Ming-Rung Tsai

Increased transforming growth factor-beta (TGF-β)-secreting T cells and IgA anti-cardiolipin antibody levels during acute stage of childhood Henoch–Schönlein purpura

Henoch–Schönlein purpura (HSP) is a small vessel vasculitis characterized by increased serum IgA and IgA‐dominant immune complex deposition in lesions. The involvement of IgA implies a probable role for TGF‐β, a major factor in IgA production, in the pathogenesis of HSP. Among IgA antibodies, serum IgA anti‐cardiolipin antibodies (aCL) have been found in many diseases, including vasculitis. In addition to the clinical presentations and laboratory parameters, we further investigated the roles of IgA aCL and TGF‐β in childhood HSP. Twenty‐six Chinese children with the diagnosis of HSP were enrolled. Blood samples from these patients were collected at both acute and convalescent stages. Intracellular staining of lymphocytes was performed to enumerate type 1 (interferon‐gamma‐secreting), type 2 (IL‐4‐secreting), and type 3 (TGF‐β‐secreting) helper T cells. Serum levels of TGF‐β were detected by ELISA. Serum IgA aCL of 21 of 26 patients at the acute stage, 11 of them at the convalescent stage, were measured by ELISA. The data showed that IgA aCL serum levels were significantly elevated in patients compared with healthy controls (P < 0·001), and those patients at the convalescent stage (P < 0·001). In addition, TGF‐β‐secreting T cells were significantly elevated during the acute stage, and decreased at the convalescent stage. Although more studies are needed, the high prevalence of IgA aCL and increased TGF‐β‐secreting T cells in children with acute HSP revealed some points which should permit a better understanding of the pathogenesis of HSP.

Comparison of serum specific IgE antibodies to staphylococcal enterotoxins between atopic children with and without atopic dermatitis

Background: The skin of patients with atopic dermatitis (AD) exhibits a striking susceptibility to colonization and infection by Staphylococcus aureus. The exotoxins secreted by S. aureus can act as superantigens and classic allergens, inducing the production of functionally relevant specific IgE antibodies. The aim of this study was to compare the levels and positive rates of serum staphylococcal enterotoxin A (SEA)‐ and staphylococcal enterotoxin B (SEB)‐specific IgE between atopic children with and without AD.

In vivo optical virtual biopsy of human oral mucosa with harmonic generation microscopy

Recent clinical studies on human skin indicated that in vivo multi-harmonic generation microscopy (HGM) can achieve sub-micron resolution for histopathological analysis with a high penetration depth and leave no energy or photodamages in the interacted tissues. It is thus highly desired to apply HGM for in vivo mucosa histopathological diagnosis. In this paper, the first in vivo optical virtual biopsy of human oral mucosa by using epi-HGM is demonstrated. We modified an upright microscope to rotate the angle of objective for in vivo observation. Our clinical study reveals the capability of HGM to in vivo image cell distributions in human oral mucosa, including epithelium and lamina propria with a high penetration depth greater than 280 μm and a high spatial resolution better than 500 nm. We also found that the third-harmonic-generation (THG) contrast on nucleus depends strongly on its thicknesses, in agreement with a numerical simulation. Besides, 4% acetic acid was found to be able to enhance the THG contrast of nucleus in oral mucosa, while such enhancement was found to decay due to the metabolic clearance of the contrast enhancer by the oral mucosa. Our clinical study indicated that, the combined epi-THG and epi-second-harmonic-generation (SHG) microscopy is a promising imaging tool for in vivo noninvasive optical virtual biopsy and disease diagnosis in human mucosa.

Second-harmonic generation imaging of collagen fibers in myocardium for atrial fibrillation diagnosis

Atrial fibrillation (AF) is the most common irregular heart rhythm and the mortality rate for patients with AF is approximately twice the mortality rate for patients with normal sinus rhythm (NSR). Some research has indicated that myocardial fibrosis plays an important role in predisposing patients to AF. Therefore, realizing the relationship between myocardial collagen fibrosis and AF is significant. Second-harmonic generation (SHG) is an optically nonlinear coherent process to image the collagen network. We perform SHG microscopic imaging of the collagen fibers in the human atrial myocardium. Utilizing the SHG images, we can identify the differences in morphology and the arrangement of collagen fibers between NSR and AF tissues. We also quantify the arrangement of the collagen fibers using Fourier transform images and calculating the values of angle entropy. We indicate that SHG imaging, a nondestructive and reproducible method to analyze the arrangement of collagen fibers, can provide explicit information about the relationship between myocardial fibrosis and AF.

Determination of chronological aging parameters in epidermal keratinocytes by in vivo harmonic generation microscopy

Skin aging is an important issue in geriatric and cosmetic dermatology. To quantitatively analyze changes in keratinocytes related to intrinsic aging, we exploited a 1230 nm-based in vivo harmonic generation microscopy, combining second- and third-harmonic generation modalities. 52 individuals (21 men and 31 women, age range 19–79) were examined on the sun-protected volar forearm. Through quantitative analysis by the standard algorithm provided, we found that the cellular and nuclear size of basal keratinocytes, but not that of granular cells, was significantly increased with advancing age. The cellular and nuclear areas, which have an increase of 0.51 μm2 and 0.15 μm2 per year, respectively, can serve as scoring indices for intrinsic skin aging.

Characterization of oral squamous cell carcinoma based on higher-harmonic generation microscopy.

