Contrast improvement in scattered light confocal imaging of skin birefringent structures by depolarization detection

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Contrast improvement in scattered lightconfocal imaging of skin birefringent structuresby depolarization detection

Babu Varghese*, Rieko Verhagen, Qiangqiang Tai, Altaf Hussain, Clemence Boudot,and Natallia Uzunbajakava

Care and Health Applications group, Philips Research Europe, High Tech Campus 34, 5656 AE Eindhoven, The Netherlands

Received 15 July 2011, revised 3 October 2011, accepted 4 October 2011Published online 21 October 2011

Key words: birefringence, hair optical properties, confocal microscopy, polarization dependent contrast

# 2011 by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Journal of

BIOPHOTONICS

Here we describe a method for enhancing the contrast inimaging skin birefringent structures. The method relieson polarization-dependent optical properties and is im-plemented using cross polarized confocal microscopy.The experimental data obtained using ex-vivo and in-vivo measurements on human scalp hairs and humanskin demonstrate a significant dependence of the changein polarization of light that interacted with the birefrin-gent hair on the orientation of the incident polarization.The polarization dependent contrast, defined as the ratioof intensity measured for different orientations of the in-cident polarization when observed using cross polarizedconfocal microscopy furthermore depends on the hairtype/degree of pigmentation and on the focusing depthinside the hair. No such dependence was observed forthe upper skin layers, including the stratum corneum andepidermis. We propose a new method for enhancing thecontrast between the skin and the birefringent hair bythe use of cross polarized confocal microscopy combinedwith the variation of the polarization of the incominglight. Potential applications of this method include imag-ing of hairs for assessing the efficacy of hair removalmethods and measurement of skin birefringence. The un-derestimation of the birefringence content resulting from

the orientation related effects associated with the use oflinearly polarized light for imaging tissues containingwavy birefringent structures could be minimized by thismethod.

CPCLSM images obtained in-vivo with the direction ofincident polarization at different angles to the orienta-tion of the hair axis.

* Corresponding author: e-mail: [email protected], Phone: +31 40 27 48054, Fax: +31 40 27 46321

J. Biophotonics 4, No. 11–12, 850–858 (2011) / DOI 10.1002/jbio.201100063

1. Introduction

Birefringence, an optical property that results fromstructure and composition of a substance or an ob-ject, can be used to identify structural orders in bothbiologic [1–5] and non-biologic materials [6–7]. Inparticular, measurement of the birefringence of bio-logical materials has the potential to provide infor-mation on dynamic changes in the tissue structure[4] and to eliminate the signal degradation and ar-tifacts commonly encountered when using opticalimaging modalities [2]. In the past decade the inter-est in studying the propagation of polarized light inbirefringent media has significantly increased [8–9].Polarization-based theoretical methods, such as time-resolved [10] and Mie theory-based [11] Monte Carloalgorithms, and experimental techniques, such aspolarization-sensitive optical coherence tomography(PS-OCT) [1–4] and cross-polarized confocal laserscanning microscopy (CPCLSM) [12–13] have ad-vanced the understanding of birefringence in biologi-cal tissues. These theoretical and experimental toolswere used to characterize a change in polarization oflight due to birefringent properties of biological tis-sues. A comprehensive summary about the use ofpolarized light for imaging of tissues has been pub-lished by Jacques et al. [14]. These reports describerandomization of polarization as polarized light pro-pagates through biological tissues. The intensity of thelight detected from the subsurface structures can beemphasized or rejected using an analyzing polarizerwith different polarization orientations [15–17]. Jac-ques et al. demonstrated that the image based on po-larization ratio of two images acquired through ananalyzing linear polarizer oriented parallel and per-pendicular to the polarization of illumination canemphasize the imaging of superficial skin structures[14]. Examples of applications of polarization-basedmethods include burn depth estimation [4], early de-tection of carious lesions, evaluation of the efficacy oflaser therapy [1], and elimination of artifacts foundin conventional OCT images [2].

Confocal laser scanning microscopy is a reliableand robust technique to provide non-invasive real-time optical imaging of biological tissue at high re-solution and contrast, approaching histology, thegolden standard for tissue inspection [12–13]. Thedetection of cross-polarized component of light canbe used to enhance the intensity of light reflected orscattered from the deeper tissue layers, while reject-ing direct reflectance from its surface [13].

