application of laser spectroscopic methods for in vivo diagnostics in dermatology

754 Laser Phys. Lett. 4, No. 10, 754–760 (2007) / DOI 10.1002/lapl.200710049

Abstract: The importance of dermatologic non-invasive imag-ing techniques has increased over the last decades. Technologicaladvancements have led to the development of various scanningtools, enabling in vivo examination of living human skin. All pro-vide a preservation of the tissue’s physical structure whilst beingstudied in its native state. Different modalities are currently beingused to investigate the skin tissue. Although many of these scan-ning instruments are still undergoing research, promising imag-ing techniques, such as high-resolution ultrasonography, opticalcoherence tomography, magnetic resonance imaging and spec-troscopic methods, may yet find a role in dermatologic diagnosisand disease monitoring. In this article, the authors demonstratetheir own results and review the influence of laser spectroscopicmethods as non-invasive diagnostic tools in dermatology.

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Fluorescence image obtained 5 min after application of a dye

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Application of laser spectroscopic methods for in vivodiagnostics in dermatologyL.E. Meyer and J. Lademann ∗

Center of Experimental and Applied Physiology, Department of Dermatology and Allergology, Charite - Universitatsmedizin Berlin,Chariteplatz 1, 10117 Berlin, Germany

Received: 26 April 2007, Accepted: 30 April 2007Published online: 9 May 2007

Key words: laser spectroscopy; confocal microscopy; biological and medical application

PACS: 42.62.Fi, 87.64.Tt, 42.62.Be

1. Introduction

Imaging the surface of the human body to detect and dis-tinguish skin disorders from benign structures is a routineprocess in the medical field of dermatology. New advancesin technology of imaging instruments and computeriza-tion have led to a variety of new dermatologic imagingdevices. They support, or even surpass, the capability oftraditional visible inspection by the human eye. The aimsof in vivo skin imaging devices are to study normal skinas well as pathological processes, and to monitor the samebody site non-invasively over a specific time interval along

with treatment. Furthermore, they offer new informationabout the living skin tissue due to special features, e.g.,x-times magnification, highlighting dyes, deep scan, etc.Digital processing of the obtained images enables reliablestorage and retrieval of the data, as well as a comfortableautomated computerized analysis of different quantitativeskin parameter [1,2].

Of all these techniques, in the last years, laser spectro-scopic methods have achieved substantial improvementsin the imaging of dermal tissue in vivo. Especially, thecombination with the optical confocal scanning system al-

∗ Corresponding author: e-mail: [email protected]

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Laser Phys. Lett. 4, No. 10 (2007) 755

lows visualization of surface and subsurface detail in liv-ing human skin with resolution similar to those of lightmicroscopy. Currently, the confocal laser scanning mi-croscopy (LSM) is the only real-time modality offering theability to perform optical sectioning from deep inside thedermal sample at such a resolution [3,4].

The first confocal laser scanning microscope was de-veloped by Marvin Minsky for studying neuronal net-works in the living brain in 1955 [5]. In the past years, thetechnique of LSM has been improved significantly. There-fore, nowadays, more and more dermatologic laser micro-scopic systems are commercially available on the openmarket. Their design has changed; huge optical imagingapparatus is either significantly reduced in size or is fibre-based with handheld scanning devices, allowing a simplein vivo application and evaluation of any region of the body[6–9]. Altogether, the modern laser microscopes are nowmore user-friendly and flexible at a lower price and with ahigh, significant resolution. Nevertheless, the general op-tical working mechanism has always remained approxi-mately the same.

The basic confocal-principle involves focusing a laserlight onto a small spot within the dermal tissue. A recur-ring light signal out of the focus point is collected simul-taneously and used to create a confocal image. The opticaldesign ensures that only the returning light directly fromthe focal point is detected, whilst prevention of any scat-tered and reflected light from out-of-focus planes increasesthe imaging contrast. A deep scan can be performed eas-ily by moving the light focus deeper into the tissue. As aresult, different cell layers can be observed, the skin archi-tecture can be studied without taking a destructive biopsy.

The high-resolution confocal images contain informa-tion about the condition of the epidermis, as well as of theupper parts of the underlying dermis. The different epider-mal layers (Stratum corneum, Stratum granulosum, Stra-tum spinosum, and Stratum basale) can be observed anddistinguished by their typical depth location, cell size andshape. In contrast to conventional skin histology, wherevertical images of the skin samples are obtained, confocalLSM provides sectioning of thin horizontal tissue planes.The sampling plane can be adjusted and positioned be-low the skin surface to offer sub-surface evaluation. Alto-gether, confocal imaging permits real-time scan sequenceswith images in microscopic resolution observed in hori-zontal en face view.

