In vivo visualization of dermal collagenfiber in skin burn by collagen-sensitive
second-harmonic-generation microscopy
Ryosuke Tanaka
Shu-ichiro Fukushima
Kunihiko Sasaki
Yuji Tanaka
Hiroyuki Murota
Takeshi Matsumoto
Tsutomu Araki
Takeshi Yasui

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Journal of Biomedical Optics 18(6), 061231 (June 2013)

In vivo visualization of dermal collagen fiber in skin burnby collagen-sensitive second-harmonic-generation
microscopy
Ryosuke Tanaka,a Shu-ichiro Fukushima,a,b Kunihiko Sasaki,a Yuji Tanaka,a Hiroyuki Murota,c Takeshi Matsumoto,aTsutomu Araki,a and Takeshi Yasuia,b,daOsaka University, Graduate School of Engineering Science, 1-3 Machikaneyama, Toyonaka, Osaka 560-8531, Japan
Nara Medical University, Department of Anatomy and Cell Biology, 840 Shijo-cho, Kashihara, Nara 634-8521, Japanc
Osaka University, Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japand
University of Tokushima, Institute of Technology and Science, 2-1 Minami-Josanjima, Tokushima, Tokushima 770-8506, Japanb

Abstract. Optical assessment of skin burns is possible with second-harmonic-generation (SHG) microscopy due toits high sensitivity to thermal denaturation of collagen molecules. In contrast to previous studies that were performed using excised tissue specimens ex vivo, in vivo observation of dermal collagen fibers in living rat burnmodels with SHG microscopy is demonstrated. Changes in signal vanishing patterns in the SHG images are confirmed to be dependent on the burn degree. Comparison of the SHG images with Masson’s trichrome-stained
images indicated that the observed patterns were caused by the coexistence of molten and fibrous structuresof dermal collagen fibers. Furthermore, a quantitative parameter for burn assessment based on the depth profileof the mean SHG intensity across the entire SHG image is proposed. These results and discussions imply a potentialof SHG microscopy as a minimally invasive, highly quantitative tool for skin burn assessment. © The Authors. Published by
SPIE under a Creative Commons Attribution 3.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of theoriginal publication, including its DOI. [DOI: 10.1117/1.JBO.18.6.061231]

Keywords: nonlinear microscopy; second-harmonic-generation; collagen; burn; thermal denaturation; skin; dermis; dermatology.
Paper 12654SSRR received Oct. 1, 2012; revised manuscript received Mar. 14, 2013; accepted for publication Mar. 19, 2013; published
online Apr. 12, 2013; corrected Apr. 18, 2013.

1

Introduction

Skin is the outer protector of the human body and is subjected toa range of environmental stimuli, such as light, mechanical, biochemical, and thermal stimuli. Exposure of the skin to excessive
heat, such as from a flame, hot liquid, or hot solid, sometimes
causes skin burns. Burns are usually categorized into threedegrees depending on the invasion depth of thermal injury inthe skin, which has a three-layer tissue structure. In a firstdegree burn, or epidermal burn, in which the injury is limited
to the epidermis, there is no need for intensive therapy because
damaged epithelial cells can regenerate themselves. Although asecond-degree burn damages both the epidermis and dermis, theskin can still be healed by reepithelialization with suitable medical treatment. Second-degree burns are further classified into
superficial dermal burn (SDB) and deep dermal burn (DDB).
On the other hand, a third-degree burn, or deep burn (DB), damages down to the subcutaneous layer and causes cellular death,requiring skin graft surgery. Furthermore, DB involving over
50% of the total body surface area may lead to death by infection. Since the appropriate therapeutic method for the burndepends on the degree of the burn, assessment of the burn
depth is a key step in determining the treatment plan.1 In particular, it is important to differentiate between SDB, DDB, andAddress all correspondence to: Takeshi Yasui, University of Tokushima, Instituteof Technology and Science, 2-1 Minami-Josanjima, Tokushima, Tokushima 7708506, Japan. Tel and Fax: +81-88-656-7377; E-mail: yasui.takeshi@tokushima-u.ac.jp

