polarization spectral imaging, reflectance-mode confocal microscopy, laser speckle imaging, spatial frequency domain imaging, photoacoustic microscopy, and polarization-sensitive optical coherence tomography.

Full text

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Review
Noninvasive assessment of burn wound severity using optical
technology: A review of current and future modalities
Meghann Kaisera,*, Amr Yafib, Marianne Cinata, Bernard Choib, Anthony J. Durkinb
aDepartment of Surgery, Division of Trauma, Burns, Critical Care and Acute Care Surgery, University of California, Irvine, Orange, CA 92806,
United States
bBeckman Laser Institute and Medical Clinic, University of California, Irvine, Orange, CA 92806, United States
Contents
1.
2.
3.
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
In vitro light microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Macroscopic imaging. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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burns 37 (2011) 377–386
a r t i c l e i n f o
Article history:
Accepted 10 November 2010
Keywords:
Burn
Optical
Laser
Perfusion
Collagen
Partial-thickness
Laser Doppler imaging
Near-infrared
Indocyanine green
Capillary microscopy
Orthogonal polarization spectral
imaging
Reflectance-mode confocal
microscopy
Laser speckle imaging
Spatial frequency domain imaging
Photoacoustic microscopy
Polarization-sensitive optical
coherence tomography
a b s t r a c t
Clinical examination alone is not always sufficient to determine which burn wounds will
heal spontaneously and which will require surgical intervention for optimal outcome. We
present a review of optical modalities currently in clinical use and under development to
assist burn surgeons in assessing burn wound severity, including conventional histology/
light microscopy, laser Doppler imaging, indocyanine green videoangiography, near-infra-
red spectroscopy and spectral imaging, in vivo capillary microscopy, orthogonal polarization
spectral imaging, reflectance-mode confocal microscopy, laser speckle imaging, spatial
frequency domain imaging, photoacoustic microscopy, and polarization-sensitive optical
coherence tomography.
# 2010 Elsevier Ltd and ISBI. All rights reserved.
* Corresponding author at: c/o Marianne Cinat, 333 The City Blvd West, Suite 705, Orange, CA 92806, United States. Tel.: +1 714 456 5840;
fax: +1 714 456 6048.
E-mail address: mkaiser@uci.edu (M. Kaiser).
0305-4179/$36.00 # 2010 Elsevier Ltd and ISBI. All rights reserved.
doi:10.1016/j.burns.2010.11.012
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/burns

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3.1.
3.2.
3.3.
Microscopic imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1. Capillary microscopy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2. Orthogonal polarization spectral imaging (OPSI) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3. Reflectance-mode confocal microscopy (RMCM). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.4. Future modalities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Laser Doppler imaging. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Indocyanine green (ICG) videoangiography. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Near infrared spectroscopy (NIRS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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4.
5.
1.Introduction
A half century ago, Dr. Zora Janzekovic and others demon-
strated that timely tangential excision and grafting of
appropriately deep burns prevented sepsis-related morbidity,
diminished the development of hypertrophic burn scars, and
radically improved cosmetic and functional outcomes [1–3].
Early excision and grafting of burn wounds thus revolution-
izedthefieldofburnsurgeryandhasbecomea centraltenetof
the field today [4,5].
The difficulty lies in determining which burn wounds will
most benefit from early excision and grafting. Red, painful,
non-blistering superficial burns—which do not require exci-
sion—are immediately apparent to most clinicians. Likewise,
most physicians can identify the pale, leathery, insensate
deep burns that must be excised. The gray zone that lies
betweentheseextremesconsistsof‘‘partial-thickness’’ burns.
Some, termed ‘‘superficial partial-thickness,’’ will heal spon-
taneously inlessthantwoweeks, withminimalornoscarring.
Others, categorized as ‘‘deep partial-thickness,’’ will require
prompt excision and grafting, without which patients suffer
prolongedexpensivehospitalizations,
dressing changes, and complications such as infections and
exacerbated scarring [6–8]. This is the daily challenge of the
burn surgeon. Overestimating burn severity could mean
unnecessary surgery, underestimation could be just as
detrimental.
