
	Version PDF

Multiphoton microscopy: a new paradigm in dermatological imaging

European Journal of Dermatology. Volume 17, Number 5, 361-6, September-October 2007, Review article

DOI : 10.1684/ejd.2007.0232

Summary

Author(s) : Sung-Jan Lin, Shiou-Hwa Jee, Chen-Yuan Dong , Institute of Biomedical Engineering, National Taiwan University, No. 1, Sec. 1, Jen-Ai Road, Taipei 100, Taiwan, Department of Dermatology, National Taiwan University Hospital and National, Taiwan University College of Medicine, No. 7, Chung-Shan South Road, Taipei 100, Taiwan, Department of Physics, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei 106, Taiwan.

Summary : In recent years, the non-linear optical imaging technique of multiphoton microscopy has gained significant popularity in biomedical imaging. Since optical imaging can provide detailed morphological information of biological structures, multiphoton microscopy holds great promise as a potential clinical diagnostic tool of dermatological conditions. In this review, we will begin by discussing the basic principles of multiphoton microscopy, including the process of fluorescence and second harmonic generation. In addition, we will present the dermatological applications of multiphoton imaging, including the diagnosis of basal cell carcinoma and the evaluation of skin photoaging. We also describe applications of this technique to transcutaneous drug delivery, melanoma imaging, skin diseases associated with extracellular matrix alterations and cutaneous microvascular observation. Finally, we will discuss the additional issues that need to be resolved before multiphoton imaging can become a major diagnostic tool in clinical dermatology.

Keywords : basal cell carcinoma, photoaging, collagen, elastic fiber, multiphoton, second harmonic generation

Pictures

ARTICLE

Auteur(s) : Sung-Jan Lin^1,2, Shiou-Hwa Jee², Chen-Yuan Dong³

¹Institute of Biomedical Engineering, National Taiwan University, No. 1, Sec. 1, Jen-Ai Road, Taipei 100, Taiwan
²Department of Dermatology, National Taiwan University Hospital and National, Taiwan University College of Medicine, No. 7, Chung-Shan South Road, Taipei 100, Taiwan
³Department of Physics, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei 106, Taiwan