Journal of Biomedical Optics

DB to prevent the spread of infection in the affected site and toachieve favorable prognosis.
Visual inspection by a medical doctor is often used in clinicalexamination. However, it is difficult for even an experienced
doctor to differentiate between SDB and DDB because thedoctor estimates the degree of the skin burn only from theappearance of the wound on the epidermis.2 The pin-prick
test is another clinical assessment often used for severeburns. Since completely damaged nerve cells lose the senseof pain to a pin-prick stimulus, this test can distinguish DB
from other burns; however, it is still difficult to know whether
the burn is an SDB or a DDB. There is therefore a significant
need for a reliable, minimally invasive, highly quantitative burnassessment method.
Optical probe methods are desirable for burn assessment
because they are simple, non-contact, and painless to the patient.
Furthermore, optical probes can be directly applied because skinis superficial tissue. Optical burn assessment will be made possible by obtaining biological parameters closely related with theskin burn by optical probe methods.1 Blood flow in the skin tissue is a useful biological parameter. Usually, there is abundant
blood flow in healthy tissue. On the other hand, a skin burncauses vascular occlusion and stops the blood flow in injured
tissue. Therefore, optical probe methods for visualizing the distribution of the blood flow in tissue can be used for burn assessment. Laser Doppler imaging (LDI) can visualize the lateral
distribution of blood flow velocity in tissue;3,4 however, it cannot provide sufficiently quantitative parameters on the burn
depth. Photoacoustic (PA) microscopy can inform us of theburn depth in the form of a temporal waveform of the PA

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signal.5,6 However, since LDI and PA microscopy measure thesignal originating from the blood in healthy tissue under the
injured tissue, the obtained information does not always directlyreflect the degree of the skin burn.
If one could utilize another biological parameter that reflects
the degree of the dermal burn more sensitively than the blood
flow, the effectiveness of optical burn assessment would begreatly improved. Collagen molecules, which are abundant in
the dermis, have the potential to provide such a biologicalparameter because thermal denaturation converts the native triple helix of three polypeptide chains to a random coil. Detecting
this structural change would enable sensitive burn assessment in
the dermis, allowing differentiation among SDB, DDB, and DB.
Polarization-sensitive optical coherence tomography has
been used to determine burn depth because the birefringencespecific to collagen molecules and their aggregates (microfibrils,
fibrils, fibers, and bundles) is reduced by thermal denaturation.7,8 However, since the burn causes only a small change
in birefringence, it is difficult to visualize the distribution in
the burn area with high image contrast.
Second-harmonic-generation (SHG) light functions as aneffective optical probe that shows high selectivity and good
image contrast to collagen molecules as well as high spatial
resolution, optical three-dimensional (3-D) sectioning, minimal
invasiveness, deep penetration, the absence of interference frombackground light, and in vivo measurement without additional
staining.9,10 Most importantly from the viewpoint of optical burnassessment, since SHG light arises from a non-centrosymmetric
triple helix of three polypeptide chains in the collagen molecule,its intensity decreases and finally disappears when thermal
denaturation changes the structure of this molecule to a centrosymmetric random coil.11,12 Therefore, thermal denaturationof collagen fibers in various tissues, such as the cornea,13tendons,14–16 and dermis,17,18 has been investigated by SHG
microscopy. These previous studies indicated that SHG light
has the potential to be an indicator for monitoring thermal denaturation. However, those experiments were performed using
excised tissue specimens ex vivo. To accelerate in vivo applications of SHG microscopy in the field of optical burn assessment,
it is essential to evaluate the technique under in vivo conditions.
In the work described in this article, we demonstrated in vivo
observation of dermal collagen fibers in rat burn models with
SHG microscopy and evaluated the potential of SHG microscopy for in vivo optical burn assessment.