In vivo higher-harmonic generation microscopy (HGM) performed on healthy human oral mucosa not only provides images with a <500 nm lateral resolution at a 280 μm penetration depth, but also leaves no photodamages in the tissues. These advantages suggest that HGM could serve as an ideal virtual biopsy tool for in vivo, in situ, and immediate histopathological diagnosis of oral cancer. However, translation of such mechanism for clinical cancer diagnosis requires evidence based algorithm capable to differentiate cancerous tissues from normal. It is thus critical to investigate if the endogenous contrast provided by the HGM would be high enough to differentiate cancerous versus normal tissues in human oral mucosa. In this report, ex vivo HGM study was performed on the cancerous mucosa from 10 patients with oral squamous cell carcinoma. Compared with histology, HGM revealed histopathological features including the cytological abnormalities, loss of differentiation, interruption of basement membrane, and irregular epithelial stratification in all 10 specimens. In addition, distinct patterns of collagen fibers and increased distribution area of actin filaments in tumor cells were noted. These results indicate HGM holds great potential for the optical biopsy screening of oral cancer lesions.

Applying Harmonic Optical Microscopy for Spatial Alignment of Atrial Collagen Fibers

Background Atrial fibrosis creates a vulnerable tissue for atrial fibrillation (AF), but the spatial disarray of collagen fibers underlying atrial fibrosis is not fully elucidated. Objective This study hypothesizes that harmonics optical microscopy can illuminate the spatial mal-alignment of collagen fibers in AF via a layer-by-layer approach. Patients and Methods Atrial tissues taken from patients who underwent open-heart surgery were examined by harmonics optical microscopy. Using the two-dimensional Fourier transformation method, a spectral-energy description of image texture was constituted and its entropy was used to quantify the mal-alignment of collagen fibers. The amount of collagen fiber was derived from its area ratio to total atrial tissue in each image. Serum C-terminal pro-collagen pro-peptide (CICP), pro-matrix metalloproteinase-1 (pro-MMP-1), and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) were also evaluated. Results 46 patients were evaluated, including 20 with normal sinus rhythm and 26 with AF. The entropy of spectral-energy distribution of collagen alignment was significantly higher in AF than that in sinus rhythm (3.97±0.33 vs. 2.80±0.18, p<0.005). This difference was more significant in the permanent AF group. The amount of collagen was also significantly higher in AF patients (0.39±0.13 vs. 0.18±0.06, p<0.005) but serum markers of cardiac fibrosis were not significantly different between the two groups. Conclusions Harmonics optical microscopy can quantify the spatial mal-alignment of collagen fibers in AF. The entropy of spectral-energy distribution of collagen alignment is a potential tool for research in atrial remodeling.

Differential diagnosis of nonmelanoma pigmented skin lesions based on harmonic generation microscopy

Abstract. In vivo harmonic generation microscopy (HGM) has been applied successfully in healthy human skin and can achieve a submicron resolution, similar to histopathologic examination, even at a penetration depth up to 270 μm. This study aims to investigate the clinical applicability of HGM imaging for differential diagnosis of nonmelanoma pigmented skin lesions. A total of 42 pigmented skin tumors, including pigmented basal cell carcinoma, melanocytic nevus, and seborrheic keratosis were evaluated by HGM ex vivo or in vivo. Based on the standard histopathologic characteristics, we established the corresponding HGM imaging criteria for each pigmented tumor. Diagnostic performance of HGM for differentiating nonmelanoma pigmented skin tumors was evaluated through the observers’ direct general assessment (overall evaluation) or the presence of two imaging criteria with the highest sensitivity and specificity (major criteria evaluation). Our results show that, based on the direct general assessment, the sensitivity is 92% [95% confidence interval (CI): 67 to 97%] and the specificity is 96% (95% CI: 83 to 99%); by major criteria evaluation, 94% sensitivity (95% CI: 70 to 99%) and 100% specificity (95% CI: 87 to 100%) are achieved. Our study indicates that HGM serves as a promising histopathological examination tool for noninvasive differential diagnostics of nonmelanoma pigmented skin tumors.

A sub-100fs self-starting Cr:forsterite laser generating 1.4W output power

Without cavity dumping or external amplification, we report a femtosecond Cr:forsterite laser with a 1.4 W output power and 2 W in continuous wave (CW) operated with a crystal temperature of 267 K. In the femtosecond regime, the oscillator generates Kerr-lens-mode-locked 84 fs pulses with a repetition rate of 85 MHz, corresponding to a high 16.5 nJ pulse energy directly from a single Cr:forsterite resonator. This intense femtosecond Cr:forsterite laser is ideal to pump varieties of high power fiber light sources and could be thus ideal for many biological and spectroscopy applications.

Applying tattoo dye as a third-harmonic generation contrast agent for in vivo optical virtual biopsy of human skin.

Abstract. Third-harmonic generation (THG) microscopy has been reported to provide intrinsic contrast in elastic fibers, cytoplasmic membrane, nucleus, actin filaments, lipid bodies, hemoglobin, and melanin in human skin. For advanced molecular imaging, exogenous contrast agents are developed for a higher structural or molecular specificity. We demonstrate the potential of the commonly adopted tattoo dye as a THG contrast agent for in vivo optical biopsy of human skin. Spectroscopy and microscopy experiments were performed on cultured cells with tattoo dyes, in tattooed mouse skin, and in tattooed human skin to demonstrate the THG enhancement effect. Compared with other absorbing dyes or nanoparticles used as exogenous THG contrast agents, tattoo dyes are widely adopted in human skin so that future clinical biocompatibility evaluation is relatively achievable. Combined with the demonstrated THG enhancement effect, tattoo dyes show their promise for future clinical imaging applications.