accepté le 25 Avril 2007

As the largest organ of the human body, the skin is responsible for a plethora of important physiological functions. The regulation of body temperature, prevention of body fluid loss, and protection against environmental harm are maintained by this vital organ. Since the skin is located on the exterior of the body, it is most accessible to imaging techniques for diagnostic purposes. Naturally, a major goal in dermatological diagnosis is the real time diagnosis and monitoring of physiological conditions. However, as in many areas of medicine, the field of dermatology continues to rely heavily on histological procedures in the diagnosis of pathological conditions. While such procedures are effective in diagnosing many pathological conditions, they have major drawbacks in that they are invasive in nature and that the fixation and processing procedures render the study of dermatological dynamic processes impossible. With advances in technical innovations, novel modalities are now been applied to the field of dermatology [1]. In particular, non-invasive or minimally invasive imaging techniques that enable real time observation of skin physiological conditions are invaluable in overcoming the drawbacks of histological examinations [2-4].The development of non-invasive imaging techniques, such as reflected confocal microscopy and optical coherence tomography (OCT), have helped to improve the clinical diagnosis of skin conditions [2, 3]. However, in recent years, multiphoton microscopy has emerged as an alternative imaging modality that holds great potential for dermatological applications in the clinics. The precursor of multiphoton imaging is two-photon fluorescence microscopy (TPFM) invented by Webb’s group in Cornell in 1990 [5]. In TPFM, a fluorescent molecule excitable by the single photon absorption of a UV or visible photon is excited by the simultaneous absorption of two less energetic, near infrared (IR) photons. A number of significant advantages associated with two-photon excitation (TPE) render this technique the preferred choice in many biomedical imaging applications. First, the high incident photon flux required for efficient TPE limits the excitation to the focal volume. As a result, the microscopic imaging achieved by scanning the excitation spot produces images with excellent axial depth discrimination without the use of confocal apertures.Furthermore, the near IR photons used for sample excitation are absorbed and scattered less by tissues, thus resulting in improved sample imaging depths. Finally, since the near IR wavelength is spectrally separated from the emission, which lies mostly in the visible range, effective detection of sample luminescence can be easily achieved [5, 6]. In addition to TPE, the non-linear polarization effect of second harmonic generation (SHG) is also of tremendous value in biomedical imaging. In general, researchers have demonstrated that SHG microscopy can be used to image a variety of biological tissues such as collagen, muscle, and microtubule [7-9]. The more specific applications of SHG microscopy include the study of the thermal denaturation of corneas and tendons [10, 11] and the prediction of heat-induced collagen shrinkage [12].SHG is a polarization effect. In general, P_i the polarization of a material is related to the external electric field E by the relation:In materials lacking an inversion symmetry, the non-vanishing second order susceptibility χ_ijk can lead to the generation of second harmonic signals at half the wavelength of the incident excitation wavelength. Tissues such as collagen and skeletal muscle have been demonstrated to be effective second harmonic generators. Since fluorescence emission is typically at longer wavelengths than the SHG signal, the two signals can be easily separated and be used for image contrast enhancement [7, 9].Over the past decade, multiphoton imaging has found a variety of applications in dermatology. In vivo imaging of human skin was achieved almost ten years ago [13, 14]. A detailed morphological comparison between multiphoton and histological images in ex vivo mouse skin shows that a variety of epithelial and dermal structures can be identified by multiphoton imaging [15]. More recently, multiphoton imaging of human skin and mucosa has been achieved [16], and time-resolved techniques have been applied to mapping the structures of human skin [17]. In addition, advances in higher harmonic imaging and spectral analysis have contributed as additional modalities in dermatological imaging [18-20]. In addition to morphological imaging, multiphoton imaging has been demonstrated to be effective in addressing specific issues in dermatology. For example, lifetime imaging has been shown to be able to characterize pH gradient within the stratum corneum [21]. The injury to skin by toxicological agents and the subsequent recovery process can also be monitored [22], and understanding wound healing is another area in which mulitphoton imaging has been successfully applied [23, 24]. The combination of multiphoton autofluorescence (AF) and SHG imaging has also been shown to be effective in the qualitative and quantitative characterization of basal cell carcinoma (BCC) and photoaging [25, 26]. Additional applications of multiphoton imaging include the characterization of skin thermal damage and the UV generation of reactive oxygen species in ex vivo human skin [27-29]. Understanding the role of spherical aberration and developing high resolution imaging apparatus for the mouse animal model are also among the advances made in multiphoton dermatological microscopy [30, 31].

Multiphoton instrumentation

Most multiphoton microscopes use the point-scanning approach in image acquisition. In our laboratory, we use a diode-pumped solid state (DPSS), titanium-sapphire (ti-sa) laser system for excitation (figure 1). The broad lasing bandwidth of the ti-sa laser allows the selection of excitation wavelengths in the 700-1,000 nm range. For imaging purposes, a modified commercial microscope is used. The excitation source is guided into the imaging microscope by a pair of galvanometer-driven mirrors (Model 6220, Cambridge Technology, Cambridge, MA). The entrance port of the microscope is modified to accommodate a pair of beam expanding lenses. Proper beam expansion is necessary to ensure overfilling of the objective’s back aperture, which results in optimal focusing. The expanded beam is reflected into the back aperture of the focusing objective by a primary dichroic and the luminescence generated at the focal volume is collected in the epi-illuminated geometry. Collected fluorescence and/or SHG signals pass through the primary dichroic and are processed by additional filters before being detected by the detectors of single-photon counting photomultiplier tubes. If three-dimensional imaging is preferred, a piezoelectric positioning objective or a sample translation stage can be used for axial imaging. Since large area images are often needed to appreciate the global tissue structure, the sample translation stage can also be used to position the specimen after each optical scan. The large area multiphoton image can then be assembled from individually acquired small area images.