2

Materials and Methods

2.1

Animal Model of Burns

The experimental protocol used in this study was approved by the
Bioethics Committee for Animal Experiment in Osaka University. An animal model of burns was prepared following thewell-established protocol of Walker and Mason.19 The accuracyof this protocol has already been confirmed by histologicalexamination.5 Male Wistar rats (Japan SLC, 8 to 10 weeksold), weighing between 250 and 300 g, were used as the animalmodel and housed in a climate-controlled animal care facility for
one week before use to acclimate themselves to a new environment. On the day before imposing the burn, we anesthetized therats with isoflurane (4% for induction, 2% for maintenance, eachwith an O2 flow rate of 1.0 l∕ min). After the animal was anesthetized, its back was shaved and epilated with scissors and hairremover lotion. On the day of imposing the burn, we anesthetized
Journal of Biomedical Optics

Fig. 1 Photograph of skin surface for (a) control, (b) SDB, (c) DDB, and(d) DB samples.

the rats by intraperitoneal injection with a combination anesthetic
containing medetomidine (0.15 mg∕kg BW), midazolam(2 mg∕kg BW), and butorphanol tartrate (5 mg∕kg BW) after
inducing anesthesia with the inhalation of isoflurane. Then, aburn 20 mm in diameter was made on each rat by immersing
its back in hot water for 10 s. The degree of the skin burnwas controlled by the temperature of the hot water: 70°C for
an SDB (n ¼ 10), 78°C for a DDB (n ¼ 10), and 98°C for a
DB (n ¼ 10). Figure 1 shows photographs of the skin surface
for different burn samples. Immediately after making the burn,saline solution (50 ml∕kg BW) was intraperitoneally injected
for resuscitation. The burned area of the skin was observed invivo under anesthesia by SHG microscopy within one hour
after the burn. Immediately after completing the experiments,
the rats were euthanized by intraperitoneal injection of sodium
pentobarbitone (75 mg∕kg BW). After the euthanasia, biopsy
samples were collected, fixed in formaldehyde, and embedded
in paraffin wax prior to sectioning. After sectioning at a thicknessof 10 μm, histological analysis of the sliced samples was performed using Masson’s trichrome (MT) staining.

2.2

Experimental Setup

Figure 2 shows the experimental setup used for our collagensensitive SHG microscopy. Since we used a reflective configuration normally employed for SHG microscopy, whose
details are given elsewhere,20–22 here we give only a brief
description of the setup. A 1250 nm mode-locked Cr:
Forsterite laser (Avesta Project Ltd., Moscow, RussianFederation, CrF-65P, pulse duration ¼ 90 fs, mean power ¼
200 mW, repetition rate ¼ 73 MHz) pumped by an ytterbium
fiber laser emitting at 1064 nm was used as a laser source.
Use of this laser wavelength, lying right in the middle ofthe optical window in biological tissue ranging from 800
to 1500 nm,23,24 leads to a drastic decrease in photodamage
caused by the laser light25,26 and a large enhancement of

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images by changing the sample position along the propagation
direction of the laser beam.

Fig. 2 Experimental setup. λ∕2: half-wave plate; PL: polarizer; λ∕4:quarter-wave plate; HS: harmonic separator; F: infrared-cut filter; L:lens; PMT: photon-counting-type photomultiplier tube; GMs: galvanometer mirrors; RL1 and RL2: relay lenses; OL: oil-immersion objective lens.