The stakes are high, and picking the ideal course of
treatment is not easy. Most clinicians make their determina-
tionon the basis of clinical examalone. But without additional
objective measurements, the judgment alone of even experi-
enced burn surgeons correlates with histology and eventual
outcome only about three-quarters of the time [9–13]. As a
result, several optical techniques have emerged in recent
years to assist in the clinical characterization of burns. Certain
modalities provide a means for macroscopic assessment of
entire anatomic regions,whereas others enable finely detailed
microscopic interrogations of tissue. All share a common
theme: visualization of intact dermal vasculature as a
measure of viability, whether by visualising the vessel
structures themselves or indirectly detecting blood flow.
Regardless, each modality offers a unique angle from which
to bolster accurate assessment of burn wound depth. Here we
review light microscopy, the gold standard of burn wound
assessment, as well as both macroscopic and microscopic
optical techniques currently in use and under development.
painful repetitive
2.
In vitro light microscopy
Punch biopsy of burn tissue with histologic analysis is still
considered the goldstandardofburndepth assessment. Thinly
sliced tissue specimens are typically stained with hematoxillin
and eosin (H&E stain) and examined under light microscopy.
Deep dermal thrombosed blood vessels, sometimes accompa-
nied by denatured collagen, are the hallmarks of deep partial
injury on H&E stain [9,13–15]. Use of Masson’s trichome stain
under polarized light can assist in differentiating normal
collagen—which stains blue—from denatured collagen—red.
Verhoeff’s stain of elastin can further demeracate injured from
uninjured dermis. Immunostaining may offer an additional
edge: anti-vimentin antibody, which labels intact melanocytes
and Langerhans’ cells, clearly delineates the zone of necrosis
[16]. Antibodies to collagenase, tissue inhibitor of metallopro-
teinases (TIMP), and collagen IV identify intact, proliferating
follicles with the capacity to heal [17,18].
However, even histologic analysis does not absolutely
predict eventual clinical outcome. Burn severity can vary
greatly over a small area of tissue, rendering punch biopsy
vulnerable to sampling errors. The process of obtaining,
sectioning, and staining a sample is expensive, time-consum-
ing (on the order of days), and requires the presence of an
experienced pathologist. Fixation inevitably leads to artifacts.
Perhaps most importantly, biopsy is invasive, guaranteeing
discomfort and scarring independent of the burn injury itself.
Primarily to address this last shortcoming, a plethora of
noninvasive in vivo optical techniques has evolved.
3. Macroscopic imaging
3.1.Laser Doppler imaging
Laser Doppler imaging (LDI) typifies the macroscopic imaging
modalities and is currently the most widely recognized of the
noninvasive, optical burn assessment techniques, with
several commercial devices in use internationally. As the
name suggests, a laser beam is directed at the tissue, and the
frequency of the reflected light, as altered by red blood cells
approaching and receding from the detector, indicates the
speed and volume of blood flow, termed ‘‘flux,’’ through an
area of interest [19,20]. Based on this information, some
commercial devices returna map of different colors calibrated
to represent a given area’s specific estimated time to healing,
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e.g., less than 14 days, 14–21 days, >21 days (Moor Instru-
ments, UK). Thus, similar to light microscopy, LDI cites patent
blood vessels as the primary determinant of burn depth,
although the focus is on flow rather than the vessels
themselves. Not surprisingly then, LDI correlates with burn
wound histology and need for surgical excision and grafting
about 95% of the time [11–13].
LDIoffersobviousadvantagesoverinvitrolightmicroscopy.
LDI is noninvasive, and in fact can be performed at distances
>1 m from the subject with no physical contact whatsoever.
The laser energy emitted is harmless. Moreover, a large area
can be evaluated, allowing for different management of areas
within the samewound [21]. A fairly large bodyof literature on
the subject of LDI speaks to a general comfort level with the
device.
LDI is not, however, without its own imperfections. The
current commercial device is expensive, large and difficult to
position [22]. In our personal experience, between positioning,
calibrating, andscanning,evaluation ofanarea 50 cm ? 50 cm
may take several minutes, during which the patient must
remain motionless to avoid artifact. This can be difficult for
anxiousorshiveringpatientsandyoungchildreninparticular,
necessitating sedation. The most commonly used commercial
device is not promoted for use during the 1st 48 h after injury,
secondary to reactive vasoconstriction, and more than 5 days
after injury, secondary to the proliferation of granulation
tissue [23,24]. Errors can result from vasomotor reactivity and
blood pooling in response to second-to-second changes in
ambient temperature, patient positioning and emotional
states [25] (see Fig. 1). The interpretation of LDI blood flow
maps is likewise unclear in the presence of anemia, cellulitis,
or peripheral vascular disease. Surface moisture, topical
medications, and transparent dressings can also all alter
[()TD$FIG]
LDI measurements [26]. Where the tissue surface slopes,
artifacts may result, making recorded signals difficult to
interpret. It is unclear to what extent all this affects the
accuracy of LDI, and which wounds are most appropriate for
assessment with this modality. Nonetheless, LDI remains an
extremely valuable clinical modality for the assessment of
burn wounds.