There are three types of confocal spectroscopic meth-ods in use: the reflectance, fluorescence and the Ramanspectroscopy [1,2]. Depending on the laser source, de-tection system and the usage of a contrast dye, differentdermatologic in vivo laser scanning microscopes are nowcommercially available.

Generally, illuminated laser light is variably absorbedand reflected by the various skin components called chro-mophores. Once absorbed, some emit radiation after awhile, these are termed fluorophores. A spectroscope sepa-rates the returned light into different wavelengths to createan individual picture.

The reflectance mode is based on differences in theabsorption and scattering properties of the various tissuemicrostructures. The laser beam is reflected irregularly bythe heterogeneous dermal chromophores. Only backscat-tered signals are captured for visualization. Generally, thehigher the differences in the refractive index of skin struc-tures, the stronger the contrast of the images. In particular,melanin and keratin have high refractive indices, produc-ing a bright contrast in the reflectance mode of LSM.

In the fluorescence mode, either endogenous fluo-rophores or applied exogenous fluorescent dyes have to bepresent for scanning a defined skin area. Prior knowledgeof the characteristics of fluorophores is therefore required.A laser source at an appropriate wavelength is used to se-lectively excite the fluorophores. A fluorescence emissionis detected and exploited to create an imaging contrast.Subsequently, the distribution of fluorosphores is analyzedby the fluorescence mode of LSM.

Raman spectroscopic CLSM is based on the detectionof Raman spectra in the focal plane. When monochro-matic light is illuminated on the skin, the returning lightcontains several new wavelengths. This is called Ramanscattering and is caused by tissue molecules or topicallyapplied substances. Molecular bonds demonstrate specificspectral signatures. Compared to fluorescence and reflec-tion LSM, Raman microscopy does not deliver an image ofthe morphological structure but it rather provides chemicalinformation with regard to the tissue.

Common commercially available confocal laser scan-ning microscopes being used for investigations are theStratum R© System (OptiScan, Ltd., Melbourne, Australia),the near-infrared VivaScope R© (Lucid, Enc., Henrietta, NJ,USA) and the Raman laser scanning microscope producedby the company River Diagnostics (Rotterdam, Nether-lands).

In the Stratum R© system, a single line argon ion laserwith a wavelength at 488 nm is used for scanning. The skinarea under investigation is 250×250 µm2. Skin structuresup to a depth of 200 µm can be analyzed. The Stratum R©

operates in the fluorescent and reflection modes [10]. Weprefer the fluorescent microscopy in combination with dif-ferent contrast dyes.

The VivaScope R© 1500 is a reflectance confocal LSMworking with a near-infrared laser at a wavelength of830 nm. Skin structures can be examined up to a depth of250–300 µm, the single test field of view is 500×500 µm2.

The Raman microscope run by the company RiverDiagnostics is based on an Argon-ion pumped titanium-sapphire laser, which irradiates in the near-infrared spec-trum at an approximate wavelength of 850 nm.

These days, diverse publications proclaim accurate re-sults and great effectiveness of the reflectance mode inthe histometric analysis of different skin structures andannexes, as well as distinction between the healthy andpathologic aspect of human skin in diagnostical matter[3,11]. In contrast, fluorescence and Raman spectroscopiclaser scanning measurements are mostly used in cosmet-ics, skin physiology and pharmacology for the analysis of

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756 L.E. Meyer and J. Lademann: Application of laser spectroscopic methods

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Figure 1 LSM is an ideal in vivo optical technique for skin tis-sue. Received images are in real-time enabling a view on the cel-lular structure. However, confocal images of the different epi-dermal cell layers (here: St. spinosum) differ in reflectance andfluorescent mode: In the fluorescence mode (a) bright shiningpapillae, embedding darker blood vessels (a, star) are surroundedby keratinocytes. Melanin-containing basal cells are slightly dark(a, arrows), whereas in the reflectance mode (b) they appear asbright dots (b, arrows). Here, the papillae are grainy grey, alsoembedding capillary structure (b, star)

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Figure 2 Analysis of skin annexes like hairs (arrows) and hairfollicles (stars) is easily performed in the fluorescence mode (a)and in the reflectance mode (b)

the distribution and the penetration process of topically ap-plied substances [12–20]. Only limited reports exist in thefield of clinical dermatology. In our research laboratoriesevery endeavour has been made to break down the obso-lete borders between the traditional fields of application.All laser microscopic imaging devices show strength andweakness in the scanning and interpretation of human skin.We aspire to expand the implementation of spectroscopicmethods for in vivo diagnosis and real time therapeuticalmonitoring, in the hope to promote their promising role inthe future of dermatology.