the probing depth.20 Circularly polarized laser light was focused
onto the sample with an objective lens [magnification ¼ 50,
NA ¼ 0.90, working distance ¼ 350 μm, and oil immersion]
after adjusting the power and polarization with a combinationof a half-wave plate (λ∕2), a polarizer (PL), and a quarterwave plate (λ∕4). A customized attachment ring (see inset of photograph in Fig. 2) was attached to the rat’s skin with double-sided
tape and was used to suppress the motion of the measurement site.
By changing the position of the attachment ring with a steppingmotor-driven translation stage, we can select the measured position inside the skin three-dimensionally. In a previous study, by
employing this type of ring, we successfully acquired SHG
microscopy images of human facial skin in vivo without movement artifacts.22 The backscattered component of the SHG
light generated in the sample was reflected by a harmonic separator (HS; reflected wavelength ¼ 625 nm) and selected by abandpass filter (F; transmission wavelength ¼ 625 nm). Then,the SHG light was detected by a photon-counting photomultiplierwith Peltier cooling (PMT; Hamamatsu, H7421-40) connected toa pulse counter.
A rapid, small-area, beam scanning system composed of galvanometer mirrors (GMs) and relay lenses (RL1 and RL2) provided SHG images of a 600 × 600 μm2 region, composed of
256 × 256 pixels, at a rate of 2 s∕image. However, this imaging
region is not always large enough for several applications of
SHG microscopy in the field of dermatology because the morphology and mechanical properties of the skin depend on themacroscopic properties of dermal collagen fibers rather thantheir microscopic properties. Furthermore, a skin burn maylack spatial uniformity, depending on the degree of damage.
Therefore, a slow, large-area, sample-scanning system wasconstructed with a combination of the skin attachment ringand the stepping-motor-driven translation stage. The sample
was divided into sixteen 600 × 600 μm2 imaged regions infour columns by four rows, and the imaged region was shiftedby moving the attachment ring with the translation stage. Each
imaged region was then scanned by using the GMs to obtain an
SHG image. The 16 SHG images obtained in this way werecombined to form a single large SHG image with a size of 2.4 ×
2.4 mm2 (total image acquisition time ¼ 64 s). This samplescanning system was also used to obtain depth-resolved SHG
Journal of Biomedical Optics

3

Results

3.1

Structural Differences of Dermal Collagen Fibers
Among Different Burns

We first performed depth-resolved SHG imaging for control,
SDB, DDB, and DB samples. To visualize structural differencesof the dermal collagen fibers at high image contrast, thepower of the laser light incident on the sample was adjusted
to 10 mW for the control, 15 mW for SDB, 25 mW for DDB,
and 40 mW for DB. The resulting series of depth-resolved SHG
images are shown in Fig. 3 (imaged region ¼ 600 × 600 μm2 ,interval of scanning depth ¼ 30 μm). Furthermore, a seriesof more precisely, depth-resolved SHG images was given inVideo 1 (imaged region ¼ 600 × 600 μm2 , interval of scanning
depth ¼ 10 μm). In this experiment, we defined the skin surface
as a depth of 0 μm. The skin surface location was determined by
confocal microscopy, which is a function included in the present
SHG microscopy system (not shown in Fig. 2). It is interestingthat the depths that the SHG light started to appear are different
among different degrees of burn: 140 μm for control, 120 μm for
SDB, 90 μm for DDB, and 40 μm for DB. This result indicatedthat the thickness of the epidermis was decreased due to the thermal shrink caused by the skin burn. If the probing depth of SHG
microscopy is determined based on the skin surface, the actual
probing depth of the dermis was different among the control,
SDB, DDB and DB. Therefore, it is important to investigate
depth dependency of the dermal collagen fiber for each burnsample. In the control sample, we observed that fine collagenfibers in the papillary dermis changed to thick collagen fibers
in the reticular dermis with increasing probing depth [seeFig. 3(a)]. On the other hand, depth-resolved SHG images of
the SDB, DDB, and DB samples indicated that each burn spread
uniformly over the whole depth of the dermis. In the SDB sample, the SHG vanishing patterns discussed later were overlaid on
the fiber structure of the dermal collagen [see Fig. 3(b)]. In the
DDB sample, the SHG vanishing patterns became finer thanthose in the SDB sample [see Fig. 3(c)]. Furthermore, thefibrous structures of the dermal collagen were almost entirely
lost and changed into amorphous structures. In the DB sample,
a little SHG light was observed only from aggregates of degenerated collagen [see Fig. 3(d)]. Thus, there was no depth dependency of the SHG vanishing pattern for each burn sample within
the probing depth range used in the present system.
We next performed large-area SHG imaging on the same
samples. The laser power incident on the samples was again
set to 10 mW for the control, 15 mW for SDB, 25 mW for
DDB, and 40 mW for DB. Figure 4 shows large-area SHG
images (image size ¼ 2.4 × 2.4 mm2 ) of these samples at a
depth of 180 μm from the skin surface, in which SHG intensity
is represented by a common gray-scale for all SHG imagesby considering the differences in the incident laser power
among samples. Imperfect matching of adjacent SHG images
at the boundary is mainly due to a slight discrepancy between
the imaged regions acquired using the GMs and the movementof the stepping motor stage. The imperfect matching will be
reduced in future works by using a method for image heterogeneity correction.27 Although we selected this probing
depthð¼ 180 μm from the skin surfaceÞ in order to obtain highcontrast SHG images for all samples, it should be considered