3.2.Indocyanine green (ICG) videoangiography
Indocyanine green is a non-toxic, protein-bound dye that is
retained within the vasculature after intravenous injection for
several minutes until rapid clearance by the liver. As a
diagnostic pharmaceutical, ICG has been in clinical use for
decades in the determination of cardiac output. ICG absorbs
and fluoresces within the near-infrared spectrum, which has
excellent skin penetration, making deeper dermal vasculature
visible using this dye [27]. Fluorescence can then be detected,
quantified, and digitally translated into color-coded regions of
relative perfusion for ease of interpretation, similar to the LDI
device (Fig. 2) [28]. Relative to the subject’s normal skin, ICG
fluorescence is markedly higher in spontaneously healing
wounds, and markedly lower in those that required surgery
[29–31]. In a few small human studies, ICG videoangiography
findings correlated with histology and/or clinical outcome
approaching 100% [30,31].
ICG videoangiography is capable of producing rapid,
macroscopic, easily interpreted scans very reminiscent of
LDI, on a more compact, less expensive device. In animal
studies, it can distinguish deep and deep partial wounds very
early, within the first few hours following injury [27]. Its utility
has been clearly demonstrated even in the presence of other
microvascular pathologies, such as diabetes and heart failure
Fig. 1 – On the left, LDI-generated blood flow map of a superficial partial thickness burn on a patient’s face. High blood flow is
manifest as a predominantly bright red region. On the right, the same area on the same patient minutes later, showing
significantly diminished perfusion, as suggested by the now dark blue facial area. Shortly after the second scan, the patient
experienced a near-syncopal episode. (For interpretation of the references to color in this figure legend, the reader is
referred to the web version of the article.)
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[28]. The primary, obvious drawback of ICG videoangiography
is the need for intravascular dye injection. While actually an
extremely safe compound, ICG is nonetheless associated with
headache, pruritis, urticaria, diaphoresis, and the ever-
present risk of life-threatening anaphylactic reaction [32].
Safety has not been well-established in pediatric, pregnant or
lactating patients. Additionally, measurements obtained via
ICGvideoangiographyarerelativetonormalskincontrols,and
anatomically equivalentregions of normalskin are not always
available in burn subjects. The differentiation between
normal, decreased, and increased fluorescence can be a very
fine line on the order of a few percent making assessment less
than clear-cut [27]. Finally, relative to LDI, the body of
literature on ICG videoangiography is somewhat small,
although a modality with such promise certainly warrants
further investigation.
3.3.Near infrared spectroscopy (NIRS)
As mentioned above, near-infrared light can penetrate further
into tissue than the visible spectrum, and several biochemi-
cally crucial constituents of the dermis absorb wavelengths
within the near-infrared spectrum (650–1100 nm). De-oxy
hemoglobin has a maximum absorption at 760 nm, whereas
absorption of oxy-hemoglobin is greatest at 900 nm. Water,
has peak absorption at 980 nm and may have implications for
identifyingedema. Near-infrared
probes, termed ‘‘point probes’’ can be geometrically tailored
to target specific depths of up to several centimeters in soft
tissue [33]. Noncontact near-infrared spectral imaging, which
currently represents the majority of NIRS burns research, can
generate a map depicting the relative prevalence of these
compounds (Fig. 3). Based on the unique reflectance spectra
spectroscopy contact
[()TD$FIG]
Fig. 2 – Near-infrared spectral imaging. (A) Digital photograph of burn wound to the leg with central area of full thickness. (B)
Map of tissue oxygen saturation with darker areas representing less oxygenation, corresponding to full thickness injury. (C)
Map of total hemoglobin concentration with darker areas representing less hemoglobin. (Reprinted from Ref. [40]. Copyright
2007 by John Wiley and Sons. Reprinted with permission.)