To demonstrate the usefulness of multiphoton imaging in characterizing physiological and pathological conditions, we present the results of multiphoton images of normal skin and the multiphoton analysis of BCC and different degrees of skin photoaging.

Multiphoton imaging of normal skin

In multiphoton imaging, the detailed morphology of epithelial and dermal structures can be obtained without fixation and staining procedures (figure 2). The stratum corneum is autofluorescent and the hexagonal morphology of single corneocytes can be clearly demonstrated (figure 2A). When the image is taken deeper, the serial transition from the granular layer (figure 2B), spinous layer (figure 2C) and basal layer (figure 2D) cells can be easily identified. Because nuclei are deficient in AF signals, the nuclei appear as dark regions in the cytoplasm. At the dermal-epidermal junction (figure 2E), SHG signals from dermal collagen start to appear. Collagen is an efficient SHG source, while elastic fibers produce intense AF. Separation of SHG and AF signals help to distinguish these two important structures in the dermis. As shown in figure 2F, fine autofluorescent elastic fibers penetrating in the network of coarse collagen fibers can be clearly demonstrated without immunofluorescent processing (figure 2F).

Multiphoton characterization of basal cell carcinoma

BCC is the most prevalent skin cancer in the world [32]. The cancer is most often seen in the head and neck area where the level of accumulative sunlight exposure is the highest. It has been shown that metalloproteinase activity is up-regulated in BCC and may account for its local invasive and destructive behavior [33]. Surgical removal is recommended in most patients. However, the margin of cancer can not be securely determined during the operation and the local recurrence rate is greater than 10% in cases undergoing simple excision. Further, wide excision can also be a problem in certain anatomical sites, such as the eyelids and nose, where preservation of the adjacent uninvolved tissue is of great functional and cosmetic significance.

For secure removal of the entire cancer tissue, the technique of Mohs’ micrographic surgery is used [34]. This technique can detect cancer margins with almost 100% certainty and the recurrence rate is only 1% [34]. However, Mohs’ micrographic surgery is time-consuming and requires the simultaneous cooperation of the specimen-processing technician and the surgeon.

To evaluate the potential of multiphoton imaging in replacing Mohs’ surgery, we applied multiphoton microscopy in discriminating BCC from the normal dermis [25]. In the multiphoton images, BCC is characterized by clumps of autofluorescent cells in the dermis (figure 3A). The cancer cells have a relatively large nuclei and a higher nucleus to cytoplasm ratio (figure 3A, yellow insert). The unique feature of BCC, peripheral palisading, also can be observed. These features can also be seen in the histological images (figure 3B). Another prominent feature revealed by multiphoton imaging is the alteration of extracellular matrix in the BCC stroma. In uninvolved dermis, SHG signal by collagen accounts for much of the optical signal (figure 3A , red insert). However, in the stroma within and surrounding the cancer clumps, SHG diminishes while AF signals increase (figure 3A, purple insert). The decrease of SHG indicates that the collagen molecule packing has been disrupted and can reflect an up-regulated collagenase activity in cancer tissue.

Further, we performed quantitative analysis in the nonlinear optical property of cancer stroma in an attempt to discriminate BCC from the normal dermal stroma (figure 3C). In our approach, the multiphoton fluorescence (MF) pixel number (a) and SHG pixel number (b) within selected regions of interest were determined and the MF to SHG index (MFSI) is defined to be (a–b)/(a+b) [25]. According to this definition, MFSI will reach the maximum value of 1 when only MF signals are present and MFSI will decrease as collagen content increases. MFSI’s lower limit of -1 occurs when MF is absent and only the SHG signal is present. The MFSI is highest within the tumor masses (mean MFSI = 0.93) where the contribution of the fluorescent signal comes from the cytoplasm (figure 3C). In normal dermal stroma, the MFSI is at its lowest, reflecting the higher content of intact collagen. In cancer stroma, the MFSI is significantly higher than that in normal dermal stroma. The decrease of SHG and increase of MF in cancer stroma accounts for the higher MFSI.