Recently, we reported on the polarization-depen-dent total attenuation coefficient of the structurescomprising human scalp hairs. In particular, we de-monstrated the polarization dependence of the totalattenuation coefficient of the hair cortex, which ori-ginates due to the birefringence of keratin fibers[18]. In addition, we have shown a difference in the

polarization-dependent attenuation coefficient fordifferent hair pigmentation.

In this manuscript, we confirm that the intensity oflight reflected or scattered from the hair indeedstrongly depends on the orientation of the polariza-tion with respect to the hair. Furthermore, we demon-strate that this polarization-dependent contrast varieswith the focusing depth inside the hair and with thehair type/pigmentation. Using this information wepropose a new method based on cross polarized con-focal microscopy to enhance the contrast betweenthe skin and hairs and we investigate its behaviorwith focusing depth and hair types. The method isfirst exemplified with ex-vivo measurements on hu-man scalp hairs and then the improved hair-skincontrast is demonstrated using in-vivo measurementson different hair and skin types.

2. Materials and methods

2.1 Confocal microscope

The polarization dependent total attenuation coeffi-cient of the cortex and the medulla of different hairtypes are measured at 830 nm using collimated trans-mittance measurements in a confocal set-up (Fig-ure 1a). This method and setup are described inmore details elsewhere [18].

2.2 Cross-polarized confocal microscope

Hair and skin imaging experiments were carriedout using a cross-polarized confocal laser scanningmicroscope (VivaScope 1000, Lucid Inc., Rochester,NY) in reflection mode with wavelength 830 nm(Figure 1b). The laser illumination beam wasscanned by polygon and galvanometer mirrors andrelayed into the water immersion microscope objec-tive that focuses the beam into the sample. A 30�numerical aperture (NA) 0.90 water immersion mi-croscope objective corrected for the cover glass wasused to focus the laser light onto the sample. Themaximum field of view was 450� 400 mm. Light re-flected from the tissue was collected by the objec-tive, descanned, and focused onto a pinhole (50 mm).Light passed through the pinhole illuminated a sili-con avalanche photodiode. The detected optical in-tensity was digitized by an 8-bit frame grabber andsaved as a bitmap image using Labview. The spatialresolution is 0.5–1.0 mm in the lateral and 3–5 mm inthe axial dimension. A zero-order half-lambda waveplate (Edmund Optics, USA) is introduced beforethe microscope objective to rotate the polarization

J. Biophotonics 4, No. 11–12 (2011) 851

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of the incoming light with respect to the optical axisof the birefringent sample.

2.3 Ex-vivo and in-vivo measurements

The lock of human hairs (DeMeo Brothers, Inc.) fora given hair type comes from one individual. Fordifferent hair types, the lock of hairs is obtainedfrom different individuals. Three European gray (hairdiameter 90 mm) and Indian black (hair diameter90 mm) scalp hairs were used for collimated transmit-tance measurements in a confocal set-up. For eachhair, the collimated transmittance was measured atthree positions along the hair shaft, and then thestandard deviation and mean were calculated.

Cross-polarized confocal images of non-medul-lated European gray scalp and Indian black scalphairs were measured at multiple depths for differentorientations of the incident polarization with respectto the optical axis of the hair. In-vivo experimentswere performed with CPCLSM with the goal ofunderstanding whether the skin and birefringentsample, such as a hair, show different behavior withrespect to the orientation of the polarization of theincoming light. To confirm that the upper layers ofskin do not show a strong dependence on polariza-

tion, we have also obtained confocal images of theskin at an adjacent position where no hairs are visible,for different incident polarization directions. Depthresolved in-vivo measurements were performed ondifferent hair and skin types (European Blond, gray,brown, black and Indian black hairs) to study thedependence of contrast on focusing depth and hairtypes.