[()TD$FIG]
Fig. 3 – Indocyanine green video angiography. (A) Digital photograph of a burn to the chest with central area of full thickness
(x) and peripheral areas of partial thickness (o). (B) Raw angiography image with brighter areas corresponding to regions of
increased perfusion. (C) Computer generated image quantifying fluorescence with areas of greater perfusion represented by
red and less perfusion by blue. (Reprinted from Ref. [29]. Copyright 2003 by Elsevier. Reprinted with permission.) (For
interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
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produced by different degrees of injury, models can be derived
to significantly predict the presence of superficial partial
versus deep partial wounds with an accuracy of 87% in animal
models [34].
TheabilityofNIRStodifferentiatebetweenoxygenatedand
deoxygenated blood confers a distinct theoretical advantage
to this modality. LDI detects active blood flow through patent
vessels, and assumes that the absence of such blood flow
correlates with thrombosed vessels and necrosis. As previ-
ously discussed, however, many physiologic states other than
thrombosis can temporarily alter blood flow. Deoxy-hemoglo-
bin—present even in thrombosed vessels—may be pivotal in
discerning thrombosis from reactive vasoconstriction. More-
over, NIRS spectra reflect not just blood flow, but also the
extraction of oxygen from this blood flow. Alterations in
cellularmetabolismmaymanifestmuchsoonerafteraninsult
than blood vessel injury and thrombosis, potentially allowing
for dramatically earlier injury grading and treatment, on the
order of hours rather than days [14,35,36]. Even the degree of
inflammation—manifest as edema secondary to capillary
leakage—may differ subtly between superficial partial and
deep partial wounds [37]. Finally, some NIRS devices have the
potential to detect denatured collagen as light scattering [38].
Integrating data on the prevalence of all these molecules—
oxy-hemoglobin, de-oxyhemoglobin, water, and denatured
collagen—improves the predictive reliability of NIRS [35].
Ontheotherhand,likeLDI,scansmaytakeseveralminutes
to complete. Scanning may entail physical probe contact with
the wound, causing discomfort for some patients, although
noncontact imaging is now commonly used for burn purposes
[34,37]. To date, NIRS has reliably differentiated only superfi-
cial from full thickness burns in human subjects, not
superficial partial from deep partial, which is a much finer
line [39]. And, while the variety of device designs, wave-
lengths,andanalysisalgorithmspossibleallowforanenviable
degree of customization, there is also a distinct lack of
standardization. The full capacity of NIRS in the diagnosis of
burn injury is promising but as yet, incompletely elucidated.
4. Microscopic imaging
4.1. Capillary microscopy
Like all the above modalities, transcutaneous in vivo capillary
videomicroscopy estimates burn wound depth based on the
presence of functioning dermal vasculature. The ability of
capillary microscopy to accurately assess dermal capillaries
has been verified in a number of other disease states affecting
skin circulation, including diabetes, chronic venous insuffi-
ciency, and psoriasis [40]. In the case of burn injuries, a 200?
lens is applied to a small (?1 mm2) area of interest and the
superficial dermal capillary plexus—filled with red, oxygenat-
ed blood cells—easily examined under the visible blue-green
spectrum. Intravascular injection of sodium fluorescein dye,
andexaminationunderaconventionalfluorescencefilterwith
excitation 450–500 nm, may serve to further accentuate the
presence or absence of viable vessels [40,41]. The presence of
[()TD$FIG]
Fig. 4 – In vivo capillary microscopy. (A) Superficial burn with preserved dermal capillary plexus visible. (B) Deep partial
thickness burn with absence of the capillary plexus apparent. (Reprinted from Ref. [22]. Copyright 2007 by Elsevier.
Reprinted with permission.)
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anintact,workingplexusimpliesonlysuperficialorsuperficial
partial injury, whereas a less robust or entirely absent
capillary network denotes deep partial or deep injuries
(Fig. 4). In one paper, a simple grading system (0–3) of relative
capillarydestructionwithoutuseoffluoresceinwasdeveloped
that correlated with both LDI and clinical outcome in 100% of
subjects tested [22].
