Transdermal drug delivery, transporting the drug molecules through epidermis to dermis, has been extensively investigated but not studied in dynamic detail. The objective of this study is to monitor the dynamical changes of drug permeation and that of polarization of skin tissue with oleic acid treatment. We utilize two-photon fluorescence microscopy (TPM) to investigate the dynamics of transdermal drug delivery in skin excised from the abdominal region of euthenized nude mice with sulforhodamine B (SRB) modeling as a drug and Laurdan serving as a polarity indicator. The treatment of oleic acid increases the permeation rate of SRB, quickly reaching to the steady state of permeation. Increases in polarity within the skin tissue (in both intercellular and intracellular region of stratum corneum) are observed in SRB permeation enhanced by oleic acid treatment. TPM has successfully demonstrated the ability to study spatial distribution of transport dynamics in oleic acid-enhanced transdermal delivery.
We discuss the recent advances in the development and applications of second-order susceptibility as a contrast mechanism in optical microscopy for biological tissues. We review nonlinear optical methods and approaches for differentiation of tissue structures and discrimination of normal and pathological skin tissues, which have been demonstrated for the potential use in clinical diagnosis. In addition, the potential of second-order susceptibility imaging, encompassing applications in differentiating various types of collagen molecules for clinical diagnosis, is demonstrated. Finally, we discuss future development and application of this technique.
Both reflected confocal and multiphoton microscopy can have clinical diagnostic applications. The successful combination of both modalities in tissue imaging enables unique image contrast to be achieved, especially if a single laser excitation wavelength is used. We apply this approach for skin and corneal imaging using the 780-nm output of a femtosecond, titanium-sapphire laser. We find that the near-IR, reflected confocal (RC) signal is useful in characterizing refractive index varying boundaries in bovine cornea and porcine skin, while the multiphoton autofluorescence (MAF) and second-harmonic generation (SHG) intensities can be used to image cytoplasm and connective tissues (collagen), respectively. In addition, quantitative analysis shows that we are able to detect MAF from greater imaging depths than with the near-IR RC signal. Furthermore, by performing RC imaging at 488, 543, and 633 nm, we find that a longer wavelength leads to better image contrast for deeper imaging of the bovine cornea and porcine skin tissue. Finally, by varying power of the 780-nm source, we find that comparable RC image quality was achieved in the 2.7 to 10.7-mW range.
Recently, multiphoton microscopy has gained much popularity as a noninvasive imaging modality in biomedical research. We evaluate the potential of multiphoton microscopy for monitoring laser-skin reaction in vivo. Nude mouse skin is irradiated with an erbium:YAG laser at various fluences and immediately imaged by a multiphoton microscope. The alterations of cutaneous nonlinear optical properties including multiphoton autofluorescence and second-harmonic generation associated with laser irradiation are evaluated morphologically and quantitatively. Our results show that an erbium:YAG laser at a low fluence can selectively disrupt the stratum corneum, and this alteration may account for the penetration enhancing effect of laser-assisted transcutaneous drug delivery. At a higher fluence, the zone of tissue ablation as well as the disruption of the surrounding stratum corneum, keratinocytes, and dermal extracellular matrix can be better characterized by multiphoton microscopy as compared with conventional histology. Furthermore, the degree of collagen damage in the residual thermal zone can be quantified by second-harmonic generation signals, which have significant difference between control skin, skin irradiated with a 1.5-, 8-, and 16-J/cm2 erbium:YAG laser (P<0.05). We show that multiphoton microscopy can be a useful noninvasive imaging modality for monitoring laser-skin reaction in vivo.
To assess laser therapies in clinical practice, histologic examinations were commonly used. But histologic
examinations were invasive and not real-time in nature. In this work, we validate multiphoton microscopy as a useful
modality in evaluating laser-tissue reaction in vivo. Multiphoton microscopy based on femtosecond titanium-sapphire
laser system were used to evaluate autoflurescence (AF) and second harmonic generation (SHG). Nude mouse skin was
irradiated with Erbium:YAG laser at low to high fluence. High resolutional images can be obtained by multiphoton
microscopy. At low fluence, Erbium:YAG laser can selectively loosen compact stratum corneum with minimal injury to
basal layer. At high fluence, ablated keratinocytes and residual debris can be imaged. The laser thermal effect on dermis
could be measured by SHG signals of collagen fibers. SHG decreased as laser fluence increased. Multiphoton
microscopy is a useful in-vivo technique in evaluating ablative and thermal effects of Erbium:YAG laser on nude mouse skin.
The aim of this study is to characterize the ability of multiphoton microscopy in monitoring the
transdermal penetration enhancing effect of a depilatory agent and the associated structural
alterations of stratum corneum. The result is compared with the electron microscopic findings. Our
results show that the penetration of both hydrophilic and hydrophobic agents can be enhanced. The
morphology of corneocytes becomes a homogenized pattern with focal detachment of surface
corneocytes. In combination with Nile red staining, multiphoton imaging also shows that the regular
motar-like distribution of lipid matrix was disrupted into a homogenized pattern of lipid distribution.
These results are well correlated with the findings of ultrastructural analysis by electron micrographs
showing disintegration of the protein envelope of coenocytes, disruption of intracellular keratin and
loss of the regular lamellar packing of intercellular lipids. We conclude that, in addition to
quantifying the permeation profiles of model drugs, multiphoton microscopy is able to detect the
penetration enhancer-induced structural alterations of stratum corneum.
Reflected confocal microscopy has been widely used in clinical application in dermatology. In recent years,
multiphoton microscopy has also emerged as an important minimally invasive bioimaging technique for the skin. In this
study, we combine reflected confocal microscopy and multiphoton microscopy for skin imaging. In the epidermis,
reflected confocal signals are expected to help in delineating cell borders while multiphoton signals provide cytoplasmic
morphologies. In the dermis, second harmonic generation signals provide the morphology of collagen fibers. When
three-dimensional images are projected, the detailed distribution of cellular component and extracellular matrix in skin
can be obtained. Properly developed, this technique is of great potential for in vivo clinical application.
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