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Fig. 3 Depth-resolved SHG images of dermal collagen fiber at a depth from 10 to 280 μm in (a) control, (b) SDB, (c) DDB, and (d) DB samples. Incident
laser power was 10 mW for control, 15 mW for SDB, 25 mW for DDB, and 40 mW for DB samples. Image size ¼ 600 × 600 μm2 . A series of more
precisely depth-resolved SHG images was given in Video 1 (imaged region ¼ 600 × 600 μm2 , interval of scanning depth ¼ 10 μm, MOV 1.5 MB)[URL: http://dx.doi.org/10.1117/1.JBO.18.6.061231.1].

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that shrinking of the burned epidermis changes the actual probing depth of the dermis depending on the degree of the skin burnas discussed above. As a result, SHG images for the burned samples visualized the dermal collagen fiber shallower than that forthe control sample. However, since there was no depth dependency of the SHG vanishing pattern for each burn sample inFig. 3 and Video 1, we did not consider this effect here.
Large-area images in Fig. 4 clearly show the structural changesof the dermal collagen fibers caused by the burns. In the case of

the control in Fig. 4(a), the fibrous structure of the dermal collagen was clearly observed in the whole field of view. Also,
pores in the skin appeared as black circles due to the absenceof collagen fiber. The thermal burn largely modified the appearance of the dermal collagen fibers. In the SDB case in Fig. 4(b),many patterns in which the SHG light had vanished appeared on
the dermal collagen fibers. In the DDB case in Fig. 4(c), the sizeof the SHG vanishing pattern decreased further; in other words,
inhomogeneity in collagen areas increased further. Similar SHG
vanishing patterns have previously been confirmed in ex vivo
measurements of excised specimens.14–16 In the case of DB
in Fig. 4(d), the SHG light almost completely vanished, indicating that thermal denaturation completed the structural transitionof collage molecules from a non-centrosymmetric triple helix toa centrosymmetric random coil. The changes in the SHG images
from Fig. 4(b)–4(d) suggests that the SHG vanishing patternspread progressively, depending on the degree of skin burn.
This is consistent with the previous reports of measurements
ex vivo, too.14–16 Therefore, we can conclude that our SHG
microscopy correctly acquires the structural features in thermally denatured collagen in vivo. A detailed discussion of
the reason for the SHG vanishing patterns will be given in
the next subsection.

3.2

Histological Analysis with Masson’s Trichrome
Staining

Next, we performed histological analysis of laterally crosssectioned samples (thickness ¼ 10 μm) to investigate thereason for the signal vanishing pattern in the SHG images.
Haematoxylin and eosin stain is normally used for burn assessment.5 Here, however, since we want to clearly reveal the relation between the thermal denaturation of dermal collagen and
the SHG vanishing pattern, we used MT staining to dye thecollagen fibers blue and the cytoplasm red. Figure 5 shows
the histological visualization of (a) control, (b) SDB, (c) DDB,
and (d) DB samples.
We first paid attention to the red dye in the stained images.
Since this dye reveals erythrocyte and/or endothelial cells, thedistribution of the capillary vessels in the tissue was visualized.