The capillary microscopy device is relatively inexpensive
(?$10,000) and portable. It is unaffected by curved skin
surfaces and does not require patients to be still for more
than seconds at a time. However, it does require direct contact
with the burn wound, and can visualize only a very small
portionofthewoundatonce,leavingitvulnerabletosampling
error,albeitlesssothan traditionalhistology. Interpretationof
microscopic findings requires a certain level of expertise, and
is to some extent subjective. Most importantly, extensive
studies of capillary microscopy in the burns arena are lacking,
with only one group having published results to date [22]. It is
unclear how soon after injury videomicroscopy is accurate, or
how infection and comorbidities might affect measurements.
Use in children younger than 13 has not been explored,
although such individuals make up a sizable portion of the
burn population. Finally capillary microscopy is not normal-
ized to account for expected regional differences in perfusion.
An area such as the face with particularly dense dermal
vasculature may undergo mild capillary damage and still
appear within normal limits. All these issues must be more
thoroughly evaluated before capillary microscopy comes to
the forefront of burn wound assessment.
4.2.Orthogonal polarization spectral imaging (OPSI)
Orthogonal polarization spectral imaging is a specialized form
of in vivo transcutaneous videomicroscopy. Polarized light of
around 548 nm (well absorbed by hemoglobin) is directed at
the tissue, and reflected light is gathered through a second
polarization filter perpendicular to the first. Any light permit-
ted through the second filter must encounter multiple
scattering surfaces, rending it no longer at right angles to
the filter. Thus superficial structures are eliminated from the
image and blood vessels around 3 mm deep—highlighted by
the presence of red blood cells absorbing the incident light—
are very apparent. With this technology, resolution is
sufficient to image the movement of a single red blood cells
transversing a capillary in real-time. Circulatory patterns
distinct for healing and nonhealing burn wounds can be
observed (Fig. 5) [43]. OPSI visualizes both the form and
function of dermal capillaries to determine an index of
‘‘functional capillary density’’ (FCD), or the length of perfused
vesselsincmpercm2ofwoundexamined[44,45].Inonestudy,
setting the threshold for deep partial thickness burns at an
FCD of 100 cm/cm2detected need for operative intervention
with a sensitivity of 93% [45].
Like trancutaneous videomicroscopy, OPSI is relatively
inexpensive, portable, unaffected by skin curvature and does
not require the subject to remain completely still [45]. FCD is
arguably a much more objective and reproducible measure of
the capillary plexus than the relative grading system used for
transcutaneous videomicroscopy. Given the unique optics of
this technology, the injection of fluorescein—with any small
but real associated risks—is not necessary to attain an
impressive level of contrast. Thrombosis can be physically
distinguished from vasoconstriction [42], allowing different
causes of low-flow states to be discerned. Capillaries can be
differentiated from larger venules and arterioles, theoretically
adding a level of precision to the microscopic determinations
of burn wound depth.
However, it is not at all clear that such detail and precision
is necessary, and the extra information adds layers of
complexity to data analysis. Fields of view are small
(?1 mm2). Since not every functioning capillary will contain
a red blood cell at any given time, thorough examinations take
around 15 min to complete, and thereafter must be replayed
and meticulously reviewed to accurately assess [45]. FCD
improves dramatically between 1 and 4 days post-injury, and
the ideal measurement interval has not been established
[42,45]. The probe is in direct contact with the burn causing
potential discomfort. One can assume that OPSI is affected by
comorbidities such as anemia, infection and diabetes [43,44],
although it has not been extensively studied in a variety of
patient populations. Most importantly, while sensitivity for
deep partial and deep burns is high, specificity is low at only
around 45% in one study [45]. Furthermore, in this samestudy,
20% of wounds could not be accurately evaluated due to the
presence of edema. Presumably, the extraordinary resolution
of OPSI is also its downfall: even slight variations in pressure,
[()TD$FIG]
Fig. 5 – Orthogonal polarization spectral imaging. (A) Normal skin, in which the dermal capillary plexus is apparent as faint
gray loops. (B) Superficial burn, appearing very similar to normal skin. (C) Deep burn, in which the destruction of the
overlying capillary plexus allows for visualization of the larger thrombosed dermal vessels beneath. (Reprinted from Ref.
[43]. Copyright 2005 by Elsevier. Reprinted with permission.)
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whether from interstitial hydrostatic forces or the application
of the OPSI probe itself, may cause substantial variation in
perceived perfusion. With this sort of inflexibility, OPSI is
likely best suited for the research arena at this time.