2.4 Data analysis

The image analysis is performed in Matlab. In eachimage, three areas corresponding to 25� 25 mm2

(35� 30 pixels) were randomly selected for cal-culating the average intensity in the selected area.The data were normalized with respect to the opticalpower used during the experiment. The opticalpower was measured using a power meter after themicroscope objective without the sample. The opticalpower will be different at different depths due to ab-sorption and scattering. We have normalized the in-tensity detected at different depths with the sameoptical power that we measured after the microscopeobjective. The mean and the standard deviation ofthe intensity detected for three samples were thencalculated. The polarization dependent contrast is

Laser815 nm

Power meter

Sample

Filter

l/2 wave plate

DiaphragmDiaphragm

Objec�ve20x 0.4 Pinhole

Cuve�e filled with cinnamate

Lens 1 Lens 2

Laser diode834 nm

APD

Sample

PBS

Objec�ve 30x 0.9

Polygon mirror

Pinhole

Lens 6 Lens 2

Lens 3

Lens system 1

fast x-scan

slow y-scan Mirror

Mirror

Diaphragm

l/2 wave plate

Detector

Lens 4 Lens 5

a)

b)

Figure 1 (online color at:www.biophotonics-journal.org)Schematic diagram of the confo-cal setup used for polarization de-pendent attenuation coefficientmeasurements (a) and cross po-larized confocal laser scanningmicroscope used for ex-vivo andin-vivo measurements (b).

B. Varghese et al.: Contrast improvement in scattered light confocal imaging852

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BIOPHOTONICS

# 2011 by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.biophotonics-journal.org

defined as the ratio of the normalized intensity de-tected when the incident polarization was at 45� tothat at 0 or 90�. The hair-skin contrast is defined asthe ratio of the intensity detected from the hair tothat from the skin. By measuring the images withtwo different polarization states and subtractingthese images, the enhanced contrast is calculated asfollows:

Enhanced contrast ¼ ðIP1H � IP2

H Þ=ðIP1S � IP2

S Þ ð1Þwhere IH and IS are the intensity of the light de-tected from hair and skin for two incident polariza-tion orientations (P1 and P2) respectively.

3. Results

3.1 Collimated transmittance measurements

The results of the measurements of the total attenua-tion coefficient of the cortex and medulla of Euro-pean blond, gray, brown, black and Indian blackhuman scalp hair are presented in Table 1. The po-larization dependent attenuation coefficient dependson the hair types and show significant difference be-tween the attenuation coefficient of the cortex for thepolarizations parallel and perpendicular to the longhair axis. No such dependence on light polarizationwas found for the total attenuation coefficient of themedulla of hairs. The total attenuation coefficient in-creases with increase in hair pigmentation.

3.2 Polarization dependent contrastand enhancement of hair-skin contrast

Cross polarized confocal images of non-medullatedEuropean gray and Indian black scalp hairs meas-ured at a depth of 30 mm with the orientation of theincident polarization at 45� and 0� with respect tothe optical axis of the hair are shown in Figure 2. Wehave used different optical power for measuringthese images and the images shown in Figure 2 arenot normalized to the optical power. As it can beseen from Figure 2, the intensity of the light re-flected or scattered from the hair strongly dependson the orientation of the incident polarization. Inparticular, the intensity reached the minimum and

Table 1 Polarization dependent total attenuation coeffi-cient of the cortex and the medulla of different scalp hairsat 830 nm wavelength (the data are given in mm�1). EII

and E? represents the orientation of the incident polariza-tion parallel and perpendicular to the long hair axis.

Polarizationstate

Cortex Medulla

EII E? EII E?

Europeanblond

0.25� 0.1 0.22� 0.1 79.8� 23 75� 21

Europeangray

0.83� 0.3 0.35� 0.1 114.6� 5 102.9� 17

Europeanbrown

1.09� 0.1 0.53� 0.2 150.9� 54 134� 40

Europeanblack

7.7� 0.7 6.2� 0.6 65.8� 12 52.3� 4.5

Indianblack

40.8� 3.4 37� 3.8 117.2� 42 116.8� 38

Figure 2 Cross polarized confocalimages of non-medullated Euro-pean gray (a, b) and Indian black(c, d) scalp hairs measured withthe orientation of the incident po-larization at 45� (a, c) and 0� (b,d) with respect to the optical axisof the hair.