Fig. 4 Large-area SHG images of dermal collagen fiber at a depth of
180 μm from the skin surface for (a) control, (b) SDB, (c) DDB, and(d) DB samples. SHG intensity is represented by the common grayscale for all SHG images by considering differences of the incident
laser power among samples. Image size ¼ 2.4 × 2.4 mm2 .
Journal of Biomedical Optics

Fig. 5 Histological images of excised tissue specimens(thickness ¼ 10 μm) with Masson’s trichrome staining: (a) control, (b)SDB, (c) DDB, and (d) DB samples. Image size ¼ 240 × 180 μm2 .

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There were no large differences in the capillary vessel distributions among Fig. 5(a)–5(c). For example, the numbers andsizes of the capillary vessels in the SDB and DDB samples[see Fig. 5(b) and 5(c)] did not change compared with thosein the control sample [see Fig. 5(a)], implying little structuralchange of blood vessels caused by SDB and DDB. However,
the image of the blood vessels in Fig. 5(d) changed considerably.
The amorphous structure shown in Fig. 5(d) indicated that cells
in capillary vessels were disrupted and then leaked out to thesurrounding collagen.
Here, we consider whether the SHG vanishing patterns are
due to the capillary vessels. One probable reason is that thermally damaged capillary vessels form a network of shadowsin the SHG image. Usually, the diameter of capillary vessels[several micrometers; see Fig. 5(b) and 5(c)] was much smaller
than that of the shadowed lines [several tens of micrometers; see
Fig. 4(b) and 4(c)]. However, thermal damage due to the burncauses water to leak from capillary vessels, which then causes
edema to appear around the capillary vessels. The coexistence ofthe capillary vessels and edema may increase the diameter ofthe shadowed lines, forming a network of shadowed lines in thelarge-area SHG image. However, the similar appearance of thecapillary vessels in Fig. 5(a)–5(c) cannot explain the fact that the
SHG vanishing pattern became finer and denser depending on
the degree of skin burn [see Fig. 4(b) and 4(c)] because the number of capillary vessels did not change from Fig. 5(a)–5(c).
Therefore, we concluded that the capillary vessels did not contribute to forming the SHG vanishing patterns.
Next, we focused on the distribution and structure of collagen fibers and bundles dyed in blue. As shown in the control
sample in Fig. 5(a), collagen bundles were distributed along
the skin surface. Furthermore, the collagen fibers were wellorganized and aligned in each bundle. Such a fibrous structure
was reflected at high image contrast in the optical-sectioning
SHG images in Figs. 3(a) and 4(a). The stained image for
the SDB sample in Fig. 5(b) was more blurred and less blue
than that in Fig. 5(a) in which individual bundles were clearlyvisualized as a bluish image. In particular, individual fibers ineach bundle seem to have been altered to another structure. In
the DDB sample in Fig. 5(c), the stained image became moreblurred and less blue. A series of these blurred images indicatesthat thermal shrinkage and/or aggregation of dermal collagenprogress at the level of fibers. The decrease in blueness wascaused by a gradual change in the microscopic structure froma triple helix (collagen molecule) to a random coil (gelatinmolecule). In the DB sample in Fig. 5(d), boundaries between
collagen bundles as well as collagen fibers almost completely
disappeared and showed a further decrease in blueness. This
image reflects the altered morphological appearance of the dermal collagen fibers and bundles, showing their widespread hyalinization. Therefore, SHG light almost completely disappearedin Figs. 3(d) and 4(d).
We now consider the reason for the signal vanishing patterns
in the large-area SHG images from the viewpoint of thermal
denaturation of dermal collagen. The results of MT stainingof SDB skin [see Fig. 5(b)] showed a partial distribution of
thickened collagen bundles (diameter ¼ several tens of micrometers) in the dermis compared with that of the control skin [seeFig. 5(a)], implying the coexistence of both molten and fibrous
structures of dermal collagen. The thickened collagen bundles
may be explained by the fact that thermal treatment breaks thehydrogen bonds that stabilize the native triple helical molecular
Journal of Biomedical Optics