4.3. Reflectance-mode confocal microscopy (RMCM)
Reflectance-mode confocal microscopy is yet another varia-
tion on the transcutaneous videomicroscopy theme. In this
version, light from a near-infrared laser is projected at an area
of interest, and reflected light received through an aperture of
specific diameter. This aperture selects for a specific focal
depth by screening out non-focused light and is responsible
for a unique feature of RMCM: the ability to view tissue in
multiple planes of depth to a maximum of about 350 mm [47].
By combining multiple planes, or ‘‘optical sections,’’ a three-
dimensional map of the burn wound can be obtained [47,48].
Dermal vessels appear as dark spaces through which bright
erythrocytespassinrealtime.Otherstructureshelpfulinburn
wound assessment can also be visualized. Melanin pigment
increases reflection and adds contrast, allowing for rapid
identificationoftheepidermal–dermaljunction,assumingitis
still present post-burn. White blood cells, which proliferate
considerably more in deep and deep partial wounds, can be
discerned and quantified [46,49]. Also apparent are dermal
appendages such as hair follicles, from which epidermal cells
regenerate and migrate in the healing wounds [47,48]. Many of
these features are significantly different when comparing
spontaneously healing and non-healing wounds (Fig. 6)
[46,49].
RMCM is a true means of ‘‘optical biopsy.’’ The degree of
histologic detail possible may add to the accuracy of depth
determination. Serially adjusting the depth of focus allows for
a precise determination of the injury’s extent. However, like
other forms of transcutaneous videomicroscopy, RMCM
requires direct contact with the wound. Miniscule fields
(500 mm ? 500 mm) are imaged, requiring repetitive measure-
ments at multiple tissue levels to establish a truly representa-
tive sampling of the wound—80 fields in one study [46]. This
results in a relatively protracted exam of around 10 min,
althoughsubjectsneednotbecompletelystillfortheduration.
Afterwards, review of the images requires even more time, as
well as considerable expertise. Optical sectioning in a plane
parallel to the skin surface, as opposed to the perpendicular
sectioning of traditional biopsies, makes interpretation more
difficult. RMCM is expensive, at around $130,000 for a
commercially developed device, almost half again the cost
of LDI. Most importantly, this modality has not been
extensively studied in human burn subjects. No absolute
thresholds for differentiating superficial partial thickness
burns from deep partial thickness burns are yet established,
only relative changes over time in blood flow and white cells.
Accuracy, sensitivity and specificity are unknown, under-
mining its practical applications in decision-making for the
average burn surgeon. Like OPSI, RMCM raises some fascinat-
ing possibilities but is far from ready for widespread routine
clinical use.
4.4.Future modalities
Many other promising optical techniques for the assessment
of burn wound depth await human trials, and though clinical
data is sparse at this time, it behooves dedicated burn
surgeons to familiarize themselves with emerging modalities
ofsuchpotential.Somerepresentconceptualimprovementon
current modalities that measure tissue perfusion. Laser
speckle imaging (LSI) creates a speckle image with laser light
reflecting off wound bed structures. By capturing two images
within milliseconds of one another, speckles appear to
‘‘smear’’ in a manner analgous to increasing exposure time
on a conventional camera. The degree of smearing corre-
sponds to the speed and volume of red blood cells. As a result,
LSI creates a color-coded map similar to LDI without the
prolonged scanning time. LSI assessment of microvasculature
has been documented in port-wine stains and other patho-
logic and physiologic states [50–52]. Our group is currently
investigating the use of LSI in human subjects with burns
(Fig. 7).
Spatial frequency domain imaging (SFDI) represents the
next generation of NIRS. ‘‘Spatial frequencies’’ refer to
different patterns of near-infrared light used to illuminate
the tissue and specify particular depths of penetration,
analogous to the aperture of confocal microscopy. Using
these patterns, absorbance of certain relevant wavelengths,
[()TD$FIG]
Fig. 6 – Reflectance confocal microscopy of a human subject at 24 h post-injury showing (a) normal skin, with dermal
capillaries manifested as black punctuate areas within dermal papillae (white rings), (b) superficial burn, with apparent
enlargement of the dermal capillaries secondary to edema, (c) superficial partial-thickness burn, showing incomplete
destruction of the dermal papillae, and (d) deep partial-thickness burn, showing complete destruction of the papillae.
(Reprinted from Ref. [47]. Copyright 2009 by Elsevier. Reprinted with permission.)