2. Laser spectroscopic findings in normalhuman skin

To distinguish and interpret images from skin disease, inthe first place, one must be familiar with the laser micro-scopic appearance of normal human skin. For this reason,

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Figure 3 Fluorescence images obtained 5 and 20 min after appli-cation of a dye; immediately after application the dye is locatedat the intercellular space (a); after 20 min, the dye has penetratedinto the cells staining the nuclei (b)

several studies have focused on healthy skin analysis pre-senting outstanding results for all spectroscopic modali-ties. Briefly, the reflectance and fluorescent modes allowin vivo cross-sectional morphologic images of the skinand consequently picture the cutaneous histoarchitecture[4,7,21], whereas the confocal Raman spectroscopy ob-tains information about the molecular composition of theskin tissue; e.g., such as hydration, mapping the concen-tration profiles of water in the epidermis [22,23].

Differences in the reflectance and fluorescence confo-cal LSM exist predominately in the preparation protocol,in the maximum imaging depth and in the gain of con-trast extraction. The reflectance mode demonstrates natu-rally occurring skin microstructures, whereas the fluores-cent mode normally represents the distribution pattern ofan exogenous contrast agent. Both methods allow reliablehigh-resolution images demonstrating the cellular archi-tecture of the epidermis and upper dermis including skinannexes like hairs and sweat glands (Figs. 1 and 2). Com-parison to routine histology has been drawn successfully[4,21], measuring of different body sites has been per-formed convincingly [11,24]. Nevertheless, advantages ofthe reflectance confocal LSM in the near-IR spectrum in-volve deeper scan values, totally non-invasive usage, and astrong contrast to melanin-containing cells, promising anintensified application for the investigation of pigmentedlesions [25,26].

For optimal scanning, in the fluorescence mode the ad-ministration of a dye is necessary in the majority of thecases. It can be topically applied for examination of the su-perficial cell layers and/or injected intradermally into theskin tissue for subsurface investigation [21,24,26]. Whenimaging is performed continuously for several minutes af-ter the application of the dye, a diffusion from extra- tointracellular can be observed (Fig. 3). Five minutes afterapplication, the dye was found around the epidermal cells(keratinocytes), 20 minutes later the nuclei were stainedand labelled. The pharmacokinetic process of the con-trast dye provides additional functional information onthe state of the tissue and characterizes the permeability-

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properties of the cell membranes. Various diseases andtherapeutic procedures influence kinetic properties signif-icantly. Only fluorescence LSM realizes such a functionalinvestigation with a microscopic resolution in vivo. Fromone’s own experience, the hydrophilic fluorescein and thelipophilic curcumin are highly suitable fluorescent dyes forcell membrane evaluation.

Raman spectroscopy has been widely used to studyliving human skin, obtaining depth-resolved informationof in vivo concentration profiles of constituents of naturalmoisturizing factor, of sweat and of water in the stratumcorneum [22,23,28].

An intelligent technical linkage between differentspectrometric modes to overcome individual limitationsreceiving additional information and predications by onlyone scan-sequence has been proclaimed recently (re-flectance/fluorescence LSM [26]) or already conducted(reflectance/Raman spectroscopic LSM [8]).

3. Analysis of the abnormal skin structurefor diagnoses and therapy control

In vivo spectroscopic devices have been used to investigateand characterize a number of clinically relevant prolifer-ative and inflammatory skin disorders. In particular, thereflectance confocal LSM appears to be a useful devicefor studying the development of a skin disease over timeas well as response to treatment [3]. Numerous prosperstudies had been carried out demonstrating its high diag-nostic potential in dermatology. Especially, two significantmulticenter-studies presenting a high sensitivity and speci-ficity for non-invasive diagnosis of basal cell carcinomaand melanoma emphasize the supremacy of reflectanceLSM along all in vivo diagnostic tools [29,30]. Neverthe-less, it is possible to envisage different skin disorders usingthe fluorescent mode of confocal microscopy [24,27,31].In addition to the reflectance mode, the dynamic distribu-tion process and the clearance of the contrast dye providefurther information on the state of the tissue. However, theclinical implementation of the fluorescence mode mightbe restricted in the case of scanning deeper dermis or byinjecting a fluorescent dye into a malignancy for imagecontrast. Even though our early observations require fur-ther investigation and validation, with time, in vivo fluores-cence spectroscopic imaging may also prove to be usefulfor diagnosis.

