Tsung-Hua Tsai

Multiphoton microscopy in dermatological imaging

A minimally invasive imaging modality that provides both cellular and extracellular structural information with subcellular resolution is helpful for clinical diagnosis as well as basic laboratory research in dermatology. Multiphoton microscopy (MPM), using femtosecond laser as the light source, is efficient in non-linear excitation of endogenous fluorophores and induction of second harmonic generation signals from non-centrosymmetric biomolecules such as collagen. This imaging modality is minimally invasive in the sense that much of the traditional histological procedures can be bypassed en route to obtain morphological and structural information of high scattering skin tissues. This unique feature has allowed clinical dermatological diagnosis, both ex vivo and in vivo. In addition to discussing the basic principles of multiphoton microscopy, this review is aimed at emphasizing its specific applications to dermatological imaging, including characterizing stratum corneum structures, visualizing and quantifying transcutaneous drug delivery, detecting skin cancers, exploring collagen structural transitions, and monitoring laser-skin interactions.

Scalable production of controllable dermal papilla spheroids on PVA surfaces and the effects of spheroid size on hair follicle regeneration

Organ size and numbers are vital issues in bioengineering for hair follicle (HF) regeneration. Murine HF dermal papilla (DP) cells are able to induce HF neogenesis when transplanted as aggregates. However, how the preparation of murine and human DP aggregates affects HF inductivity and the size of regenerated HF is yet to be determined. Here we report a scalable method for production of controllable human and rat DP spheroids in general labs for reproducible experiments. Compared with more hydrophobic polyethylene and poly(ethylene-co-vinyl alcohol), DP cells are poorly adhesive to hydrophilic polyvinyl alcohol (PVA). Seeded in PVA-coated 96-welled commercial PCR tube arrays, DP cells quickly aggregate into single spheroids with progressive compaction. Varying seeded cell numbers and culture periods enables us to control the size and cell number of the spheroids. The spheroids obtained have high viability and preserve DP characters. A proof of principle experiment was conducted to examine the size effect on the efficiency and efficacy of HF regeneration. We found that both human and rat DP spheroids are able to induce HF neogenesis and larger DP spheroids exhibit higher HF inductivity. However, the average diameter of regenerated hair fiber did not significantly change with the increasing size of transplanted DP spheroids. The result suggests that an appropriate size of DP spheroid is essential for HF inductivity, but its size cannot be directly translated to a thicker regenerated hair. Our results also have implications on the efficiency and efficacy in the regeneration of other epithelial organs.

Quantitative analysis of multiphoton excitation autofluorescence and second harmonic generation imaging for medical diagnosis

In recent years, two-photon excitation fluorescence and second harmonic generation microscopy has become an important tool in biomedical research. The ability of two-photon microscopy to achieve optical sectioning with minimal invasiveness is particularly advantageous for biomedical diagnosis. Advances in the miniaturization of the imaging system have increased its clinical potential, together with the development of quantitative technique for the analysis of data acquired using these imaging modalities. We present a review of the quantitative analysis techniques that have been used successfully with two-photon excitation fluorescence and SHG imaging. Specifically, quantification techniques using ratiometric, morphological, and structural differences to analyze two-photon images will be discussed, and their effectiveness at evaluating dermal and corneal pathologies and cancerous tumor growth will be described.

Single-wavelength reflected confocal and multiphoton microscopy for tissue imaging

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.

Imaging of biological tissues with pixel-level analysis of second-order susceptibility

Abstract. 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.

Enhancing hair follicle regeneration by nonablative fractional laser: Assessment of irradiation parameters and tissue response.

Identification of methods to enhance anagen entry can be helpful for alopecia. Recently, nonablative laser has been proposed as a potential treatment for alopecia. However, how the laser parameters affect stem cell activity, hair cycles and the associated side effects have not been well characterized. Here we examine the effects of irradiation parameters of 1,550‐nm fractional laser on hair cycles.

The Use of Second-Order Susceptibility as Contrast Mechanism for Label-Free Imaging of Biological Tissue

This review paper conveys state-of-the art research on second-order susceptibility microscopy: physical origin, devices and instrumentation, applications in biological system, and prospects of clinical applications. The organization of this paper started with an overview of second harmonic generation (SHG) in biological medium. Illustrating with figures system architecture of second-order susceptibility and manipulation of polarization are introduced, which is the central scheme in this imaging modality. Several applications of SHG susceptibility imaging in biological and biomedical sciences are then discussed. This review paper is finally concluded with future prospects of susceptibility imaging in clinical settings.

Images in clinical medicine. Keloidal morphea.

n engl j med 364;14 nejm.org april 7, 2011 e28 A 44-year-old woman presented with a 2-year history of multiple enlarging, painful nodules. Physical examination revealed several irregularly shaped, firm nodules with hyperpigmentation and induration on the trunk and upper arms (Panel A). She reported no other concerns about her health, and physical examination showed no other abnormal findings. Neither she nor her family had a history of keloidal scars or systemic sclerosis. The results of laboratory investigations, including assays for antinuclear and anti-dsDNA antibodies and screening for extractable nuclear antigens (including anti-Jo-1, anti-SSA, anti-SSB, anti-Scl-70, anti-Sm, and anti-RNP antibodies) were unremarkable. Findings on examination of a specimen from a skin biopsy were consistent with a diagnosis of keloid formation, except for the presence of perieccrine lymphocytic infiltration. On the basis of these clinical and pathological features, the nodules were diagnosed as keloidal morphea, a rare form of localized cutaneous scleroderma. Despite multiple treatments, the lesions enlarged over a 10-year period (Panel B).

Investigation of transport dynamics in oleic acid–induced transdermal drug delivery by two-photon fluorescence microscopy: an ex-vivo study of mouse skin

Abstract. 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.

Three-dimensional skin imaging using the combination of reflected confocal and multiphoton microscopy

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.