Alteration of dermal structures associated with photoaging

In addition to BCC, pathological conditions due to skin photoaging are also among the critical issues in dermatological care. In skin photoaging, exposure to UV radiation from sunlight plays a major role in causing the associated pathological conditions [35]. Various surgical procedures and topical treatments are used to rejuvenate photoaged skin, aimed at reversing the epidermal and dermal alterations associated with photoaging. Due to the lack of a reliable and noninvasive methodology, evaluation of the treatment response usually depends on subjective clinical assessment. Nonetheless, photoaging can be evaluated by the histological changes in the extracellular matrix components of the dermis. In cases of severely photoaged skin, an increase in disorganized elastic fibers can be found in the superficial dermis (solar elastosis) [35]. As we demonstrated in BCC imaging, multiphoton microscopy is effective in monitoring the alteration in the extracellular matrix at high resolution. When the facial skin specimens from patients of different ages were investigated by multiphoton imaging, we found age-dependent changes in the dermal extracellular matrix [26]. In the case of a 20-year-old individual, AF and SHG signals are interspersed in the papillary dermis (figure 4A, A3). In the case of a 40-year-old individual, the AF signals increase and SHG signals decrease (figure 4A, B3). In the case of 70-year-old individual, SHG signals can only be detected in a very thin zone right beneath the basement membrane and large amounts of fluorescent elastic fibers (solar elastosis) can be found in the dermis (figure 4A, C3). The trend of decreasing SHG signals and increasing AF signals is well-correlated with the histological findings of the decrease of collagen fibers and an increase of elastic fibers with increasing age (figure 4A, panels of H&E and elastic stains). When the non-linear optical properties of the patients are quantitatively analyzed, we found that a radiometric approach based on the SHG and AF signals is a good indicator of the degree of photoaging (figure 4B). Similar to the MFSI ratio used to characterize the BCC specimens, the SAAID (SHG to AF aging index of dermis) ratio, defined as (b–a)/(b+a), can be used for analyzing differently photoaged skin. In severely photoaged patients, the SAAID ratio is expected to approach –1 while younger patients are expected to have higher SAAID ratios. Indeed, figure 4B shows this trend and supports the fact that analysis of AF and SHG signals can be used for the qualitative and quantitative characterization of differently photoaged skin.

After our proof-of-principle demonstration of this technique ex vivo, Koehler et al. conducted an in vivo study in 18 individuals from 21 to 84-years-old for the determination of intrinsic skin aging. They found a negative correlation between the age of the individual and the measured SAAID [36]. Hence, this technique is capable of analyzing the changes of skin extracellular matrix associated with aging and can be developed into a diagnostic tool for the minimally invasive evaluation of effects of various treatments aimed at skin rejuvenation.

Additional dermatological applications of mulitphoton imaging

There have been numerous reports applying multiphoton microscopy for drug delivery applications [37-44]. Due to the ease in accessing the fluorescence emission, the penetration pathways of model drugs in the stratum corneum can be visualized and quantified [37-41, 44]. Combination with polarization techniques enables multiphoton imaging to analyze the molecular alignment of intercellular lipids associated with the treatment of penetration enhancers [44, 45]. In addition, the dermal penetration of drugs can be visualized [43]. Finally, multiphoton imaging in vivo also allows the real time investigation of transcutaneous drug delivery in humans [42].

Melanin is an effective generator of multiphoton fluorescence [17]. In addition to the discrimination of basal cell carcinoma from normal dermis [25], benign pigmented lesions and malignant melanoma can also be highlighted by their intrinsic AF [17]. Clinical trials are needed to prove whether multiphoton microscopy can be a useful tool for the detection of early melanomas and differentiating benign pigmented lesions from malignant melanomas.