The lowest diagnostical experience exists with Ramanspectroscopy. Clinical application has been investigated instudies of vitiligo, atopic and psoriatic skin, and basal cellcarcinoma [32–34]. But the full developmental potentialhas not yet been exhausted; currently, new publications arestreaming onto the market to ascertain the future role in theclinical field.

In the following, we would like to concentrate on a fewskin disorders prioritising our own research results.

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Figure 4 Fluorescence microscopic images of yeasts obtainedfrom healthy scalp skin (a) in comparison to their pathologicform causing pityriasis versicolor (b). On the one hand, ovalyeasts with dark nuclei colonize the normal skin surface (a, ar-row), whereas in pityriasis versicolor bright shining hyphae couldbe recognized as pathologic feature (b, arrow)

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Figure 5 Psoriasis: twisted and dilated capillary loops embed-ded in oedematous and elongated papillary dermis transport tum-bling blood cells; a pathological skin phenomenon perceived inthe fluorescence (a) and reflectance mode (b) of LSM. Sim-ilar appearance, but different contrast: in both, papillae shinebrightly (stars), however, the blood vessels in the fluorescencemode are more dye-stained and contain dark blood cells(a, ar-rows), whereas, in the reflectance mode, capillaries appear dark-grey with moving bright blood cells (b, arrows)

3.1. Analysis of mycosis by confocal lasermicroscopy

Standard diagnostic procedures for mycosis include light-microscopic analyses of scrapings, fungal cultivations andskin biopsies. Therefore, traditional diagnoses can be verytime-consuming and painful. The application of fluores-cence and reflectance laser scanning microscopy short-ens and simplifies the diagnostic procedure consider-ably [27,35,36]. It allows real-time imaging of fugal mi-crostructures on the human skin in vivo. The distinction ofnormal fungal skin flora and mycosis is provided twofold:Mycotic skin lesions show resultant inflammatory infil-trate and changes in the fungal morphology. Both featurescould be observed in vivo by using confocal spectroscopicmethods. For example, Fig. 4 compares a yeast colony ofthe ubiquitous genus Malassezia forming part of the nor-




mal cutaneous microflora to their pathological conditioncausing pityriasis versicolor.

3.2. Psoriatic skin lesions observed by laserspectroscopic methods

Psoriasis is a chronic skin disorder that effects nearly 2%of the population, worldwide. Psoriatic skin lesions arecharacterized histologically by parakeratosis, scaly hyper-keratosis, thinning of the suprapapillary plates, traffickingof inflammatory cells, and elongation of the rete ridges.Altogether, laser spectroscopic methods provide a reliabletechnique for evaluating the major microscopic features invivo. Fluorescence and reflectance LSM are suitable toolsfor observation of the psoriatic changes in epidermis andthe upper dermis (Fig. 5) [3,24,27,37]. Furthermore, Ra-man spectroscopic measurements allow molecular predi-cations of the superficial stratum corneum, giving excel-lent information such as changes in hydration [33]. Up tonow, laser spectroscopy was only used for basic research;an expansion into the diagnostical field and as a tool fortreatment control can be visualized.

3.3. Application of laser scanning microscopyin diagnoses and therapy control ofnon-melanomous skin cancer

Non-melanomous skin cancer represents the most com-mon malignant neoplasia in human skin. Hereby, basal cellcarcinoma (BCC) is responsible for approximately 65%of all cutaneous malignancies, and spinal cell carcinoma(SCC) has a proportion of about 20%. This explains thegreat demand for a reliable diagnostic tool without theneed of an invasive biopsy and time-consuming histolog-ical preparation of the biopsy specimen. Early detectionwould shorten the progressive course and minimize the in-cidental costs. Hereby, LSM may facilitate the in vivo di-agnostic procedure. It represents a promising approach fornon-invasive diagnosis and therapy control in skin cancertreatment.

As mentioned above, several published studies confirmthe vast diagnostical potential of reflectance confocal LSMin skin tumors [28,38,39]. They found that the confocalfeatures correlate well with the stained section of a biopsy.Furthermore, BCC-patients were treated with a topical im-mune response modifier; the presence, as well as the clear-ance of the BCC was analyzed by reflectance microscopicmeasurements [40,41]. Another study looks at the abilityfor accurate differentiation of actinic keratoses (AK) vs.SCC, reaching the conclusion that the reflectance LSM iscurrently restricted because of the limited depth visualiza-tion [42].