maximum when the orientation of the incident polari-zation is at 0 or 90� and at 45� with respect to theorientation of the hair, respectively (Figure 3), show-ing periodic behavior (Figure 4). If one of the opticalaxes coincides with the hair itself, the maximal bire-fringence effect is expected when the polarization ofthe incoming light is at 45� with respect to the hairaxis. The results of this experiment suggest that oneof the hair optical axes is along the hair itself. Thedepth resolved polarization dependent contrast,measured for non-medullated European gray andIndian black scalp hair is shown in Figure 5. As thefocusing depth increases, the contrast, defined as theratio of the normalized intensity detected when theincident polarization was at 45� to that at 0 or 90�,increases and after the first 10–30 mm depth, thecontrast approaches 12 for the European gray hairand 9 for the Indian black hair (Figure 5). The de-tected intensity was very low for focusing depthslarger than 40 mm inside the pigmented hair and inparticular when the orientation of the hair was at 0�

or 90�.Cross polarized confocal images of the skin (In-

dian black hair, skin type V) measured with differentincident polarization orientations at a focusing depth

of 30 mm are presented in Figure 6a, b. The intensitydetected from the birefringent hair varies from max-imum to minimum. However, we did not observe thisdistinct behavior for the superficial skin layers;namely, the variation in the intensity detected fromthe superficial layers of the skin was maximum 4%(Figure 6c, d).

When the contrast is expressed as the ratio of thenormalized intensity detected from the hair to thatfrom the skin, the contrast between the hair and theskin is 1.6. When we measure the images with twopolarization states and subtract these images, thecontrast between the hair and skin is increased to32. Thus the contrast is enhanced by a factor of 20 ata focusing depth of 10 mm and this factor dependson the focusing depth inside the hair and skin (Fig-ure 7). The hair-skin contrast measured for two or-ientations of the incident polarization was averagedand is shown in Figure 7 for comparison. The errorbars indicate the standard deviation of three meas-urements. The contrast enhancement is more pro-minent in the first 10–50 mm and degrades at largefocusing depths. Additional in vivo measurementsperformed on different hair-skin types confirm thatthe contrast enhancement factor depends on the hairtypes and focusing depth inside the hair and skin(Figure 8).

Figure 3 Cross polarized confocalimages of non-medullated Euro-pean gray scalp hair measured withthe orientation of the incident po-larization at 0�, 45� and 90� withrespect to the optical axis of thehair.

Figure 4 The periodic variation in the intensity of light de-tected in the cross polarized confocal images of a non-medullated European gray hair as the orientation of theincident polarization is varied with respect to the orienta-tion of the hair axis.

Figure 5 The depth resolved polarization dependent con-trast measured for non-medullated European gray and In-dian black scalp hair.


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4. Discussion

In this manuscript, we have presented a method forenhancing the contrast in imaging skin birefringentstructures. The method is based on discriminatingthe polarization change induced by a birefringentmaterial from the non-birefringent background byusing the combination of CPCLSM and differentialpolarization detection.

We have investigated the polarization dependentoptical properties of the cortex and medulla of dif-ferent hair types using collimated transmittancemeasurements in a confocal set-up. In general, thepolarization dependence of attenuation coefficient is

less prominent for medulla because of the resultingscrambling of polarization as the polarized light pro-pagates through the highly scattering medulla. Thepolarization dependent optical properties of the haircortex originate from the longitudinal alignment ofkeratin fibers and preferential absorption by melaningranules. In heavily pigmented hairs, the polarizationdependence could be masked by scattering by themelanin granules [19]. As a result, this effect shouldbe prominent for lightly pigmented hairs.

The contrast in the images depends on the orienta-tion of birefringence and birefringence value (d), de-fined as the difference in refractive index between theextraordinary and ordinary rays traveling throughthe anisotropic material. The hair consists of a thin

Figure 6 CPCLSM images ob-tained in-vivo with the directionof incident polarization at differ-ent angles to the orientation ofthe hair axis. Skin location with(a), (b) and without hair (c), (d)imaged with the orientation ofthe incident polarization at 45�

(a), (c) and 0� (b), (d).

Figure 7 The dependence of average and enhanced hair –skin contrast on different focusing depths inside the hairand skin.