Since multiphoton imaging is able to differentiate extracellular collagen from elastic fibers, it has great potential for application in skin diseases featured by changes of dermal collagen fibers and elastic fibers, including pseudoxanthoma elasticum, elastolysis, morphea, scleroderma, and GVHD.

In addition to visualizing cells and extracellular matrix, dermal vessels can also be imaged by multiphoton imaging. By intravascular injection of fluorescent agents, the morphology of dermal vessels and even the blood cells can be observed [31]. This approach can be used to analyze the angiogenesis of skin cancers and the microvascular hemodynamics associated with various skin disorders in animal models.

Conclusion

Since its introduction, multiphoton microscopy has contributed to a number of areas in biology and medicine. The non-invasive nature, reduced photodamage, and enhanced penetration depths make multiphoton microscopy the preferred imaging modality in many areas of biomedical applications. In dermatology, applications such as the imaging and identification of BCC and skin photoaging may be extended into clinical practice. For this approach to become widely applicable in clinical settings, a number of issues remained to be resolved. First, clinical applications would need the availability of high speed (real-time) scanning systems in which disturbances associated with patient movements can be minimized. Furthermore, an ex vivo, and eventually in vivo image database of the various types of pathological skin conditions need to be established and analyzed in order to understand the effectiveness of multiphoton imaging in discriminating diseased states of the skin from normal ones. Finally, the potential health hazards associated with the multiphoton excitation processes need to be well characterized. With additional development, multiphoton imaging has potential to become widely applied in the clinical diagnosis of skin pathological conditions in vivo.

Acknowledgements

The authors want to express their gratitude for the financial support from National Research Program for Genomic Medicine and National Science Council, Taiwan (NSC94-3112-B-002-015-Y and NSC95-3112-B-002-019 for S. J. L. and S. H. J.; NSC94-3112-B-002-015-Y for C.Y.D.). Conflict of Interest: none.

References

1 Serup J. High Tech dermatology. Eur J Dermatol 2007; 17: 178-80.

2 Rajadhyaksha M, Grossman M, Esterowitz D, Webb RH. In-Vivo Confocal Scanning Laser Microscopy of Human Skin - Melanin Provides Strong Contrast. J Invest Dermatol 1995; 104: 946-52.

3 Welzel J, Lankenau E, Birngruber R, Engelhardt R. Optical coherence tomography of the human skin. J Am Acad Dermatol 1997; 37: 958-63.

4 Leveque JL, Xhauflaire-Uhoda E, Pierard GE. Skin capacitance imaging, a new technique for investigating the skin surface. Eur J Dermatol 2006; 16: 500-6.

5 Denk W, Strickler JH, Webb WW. 2-photon laser scanning fluorescence microscopy. Science 1990; 248: 73-6.

6 So PTC, Dong CY, Masters BR, Berland KM. Two-photon excitation fluorescence microscopy. Annu Rev Biomed Eng 2000; 2: 399-429.

7 Zoumi A, Yeh A, Tromberg BJ. Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence. Proc Natl Acad Sci USA 2002; 99: 11014-9.

8 Campagnola PJ, Loew LM. Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms. Nat Biotechnol 2003; 21: 1356-60.

9 Zipfel WR, Williams RM, Christie R, Nikitin AY, Hyman BT, Webb WW. Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation. Proc Natl Acad Sci USA 2003; 100: 7075-80.

10 Tan HY, Teng SW, Lo W, Lin WC, Lin SJ, Jee SH, Dong CY. Characterizing the thermally induced structural changes to intact porcine eye, part 1: second harmonic generation imaging of cornea stroma. J Biomed Opt 2005; 10; (054019).

11 Lin SJ, Hsiao CY, Sun Y, Lo W, Lin WC, Jan GJ, Jee SH, Dong CY. Monitoring the thermally induced structural transitions of collagen by use of second harmonic generation microscopy. Opt Lett 2005; 30: 622-4.