Raman LSM was used to distinguish basal cell carci-noma from the surrounding tissue by identifying different

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Figure 6 Changes of morphological structures in the case ofBCC (a, fluorescence LSM) and SCC (b, reflectance LSM). Aluminous, superficial blood vessel (a, arrow), and loss of the nor-mal epidermal pattern is evident for BCC. Parakeratosis with nu-clear retention in the corneocytes (b, arrows) was seen in mosttypes of skin cancer and is slightly unspecific. In this case, thepatient was suffering from SCC

chemical composition in healthy tissue and basal cell car-cinoma [34]. Furthermore, currently ongoing studies areconcentrating on skin malignancies.

Our research group demonstrated that fluorescencelaser scanning microscopy could also be used successfullyfor the diagnosis and therapy control with Imiquimod forbasal cell carcinomas [27,31]. We defined in vivo histo-logical features for BCC, SCC and AK in the fluores-cent mode and compared our results with other widelyaccepted imaging techniques. A number of histologicalfindings: BCC, SCC and AK show hyperkeratosis as wellas parakeratosis. Horizontal, elongated vascular loops arecharacteristic in the case of BCC. Pleomorphic nuclei canbe detected in the case of the AK and SCC, whilst BCCshow predominately elongated and polarized cell nuclei.The differences in morphology can be used as features todistinguish between basal cell carcinoma, spinal cell car-cinoma and actinic keratosis, using fluorescence LSM.

Typical examples of non-melanomous skin tumors arepresented in Fig. 6.

4. Summary

Significant advances have been made in the last decadesin regard to direct laser spectroscopic imaging of the hu-man skin. No longer is the dermatologist limited to inva-sive biopsy for microscopic investigation of skin disorders.Depending on the spectroscopic modality, different assess-ments and predications on the skin condition are possible.

The fluorescence LSM is widely established in thefield of penetration research and distribution studies oftopically applied substances labelled with a contrast dye.We could demonstrate that it might be a valuable tool forimaging skin morphology. Although it is still being usedmostly in research settings, fluorescent LSM shows con-siderable promise as a diagnostical tool in dermatology.



However, the necessity of a dye represents the main lim-itation for this method: Firstly, all dye-labelling of med-ical skin-agents is a complicated process, and further-more, dye injections are not always desired, as in ma-lignant skin lesions. However this weakness could gainin strength soon. The development of new contrast dyesstaining specific microstructures would expand its role sig-nificantly in a clinical setting. Preliminary in vivo studiesusing fluorescein-labelled antibodies to detect melanomahave demonstrated promising results in the mice model[43]; validation on the human skin is still awaiting investi-gation.

LSM measurement in the reflectance mode does nothave limitations caused by the administration of a dye.Therefore, it is totally non-invasive and furthermore itshows deeper scan values working with a near-IR laser.The application of the reflectance LSM is strongly de-termined for imaging skin morphology. Numerous stud-ies ascertain a major impact on the traditional practice ofdermatology, in the future. Epidermal components, suchas melanin provide a strong contrast. Accordingly, it willbe a promising tool in the investigation of pigmented skinlesions and for the management of different skin tumors.Nevertheless, the imaging contrast is less compared to thefluorescence LSM measurements. Furthermore, in the ab-sence of a contrast dye, there is only a limited potential toextract additional information from the tissue by stainingspecific structures or interpretation of the dye’s pharma-cokinetic processes.

The Raman LSM is generally the first choice for thedetection of chemical compounds in the skin [44,45]. Oneof the main applications of Raman LSM is the analysis ofthe water distribution in the stratum corneum and deeperskin layers. For diagnostical purposes, the combination ofRaman spectroscopy with other spectrometric modalitiesin only one imaging tool seems to be a promising ap-proach. It enables information on molecular compositionin relation to skin architecture, giving additional predica-tions on the state of the tissue.

Finally, the considerable developments and improve-ments in LSM in the light source, computer technologyand optical system show new possibilities in the fieldsof dermatological research and diagnosis. The confocalspectroscopic systems have become smaller, cheaper andachieve a higher resolution. The non-invasive character ofthese methods guarantees an increased use for the diagno-sis and management of skin disease in the future.

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application of laser spectroscopic methods for in vivo diagnostics in dermatology

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