structure of collagen, leading to an irreversible transformation ofcollagen molecules into random coils.28,29 We consider that such
thickened collagen bundles cause the SHG vanishing patterns
observed in Fig. 4(b). In the DDB skin in Fig. 5(c), the densityof thickened collagen bundles increased, indicating the
progressive replacement of the fibrous structure with a molten
amorphous structure. This result is consistent with the finerand denser distribution of SHG vanishing patterns shown in
Fig. 4(c). As for the DB skin, dermal collagen bundles showedmetachromatic staining, which indicates the degeneration of collagen bundles [see Fig. 5(d)]. Since the degenerated collagenbundles were composed of random coils of polypeptide chains,
SHG light disappeared due to their centrosymmetric structure,
as shown in Fig. 4(d).

3.3Depth Dependence of Mean SHG Intensity

In vivo SHG microscopy could qualitatively visualize structuralchanges in dermal collagen fibers caused by skin burns in theform of SHG vanishing patterns (see Fig. 4). Furthermore, aquantitative parameter for burn assessment may be obtained
from image analysis of SHG vanishing patterns. Recently,
image analysis based on Fourier transformation has beenused for SHG images in order to assess the structure of collagenfibers.22,30,31 However, the inhomogeneous structure of dermal
collagen fibers sometimes hinders image analysis. On theother hand, many previous papers have reported that SHG intensity rapidly decreases around the thermal denaturation temperature when the sample temperature is increased.11,14,17,18
However, only use of the absolute value for SHG light intensity
tends to be influenced by the conditions of the laser source andother factors, although it is related to the degree of thermal denaturation in dermal collagen. Here, we tried to extract a morereliable, quantitative parameter based on a simpler method.
We performed depth-resolved SHG imaging for control,
SDB, DDB, and DB samples when the incident laser power
was set to 10 mW for all burns. Then, we calculated themean value of SHG intensity across the entire image at eachprobing depth. After such the procedure was repeated to fiverats for the control, SDB, DDB, and DB samples (n ¼ 5 foreach burn), we calculated the mean and standard deviationof the SHG intensity with respect to the probing depth asshown in Fig. 6. In this figure, the skin surface was defined
as a probing depth of 0 μm. On the other hand, since the
SHG images started to be blurred deeper than 180 μm due toFig. 6 Dependence of the mean SHG intensity on the probing depth forcontrol, SDB, DDB, and DB samples. Incident laser power was set to
10 mW for each sample.