12 Lin SJ, Lo W, Tan HY, Chan JY, Chen WL, Wang SH, Sun Y, Lin WC, Chen JS, Hsu CJ, Yu HS, Jee SH, Dong CY. Prediction of heat-induced collagen shrinkage by use of second harmonic generation microscopy. J Biomed Opt 2006; 11; (034020).

13 Masters BR, So PTC, Gratton E. Multiphoton excitation fluorescence microscopy and spectroscopy of in vivo human skin. Biophys J 1997; 72: 2405-12.

14 Masters BR, So PTC, Gratton E. Optical biopsy of in vivo human skin: Multi-photon excitation microscopy. Lasers Med Sci 1998; 13: 196-203.

15 So PTC, Kim H, Kochevar IE. Two-photon deep tissue ex vivo imaging of mouse dermal and subcutaneous structures. Opt Express 1998; 3: 339-50.

16 Malone JC, Hood AF, Conley T, Nurnberger J, Baldridge LA, Clendenon JL, et al. Three-dimensional imaging of human skin and mucosa by two-photon laser scanning microscopy. J Cutan Pathol 2002; 29: 453-8.

17 König K, Riemann I. High-resolution multiphoton tomography of human skin with subcellular spatial resolution and picosecond time resolution. J Biomed Opt 2003; 8: 432-9.

18 Sun CK, Chen CC, Chu SW, Tsai TH, Chen YC, Lin BL. Multiharmonic-generation biopsy of skin. Opt Lett 2003; 28: 2488-90.

19 Palero JA, de Bruijn HS, van der Ploeg-van den Heuvel A, Sterenborg HJCM, Gerritsen HC. In vivo nonlinear spectral imaging in mouse skin. Opt Express 2006; 14: 4395-402.

20 Pena AM, Strupler M, Boulesteix T, Schanne-Klein MC. Spectroscopic analysis of keratin endogenous signal for skin multiphoton microscopy. Opt Express 2005; 13: 6268-74.

21 Hanson KM, Behne MJ, Barry NP, Mauro TM, Gratton E, Clegg RM. Two-photon fluorescence lifetime imaging of the skin stratum corneum pH gradient. Biophys J 2002; 83: 1682-90.

22 Werrlein RJ, Madren-Whalley JS. Multiphoton microscopy: an optical approach to understanding and resolving sulfur mustard lesions. J Biomed Opt 2003; 8: 396-409.

23 Navarro FA, So PTC, Nirmalan R, Kropf N, Sakaguchi F, Park CS, et al. Two-photon confocal microscopy: A nondestructive method for studying wound healing. Plast Reconstr Surg 2004; 114: 121-8.

24 Yeh AT, Kao BS, Jung WG, Chen ZP, Nelson JS, Tromberg BJ. Imaging wound healing using optical coherence tomography and multiphoton microscopy in an in vitro skin-equivalent tissue model. J Biomed Opt 2004; 9: 248-53.

25 Lin SJ, Jee SH, Kuo CJ, Wu RJ, Lin WC, Chen JS, Liao YH, Hsu CJ, Tsai TF, Chen YF, Dong CY. Discrimination of basal cell carcinoma from normal dermal stroma by quantitative multiphoton imaging. Opt Lett 2006; 31: 2756-8.

26 Lin SJ, Wu RJ, Tan HY, Lo W, Lin WC, Young TH, et al. Evaluating cutaneous photoaging by use of multiphoton fluorescence and second-harmonic generation microscopy. Opt Lett 2005; 30: 2275-7.

27 Masters BR, So PTC, Buehler C, Barry N, Sutin JD, Mantulin WW, et al. Mitigating thermal mechanical damage potential during two-photon dermal imaging. J Biomed Opt 2004; 9: 1265-70.

28 Lin MG, Yang TL, Chiang CT, Kao HC, Lee JN, Lo W, Jee SH, Chen YF, Dong CY, Lin SJ. Evaluation of dermal thermal damage by multiphoton autofluroescence and second harmonic generation (SHG) microscopy. J Biomed Opt 2006; 11; (064006).