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such as the peak absorbance of oxy-hemoglobin, deoxy-
hemoglobin, and water, can be measured at different depths,
creating a three-dimensional map of both the perfusion and
metabolic activity within a tissue. Changes in scattering that
result from the denaturation of collagen can also be quanti-
fied.SFDIcanbeadjustedtomeasureareasrangingfrom1 cm2
[()TD$FIG]
to in excess of 100 cm2and, unlike many other NIRS-based
devices, does not require any physical contact with the
wound. Animal studies with SFDI show clear distinction
between superficial and deep wounds [36,53]. A study of SFDI
now underway in human burn wounds has produced
preliminary findings echoing those of animals (Fig. 8).
[()TD$FIG]
Fig. 7 – On the left, a digital photograph of a hot oil burn to the hand with areas of deep (white) and partial (pink) thickness.
On the right, the laser speckle image (LSI) of the same burn. Areas of high perfusion are represented by brighter colors (red,
yellow, green, in order of decreasing perfusion) and lower perfusion is represented by blue. Deep thickness regions on the
proximal second, third, and fourth digits correspond to light blue areas on the speckle image, whereas the dorsum of the
hand overlying the metacarpals is partial thickness on clinical exam green/yellow/red on the speckle image. Uninjured,
non-inflamed skin is blue. (Patient permission was obtained to publish photograph.) (For interpretation of the references to
color in this figure legend, the reader is referred to the web version of the article.)
Fig. 8 – In the upper left corner, a digital photograph of mechanical burn (road rash) to the forearm with areas of full (oval),
deep partial (dashed oval), and superficial partial (circle) thickness injury. Regional maps of hemoglobin, oxygen saturation,
and light scattering can differentiate between different depths within the wound as shown (patient permission was
obtained to publish photograph).
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Photoacoustic microscopy (PAM) is based on the principal
that objects reflecting light energy vibrate as the energy
undergoes conversion to heat, emitting sounds waves. These
sounds waves can be picked up by an ultrasonic detector. By
projecting a wavelength of light at hemoglobin’s peak
absorption, inflamed, hyperemic tissue appears dark (hypoe-
choic) on PAM, while surrounding tissues reflecting such light
waves appearbright (hyperechoic). The result is an image very
similar to conventional sound-based ultrasonography, but on
a microscopic scale proportional to the much shorter
wavelength of light [54]. In an experimental model of burns,
PAM was able to distinguish different durations of thermal
exposure within minutes of injury [55].
Collagen in its native state is birefringent, or capable of
splitting light into two rays polarized perpendicular to one
another. When collagenis denatured by thermal injury,itloses
this property [56]. Polarization-sensitive optical coherence
tomography (PSOCT) quantifies tissue damage according to
the degree of polarization in reflected light, detected as phase
retardance. The phase retardance of tissues at different tissue
levels can be quantified to demarcate injured from uninjured
tissues. Animal studies show a statistically strong mathemati-
cal correlation between PSOCT measurements and absolute
burn depth as determined by histology [57,58]. This last
modality thus differs from the previous modalities discussed
in that it is entirely dependent on the structure of collagen, not
the microvasculature, for burn depth assessment.
5.Conclusion
The ability to predict which burn wounds will heal spontane-
ously and which will require surgical intervention is a critical
component of clinical treatment algorithms. Gross clinical
exam alone is accurate only about three-quarters of the time.
Current optical techniques to complement clinical exam
operate on the premise that functioning blood vessels are
retained in viable tissue. Both macroscopic and microscopic
modalities are available, offering different advantages and
disadvantages. While many questions remain to be answered,
experience to date indicates that the field of optics will
contribute an invaluable degree of accuracy and insight to the
field of burn assessment.
Acknowledgements
Drs. Durkin and Yafi acknowledge salary support provided by
the NIH NCRR Laser and Medical Microbeam Program,
(LAMMP: 5P-41RR01192); the Military Photomedicine Program,
(AFOSR Grant # FA9550-08-1-0384), the Beckman Foundation
and the Hazem Chehabi BLI Research Fellowship.
Conflict of interest
Dr. Anthony Durkin has a financial interest in Modulated
Imaging Inc., a company with interests related to spatial field
domain imaging (SFDI). Dr. Durkin is a cofounder of the
company and owns equity interests in Modulated Imaging.
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