Figure 8 The contrast enhancement factor measured fordifferent hair-skin types as a function of focusing depth in-side the hair and skin.




outermost layer, the cuticle, a porous central part,the medulla, and the cortex. We explain the observ-ed different behavior of hair and skin on the basis ofbirefringence. The cortex of the human hair is bire-fringent, where the ordinary and extraordinary refrac-tive indices, n0 and nE, are 1.541 and 1.548, respec-tively and the corresponding value of birefringence,d, is equal to 0.007. This anisotropy of the refractiveindex is due to the intrinsic and form birefringence[20], resulting both from the a-helix structure ofkeratin fibers and the refractive index differencebetween the keratin fibers and the surroundingamorphous matrices, respectively [21]. If the anglebetween the hair axis and the incoming light polari-zation is 0� or 90�, the light traversed through thehair experiences no birefringence and consequentlyno change in polarization (apart from that inducedby light reflectance at the hair interface, which isdescribed by the Fresnel equations). Hence, theamount of light originating from a hair detected in across-polarized mode will be minimal. But if the po-larization of the incoming light is 45� with respect tothe hair axis, light traversing through a hair will ex-perience birefringence, which results in a change inpolarization. The change in polarization due to bire-fringence, expressed as a phase retardation betweenthe orthogonal components of the light wave is theproduct of birefringence and optical path length in-side the hair and thus increases with focusing depth(optical path length ¼ 2 � focusing depth) inside thehair. However, for large path lengths inside the hair,the contrast increases to 10 and decreases for largefocusing depth inside the hair. This may be attribut-ed to the randomization of polarization and the de-crease in the detected intensity for large optical pathlengths inside the hair.

In contrast to hair, the upper skin layers, such asstratum corneum and epidermis, are not birefringentor the birefringence is small. Therefore, the intensityof light detected from the skin does not show a de-pendence on the orientation of the incoming polari-zation. Even though the stratum corneum may havesome birefringent properties, it is very thin. And theepidermis is not birefringent. Thus, the intensity oflight detected from the birefringent hair has a highdependency on the polarization state of the incom-ing light, while no such dependency is expected fromskin. In general, the proposed method can be usedfor imaging hair shafts with high contrast. Howeverwhen used for identifying hair follicles, the contrastwill be reduced.

Other methods, such as confocal auto fluorescenceimaging, based on the detection of auto fluorescenceof keratin [21] and melanin [22] can also be utilizedfor imaging hairs. On practice this can be realized byusing one- and two photon-excited auto fluorescenceprocesses [23]. However, in two-photon-excited autofluorescence measurements we have observed ab-

sence of two-photon-excited auto fluorescence inten-sity from the medulla due to high light scattering,light re-absorption and thermal damage in dark hairsand limited contrast between hair and skin.

The dependence of degree of polarization ofbackscattered light from birefringent medium on dif-ferent incident polarizations was reported by Wanget al., based on Monte Carlo algorithms [10, 11]. Fora linearly birefringent turbid medium, they showedthe periodic variations in the degree of polarizationof backscattered light versus the orientations of thepolarizations [11]. For birefringence, d > 0.001, largedifference in the degree of polarization of backscat-tered light was obtained with the orientation of thepolarization parallel, perpendicular and at 45� to theslow axis of the birefringent material. For birefrin-gence, d < 0.0005, the difference in the degree ofpolarization measured for different orientations wassmall and this implies that the contrast enhancementby this method will not be significant for materialswith low birefringence.

In the previous report Jacques et al. demonstrateddifferential polarization imaging between two imageshaving orthogonal states of polarization for imagingskin with polarized light [14]. Compared to thismethod, we have implemented differential polariza-tion imaging in a confocal set-up in which polariza-tion difference was 45� separated, resulting in greatercontrast in imaging of hair birefringence. The possi-bility of changing the incident polarization to anydesired orientation offers the feasibility to image tis-sues containing wavy birefringent structures with en-hanced contrast.

