Hsin-Yuan Tan

Evaluating cutaneous photoaging by use of multiphoton fluorescence and second-harmonic generation microscopy

The photoaging process of facial skin is investigated by use of multiphoton fluorescence and second-harmonic generation (SHG) microscopy. We obtain the autofluorescence (AF) and SHG images of the superficial dermis from the facial skin of three patients aged 20, 40, and 70 years. The results show that areas of AF increase with age, whereas areas of SHG decrease with age. The results are consistent with the histological findings in which collagen is progressively replaced by elastic fibers. The AF and SHG changes in photoaging are quantified by a SHG to autofluorescence aging index of dermis (SAAID). Our results suggest that SAAID can be a good indicator of the severity of photoaging.

Characterizing the thermally induced structural changes to intact porcine eye, part 1: second harmonic generation imaging of cornea stroma

We characterize the structural changes of porcine corneal structures from 25 to 90 degrees C using second harmonic generation (SHG) microscopy. Our results show that porcine stroma undergoes several distinct stages of structural changes between 25 and 90 degrees C. A decrease in SHG intensity from 30 to 45 degrees C and the existence of SHG intensity peaks at 53, 65, and 77 degrees C correlate to distinct structural alterations of the corneal stroma. At higher temperatures, the SHG intensity decreases and a baseline in SHG signal is reached at 90 degrees C. Our results demonstrate that SHG microscopy is a useful technique for obtaining qualitative and quantitative information of thermally treated corneal fibers without histological or labeling procedures. With additional developments, SHG imaging may be developed into an effective imaging technique for in vivo characterization of cornea structural changes.

Prediction of heat-induced collagen shrinkage by use of second harmonic generation microscopy

Collagen shrinkage associated with denaturation from thermal treatment has a number of important clinical applications. However, individualized treatment is hindered by the lack of reliable noninvasive methods to monitor the process of collagen denaturation. We investigate the serial changes of collagen denaturation from thermal treatment of rat tail tendons at 58 degrees C by use of second harmonic generation (SHG) microscopy. We find that rat tail tendon shrinks progressively from 0 to 9 min of thermal treatment, and remains unchanged in length upon further thermal treatment. The SHG intensity also decreases from 0 to 9 min of thermal treatment and becomes barely detectable from further thermal treatment. Collagen shrinkage and the SHG intensity are well correlated in a linear model. In addition, SHG imaging reveals a tiger-tail-like pattern of collagen denaturation. The bands of denatured collagen progressively widen from increased thermal treatment and completely replace the adjacent bands of normal collagen after 9 min of thermal treatment. Our results show that collagen denaturation in rat tail tendon from thermal treatment is inhomogeneous, and that SHG intensity can be used to predict the degree of thermally induced collagen shrinkage. With additional development, this approach has the potential to be used in biomedical applications.

Amniotic membrane transplantation for persistent corneal ulcers and perforations in acute fungal keratitis.

Purpose: To report the therapeutic effect and complications of amniotic membrane transplantation (AMT) in acute fungal keratitis. Methods: Diagnosis of fungal keratitis was confirmed by cultures in 23 eyes of 23 patients. The indications to perform AMT were to promote reepithelialization in non-healing ulcers or to prevent corneal perforation. Antifungal agents were administered throughout the whole course of hospitalization. Repeated cultures were performed immediately before AMT. The main outcome measurements were epithelial healing rate, necessity of therapeutic penetrating keratoplasty (TPK), and persistence of infection. Results: During a mean follow-up time of 20.6 months ± 23.22 (6-65 months) AMT was performed during the active phase of the keratitis (fungal culture was still positive) in 16 patients (69.6%), and during the inactive phase (fungal culture negative) in 7 patients (30.4%). Single-layer AMT was performed in 17 patients, and double-layer AMT was performed in 6 patients with corneal perforation and anterior chamber collapse. Complete epithelialization was observed in 12 patients (75%) in the active group and in 7 patients (100%) in the inactive group. Treatment failure requiring TPK was experienced in 4 patients (25%) in the active group. Persistent fungal keratitis was noted in 2 patients (8.7%) in that group. The final visual acuity improved in 17 cases, worsened in 2 cases, and remained unchanged in 4 cases. Twelve of the 23 eyes (52.2%) in this study preserved useful vision (20/400 and better) with or without subsequent surgeries. Conclusion: AMT is effective in promoting epithelialization and preventing corneal perforations in acute fungal keratitis, and there is no risk of rejection. However, the risk of persistent or recurrent infection necessitates continued antifungal treatment and patient monitoring.

Infectious keratitis related to overnight orthokeratology.

PURPOSE To report the microbial culture results, clinical course, and visual outcomes for infectious keratitis related to overnight orthokeratology. METHODS The records of patients with infectious keratitis related to overnight orthokeratology who presented to a tertiary referral center from April 2000 to March 2003 were retrospectively reviewed. RESULTS Twenty patients (21 eyes) were included; 1 patient had bilateral infections. The average age of the patients was 14 years. The average period between the time the patient started the overnight orthokeratology program and the onset of infectious keratitis was 23 months. Thirteen of the 21 eyes were culture positive. Organisms cultured were Pseudomonas aeruginosa (n = 9), coagulase-negative Staphylococcus species (n = 2), Serratia marcescens (n = 1), and Acathamoeba species (n = 1). All patients responded well to medical antimicrobial treatment. Final best spectacle-corrected visual acuity ranged from 20/20 to 20/100. CONCLUSIONS Infectious keratitis is a potential complication of overnight orthokeratology that may cause significant visual impairment. Parents of children who consider overnight orthokeratology should evaluate the benefit of temporary myopia reduction and the risk of infection.