29 Hanson KM, Clegg RM. Observation and quantification of ultraviolet-induced reactive oxygen species in ex vivo human skin. Photochem Photobiol 2002; 76: 57-63.

30 Lo W, Sun Y, Lin SJ, Jee SH, Dong CY. Spherical aberration correction in multiphoton fluorescence imaging using objective correction collar. J Biomed Opt 2005; 10; (034006).

31 Li FC, Wang CC, Lin SJ, Jee SH, Lo W, Dong CY. Dorsal skin fold chamber for high resolution multiphoton imaging. Opt Quantum Electron 2005; 37: 1439-45.

32 Rubin AI, Chen EH, Ratner D. Basal-cell carcinoma. N Engl J Med 2005; 24(353): 2262-9.

33 Yucel T, Mutnal A, Fay K, Fligiel SE, Wang T, Johnson T, Baker SR, Varani J. Matrix metalloproteinase expression in basal cell carcinoma: relationship between enzyme profile and collagen fragmentation pattern. Exp Mol Pathol 2005; 79: 151-60.

34 Rowe DE, Carroll RJ, Day Jr. CL. Long-term recurrence rates in previously untreated (primary) basal cell carcinoma: implications for patient follow-up. J Dermatol Surg Oncol 1989; 15: 315-28.

35 Yarr M, Gilchrest B. In: Freedberg IM, Eiser AZ, Wolff K, Austen KF, Goldsmith LA, Katz SI, eds. Fitzpatrick’s Dermatology in General Medicine, 6th edn. New York: McGraw-Hill, 2003: 1386-98.

36 Koehler MJ, Konig K, Elsner P, Buckle R, Kaatz M. In vivo assessment of human skin aging by multiphoton laser scanning tomography. Opt Lett 2006; 31: 2879-81.

37 Grewal BS, Naik A, Irwin WJ, Gooris G, de Grauw GJ, Gerritsen HG, et al. Transdermal macromolecular delivery: Real-time visualization of iontophoretic and chemically enhanced transport using two-photon excitation microscopy. Pharm Res 2000; 17: 788-95.

38 Yu B, Dong CY, So PTC, Blankschtein D, Langer R. In vitro visualization and quantification of oleic acid induced changes in transdermal transport using two-photon fluorescence microscopy. J Invest Dermatol 2001; 117: 16-25.

39 Yu B, Kim KH, So PTC, Blankschtein D, Langer R. Topographic heterogeneity in transdermal transport revealed by high-speed two-photon microscopy: Determination of representative skin sample sizes. J Invest Dermatol 2002; 118: 1085-8.

40 Yu B, Kim KH, So PTC, Blankschtein D, Langer R. Visualization of oleic acid-induced transdermal diffusion pathways using two-photon fluorescence microscopy. J Invest Dermatol 2003; 120: 448-55.

41 Yu B, Kim KH, So PTC, Blankschtein D, Langer R. Evaluation of fluorescent probe surface intensities as an indicator of transdermal permeant distributions using wide-area two-photon fluorescence microscopy. J Pharm Sci 2003; 92: 2354-65.

42 König K, Ehlers A, Stracke F, Riemann I. In vivo drug screening in human skin using femtosecond laser multiphoton tomography. Skin Pharmacol Physiol 2006; 19: 78-88.

43 Stracke F, Weiss B, Lehr CM, Koenig K, Schaefer UF, Schneider M. Multiphoton microscopy for the investigation of dermal penetration of nanoparticle-borne drugs. J Invest Dermatol 2006; 126: 2224-33.

44 Sun Y, Lo W, Lin SJ, Jee SH, Dong CY. Multiphoton polarization and generalized polarization (GP) microscopy reveals oleic-acid-induced structural changes in intercellular lipid layers of the skin. Opt Lett 2004; 29: 2013-5.

45 Sun Y, Su JW, Lo W, Lin SJ, Jee SH, Dong CY. Multiphoton polarization imaging of the stratum corneum and the dermis in ex-vivo human skin. Opt Express 2003; 11: 3377-84.