In general, change in the degree of polarizationcan be utilized to enhance the contrast and thus en-ables to provide information on the birefringentstate and to image tissue birefringence in biologicalsamples with an improved specificity. When tissuescontaining wavy collagen fibers are viewed with line-arly polarized light, some portion of each fiber willappear dark if the optic axis is aligned parallel to theorientation of the incident polarization [24, 25]. Theproposed method has the ability to enhance histo-logical assessment of tissue by providing additionalinsights into the composition and structure of col-lagen. Further research is required for the character-ization of complicated heterogeneous birefringenttissue structures, having different birefringent valueand orientation at different layers.

5. Conclusion

To summarize, we have shown that cross-polarizedconfocal laser scanning microscopy when combinedwith variation of the polarization of the incominglight can be used to distinguish between birefringent


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and non-birefringent objects. Furthermore, the depth-resolved polarization-dependent measurements showthat the polarization-dependent contrast depends onthe hair type and on the focusing depth inside thehair. We demonstrated that this information can beused to enhance the contrast between the birefrin-gent hair and the non-birefringent skin background.Based on the experimental results obtained using exvivo scalp hairs and in vivo measurements, we de-monstrate that the proposed method enhances thecontrast between the skin and birefringent hair by afactor of 20 and shows its dependence on hair typesand different focusing depths inside the hair andskin.

In many biomedical applications the presence ofhair and its movement is a critical issue affecting themeasured signal and its stability. These applicationsinclude functional near-infrared spectroscopy imag-ing [27], skin hydration measurements using corne-ometer [28], and non-invasive optical diagnosis, inparticular with fiber optic probes [18], and lasertreatment of skin diseases. The signal degradationand artifacts caused by the presence of hairs andtheir movements when using these imaging modali-ties can be surmounted by detecting and avoidingtheir presence in the measurement volume using theproposed technique. Furthermore, detecting the pre-sence of hairs and measurement of stubble lengthsare important for assessing the efficacy of hair re-moval methods. In general, the presented method isof great interest in measuring birefringence in biolo-gical samples having measurable polarization proper-ties (e.g. collagen) where the detected light level islow and thus the contrast is low and needs enhance-ment.

Babu Varghese received theMaster of Science degree inPhysics from Calicut Uni-versity, in 1997, the Masterof Technology degree inOptoelectronics and LaserTechnology from CochinUniversity, in 2000. In 2007,he obtained Ph.D. degreein Biomedical Optics fromUniversity of Twente, theNetherlands. Currently, he isa Senior Scientist in the De-

partment of Care and Health Applications at PhilipsResearch Laboratories, the Netherlands.

Rieko Verhagen receivedthe Master of Science de-gree in Technical Physicsfrom Delft University, theNetherlands in 1997 and aPh.D. degree (highest hon-ours) in Chemical Physicsfrom Radboud UniversityNijmegen, in 2002. Cur-rently, he is a Senior Scien-tist in the Department ofCare and Health Applica-

tions at Philips Research Laboratories, the Nether-lands.

Qiangqiang Tai received theBachelor of Science degreein Optics from ShandongUniversity, China in 2008and Master of Science de-gree in Photonics fromRoyal Institute of Technol-ogy, Sweden in 2011. Cur-rently, he is a Graduate stu-dent in the Department ofApplied and EngineeringPhysics, Cornell University,U.S.

Altaf Hussain received theMaster of Science degree inPhotonics from Royal Insti-tute of Technology, Swedenin 2010. Currently, he is doingPh.D in the Biomedical Pho-tonics Imaging Group, Uni-versity of Twente, the Neth-erlands.

Clemence Boudot receivedPolytechnic Diploma in Phy-sics from Universite JosephFourier in France in 2007and Masters degree in Ma-terials Sciences and Funda-mental Physics from INSAToulouse, France in 2011.




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Natallia Uzunbajakava re-ceived the M.S. and Ph.D.degrees in Applied Physicsfrom the University ofTwente, Enschede, TheNetherlands. In 2004, shejoined the Department ofBiomedical Photonics, andlater moved to the Depart-ment Care and Health Ap-plications at Philips Re-

search Laboratories, Eindhoven, The Netherlands.Currently, in her role as a Senior Scientist, she focuseson application oriented research in the fields of biophy-sics, tissue optics, and non-invasive diagnostics.


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Contrast improvement in scattered light confocal imaging of skin birefringent structures by depolarization detection