Medical treatment for combined Fusarium and Acanthamoeba keratitis

Purpose:  Acanthamoeba and fungal keratitis are rare ocular infections. We report cases of combined Fusarium and Acanthamoeba keratitis and the clinical course of medical treatment.

Fast Fourier transform-based analysis of second-harmonic generation image in keratoconic cornea.

PURPOSE To qualify and quantify structural alterations in keratoconic corneas ex vivo by the use of second-harmonic generation (SHG) microscopy and two-dimensional fast Fourier transform (2D-FFT) analysis. METHODS Two keratoconic and three normal human corneal specimens were imaged with a multiphoton microscope. Forward and backward second-harmonic generation images (FSHG and BSHG) of corneal stroma were obtained at high resolution and at different depths. The SHG images were further analyzed with a 2D-FFT algorithm to quantify the texture and orientation changes of collagen fibers. RESULTS The results showed that the abnormality of collagen architecture was found through the whole layer of stroma. 2D-FFT analysis of SHG cornea images provided fiber orientation direction and an aspect ratio (AR) as a quantitative measure of fiber direction determination. It was found that for keratoconic cornea the average AR values are statistically greater than those of the normal cornea: 1.66 ± 0.42 (case I keratoconic cornea), 1.72 ± 0.44 (case II keratoconic cornea), and 1.34 ± 0.16 (average of three normal human corneas). Furthermore, the fiber directions determined by 2D-FFT analysis of BSHG and FSHG images were strongly correlated for large AR values (AR > 2). CONCLUSIONS The high correlation between FSHG and BSHG fiber direction for large AR values shows that BSHG imaging can provide qualitative and quantitative information of the extent of structural changes in a keratoconic cornea. The ability to use BSHG for diagnosing and monitoring stroma abnormalities, even when cornea transparency is retained, demonstrates the clinical potential of this method:

Pediatric herpes simplex virus keratitis.

Purpose: To report the clinical characteristics and visual outcomes of pediatric herpes simplex virus (HSV) keratitis. Methods: The medical records of 29 patients younger than 16 years with HSV keratitis who were diagnosed and treated at Chang Gung Memorial Hospital, Taoyuan, Taiwan, between 1996 and 2004 were retrospectively reviewed. The diagnosis of HSV keratitis was proven by a positive viral culture and/or real-time quantitative polymerase chain reaction or by a clear history of dendritic keratitis or herpetic kerato-uveitis. Type of HSV keratitis, recurrence rate, and visual outcome were analyzed. Results: The average age at the entry into the study was 5.7 years (range: 7 months to 15 years). Mean follow-up time was 35.3 months (range: 2-69 months). Epithelial keratitis including dendritic and geographic ulcers was noted in 14 eyes, stromal keratitis in 2 eyes, stromal keratitis concurrent with epithelial keratitis in 8 eyes, and endotheliitis in 6 eyes. One patient had sequential involvement of both eyes. Thirteen patients (45%) developed recurrent HSV keratitis after the first documented episode. Female gender (but not age or the type of keratitis) was significantly associated with recurrences. Five patients who were maintained on oral valacyclovir prophylaxis up to 1 year had no recurrence during the period. Fifteen of 21 patients younger than 8 years of age had best-corrected visual acuity available at last follow-up, and 10 patients developed amblyopia. Conclusions: Children with HSV keratitis are at risk for recurrent keratitis and amblyopia. Prolonged systemic antiviral prophylaxis may help to prevent such consequences.

The successful medical treatment of a case of Paecilomyces lilacinus keratitis.

Purpose: To report the successful medical treatment of Paecilomyces lilacinus keratitis case with topical voriconazole. Methods: A 72-year-old male farmer developed a corneal infection in the left eye and corneal culture revealed Paecilomyces lilacinus. Infection was progressive despite initial treatment with hourly topical amphotericin B (1.5 mg/mL) and topical 5% natamycin. Treatment was switched to hourly topical voriconazole (10 mg/mL). Results: The infection responded well to topical voriconazole and resolved within 1 month without any surgical intervention. The patients best-corrected visual acuity was 20/200 secondary to central corneal scarring and a cataract in the left eye at the 10-month follow-up. Conclusion: Voriconazole may be effective in the treatment of Paecilomyces lilacinus keratitis refractory to standard antifungal agents. Early organism detection and appropriate treatment are necessary to eradicate such an infection.

Structural Characterization of Edematous Corneas by Forward and Backward Second Harmonic Generation Imaging

The purpose of this study was to image and quantify the structural changes of corneal edema by second harmonic generation (SHG) microscopy. Bovine cornea was used as an experimental model to characterize structural alterations in edematous corneas. Forward SHG and backward SHG signals were simultaneously collected from normal and edematous bovine corneas to reveal the morphological differences between them. In edematous cornea, both an uneven expansion in the lamellar interspacing and an increased lamellar thickness in the posterior stroma (depth > 200 microm) were identified, whereas the anterior stroma, composed of interwoven collagen architecture, remained unaffected. Our findings of heterogeneous structural alteration at the microscopic scale in edematous corneas suggest that the strength of collagen cross-linking is heterogeneous in the corneal stroma. In addition, we found that qualitative backward SHG collagen fiber imaging and depth-dependent signal decay can be used to detect and diagnose corneal edema. Our work demonstrates that SHG imaging can provide morphological information for the investigation of corneal edema biophysics, and may be applied in the evaluation of advancing corneal edema in vivo.