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the multiple scattering, we limited the range of the abscissa
below 180 μm. We consider that the reason why the mean
SHG intensities in all of the curves did not start from 0 isdue to the dark noise of the PMT and/or the backgroundnoise of the fundamental light and/or the SHG light comingfrom the sample. In the control sample, as the probing depth
increased, the SHG intensity increased, reaching its peak arounda depth of 130 μm, and then decreased. In the SDB sample, thepeak value of the mean SHG intensity was greatly decreased,
and its position was shifted to a deeper portion comparedwith the control sample. In the DDB sample, the peak valueof the mean SHG intensity was further decreased, whereas
the peak position was almost the same as that of the SDB sample. The mean SHG intensity for the DB sample was almost constant at the noise level of the detector, indicating the vanishing of
SHG light due to complete thermal denaturation of the collagenmolecules. In this way, the depth profile of the SHG lightsignificantly varied depending on the degree of the burn.
Let us consider the reason for the form of the depth profile.
First, SHG light starts to be generated when the focal spot moves
from the epidermis to the dermis, and then the generated SHG
light reaches a plateau when the focal spot probes a deeper portion in the dermis. Second, the laser light arriving at the focal
spot is attenuated due to scattering and absorption, depending on
the probing depth. Third, the backscattered SHG light from the
focal spot is attenuated in proportional to the probing depth until
reaching the skin surface. The balance of these three effects
determines the depth profile of the control in Fig. 6, where
the SHG light increased, reached the peak, and decreased.
Next, we consider the depth profile in the case of SDB,
DDB, or DB. Thermal denaturation of dermal collagen fiber
largely reduces the generation efficiency of SHG light, namely,
the second-order nonlinear-optical susceptibility χ ð2Þ, due tothe structural transition from a triple helix to a random coil.
Simultaneously, the burn changes the optical scattering andabsorption properties in the epidermis and dermis. These leadto the observed differences among the rising slopes shownin Fig. 6.
One interesting feature in these depth profiles is the difference of the depth, indicating the peak of mean SHG intensity,
among different burn samples. However, since the depths that
the SHG light started to appear were different among them
due to the shrink of the burned epidermis as shown in Fig. 3
and Video 1, this parameter may include the influence of the
shrunk epidermis. Another interesting feature is the differenceof the rising slope among them. To extract this rising slope as aquantitative parameter relating to the skin burn, we performed
the curve fitting analysis using an error function (erf) as followsIðxÞ ¼

I0x − xc1 þ erf
2
d


;

(1)

where I 0 is the maximum intensity of the SHG light, x is the
probing depth, xc is the depth as the SHG light indicates ahalf of I 0 , and d is a depth range of the rising slope. Before
performing the curve-fitting analysis, the background noise
signal was subtracted. Also, the data on the falling slope
after the peak of the SHG depth profile was eliminated in the
curve fitting analysis. We here defined a ratio of I 0 to d as arising slope parameter. Large I 0 ∕d means the steeply risingslope whereas small I 0 ∕d indicates gently rising slope.
Figure 7 shows a comparison of the rising slope parameter
Journal of Biomedical Optics

Fig. 7 Comparison of rising slope in SHG depth profile among control,
SDB, DDB, and DB samples.

I 0 ∕d among the control, SDB, DDB, and DB samples(n ¼ 5). The rising slope parameter I 0 ∕d decreased according
to the degree of the skin burn. A significant difference(p < 0.01) was confirmed between all sample groups except
between DDB and DB samples. The reason for no significant
difference between DDB and DB samples is mainly due tolarge error bar of DB caused by too weak SHG light.
However, DB will be easily distinguished from other burns
by existence of SHG light (see Fig. 6). These results imply apotential of this parameter for optical burn assessment.

4

Conclusion

We demonstrated in vivo SHG imaging of dermal collagen fibers
in skin burns using a rat burn model under anesthesia. Similar toprevious studies of ex vivo measurement of excised tissue specimens,14–16 the influence of thermal denaturation of collagenmolecules was visualized in the form of signal vanishing patterns in the SHG image. From a comparison of the SHG images
with MT-stained images, the SHG vanishing patterns werecaused by the coexistence of molten and fibrous structures ofdermal collagen fibers. Furthermore, since the depth profileof the mean SHG intensity across the entire SHG image significantly varied depending on the degree of the burn, we proposed
a quantitative parameter for burn assessment based on this SHG
depth profile. The demonstrated results show a high promise in
SHG microscopy for optical burn assessment in vivo.

Acknowledgments
The authors acknowledge Dr. Mamoru Hashimoto of Osaka
University and Dr. Shun-ichi Sato of the National Defense
Medical College, Japan, for fruitful discussion. This work
was supported by Grants-in-Aid for Scientific Research
Nos. 22300154, 23240069, 23650260, and 23300165 from
the Ministry of Education, Culture, Sports, Science, andTechnology of Japan.

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June 2013 • Vol. 18(6)