Shu-Wen Teng

Multiphoton fluorescence and second harmonic generation microscopy for imaging infectious keratitis.

The purpose of this study is to demonstrate the application of multiphoton fluorescence and second harmonic generation (SHG) microscopy for the ex-vivo visualization of human corneal morphological alterations due to infectious processes. The structural alterations of both cellular and collagenous components can be respectively demonstrated using fluorescence and SHG imaging. In addition, pathogens with fluorescence may be identified within turbid specimens. Our results show that multiphoton microscopy is effective for identifying structural alterations due to corneal infections without the need of histological processing. With additional developments, multiphoton microscopy has the potential to be developed into an imaging technique effective in the clinical diagnosis and monitoring of corneal infections.

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.

Imaging the bone marrow stem cells morphogenesis in PGA scaffold by multiphoton autofluorescence and second harmonic (SHG) imaging

The ability to image tissue engineering products without damaging histological procedures is important for the understanding of the dynamics of tissue reorganization and formation. In this work, we test the ability of multiphoton autofluorescence and second harmonic generation microscopy to image engineered tissues following chrondrogenic induction. The system we used is human bone marrow stem cells seeded in the scaffold polyglycolic acid (PGA). Our results show that autofluorescence can be used to image cells while second harmonic generation signal can be used to visualize the synthesis of extracellular matrix. This approach demonstrates the ability of multiphoton imaging in the study of tissue engineering products.

Multiphoton imaging of porcine eye: cornea, limbus, conjunctiva, and sclera

This purpose of this study is to demonstrate the feasibility of using multiphoton microscopy in imaging eye surface. Specifically, the cornea, limbus, conjunctiva, and sclera were imaged using multiphoton induced fluorescence and second-harmonic generation (SHG) imaging.

In vitro characterization of corneal wound healing using multiphoton autofluorescence and second harmonic generation (SHG) microscopy

The purpose of this investigation is to characterize corneal wound healing under in vitro conditions. Multiphoton autofluorescence and second harmonic generation (SHG) microscopy will be used to visualize cells and collagen fibers associated with corneal wound healing. Using the near-infrared excitation source from a titanium-sapphire laser pumped by a diode-pumped, solid state (DPSS) laser system, we can induce and simultaneously acquire multiphoton autofluorescence and SHG signals from the cornea specimens. A home-modified commercial microscope system with specified optical components is used for optimal signal detection. To acquire both high resolution and tissue-level information of the specimen, a sample positioning stage is used in conjunction with the beam scanning system. Finally, the organ level image can be assembled from individual area scans. The in vitro samples we used are cornea buttons acquired from porcine eyes. Localized wounds will be induced by #11 blade and imaged using multiphoton microscopy. Based on these results, we envision the in vitro imaging chamber to be able to follow the wound healing process without damaging histological procedures. We envision this approach will enable us to further understand wound healing process associated with corneal scar and can lead to in vivo methodology for diagnosing cornea damage.

Two-photon and second harmonic generation microscopy of porcine eye: implications to conductive keratoplasty

Two-photon autofluorescence and second harmonic generation (SHG) microscopy are useful imaging techniques for studying tissue components. In this work, we apply this imaging modality to study porcine eye. In particular, we use SHG microscopy to investigate the structural changes to excised porcine corneas and found that this technique is useful for studying its structural changes under thermal treatment.

Ophthalmic imaging using multiphoton microscopy

This purpose of this study is to demonstrate the feasibility of using multiphoton microscopy in ophthalmologic imaging. Without the introduction of extrinsic fluorescence molecules, multiphoton induced autofluorescence and second harmonic generation signals can be used to obtain useful structural information of normal and diseased corneas. Our work can potentially lead to the in vivo application of multiphoton microscopy in investigating corneal physiology and pathologies.

Characterization of thermally induced transitions of collagen using second harmonic generation (SHG) microscopy

As a major component of the connective tissues, collagen fibers are responsible for various physiological functions inside the body. They provide support for the skin, partial focusing through the cornea, and coordinate movements via tendons, ligaments, and cartilages. In many medical procedures, thermal reorganization of the collagen structure is inevitable or desired. Therefore, the optimization of the therapeutic values of these procedures requires the characterization of thermal changes to collagen fibers. In this presentation, we use multiphoton microscopy to achieve this task. We will show that second harmonic generation (SHG) microscopy can characterize the thermally altered states of collagen and that they have potentials to be used in imaging applications in vivo.

Multiphoton characterization of tissue engineering scaffolds

The use of seeding scaffolds has facilitated advances in tissue engineering. We found that multiphoton microscopy is useful in imaging commonly used tissue engineering scaffolds such as polyglycolic acid (PGA).

Multiphoton optical biopsy

Multiphoton microscopy is to a powerful imaging modality in the life sciences. The improved image contrast, greater imaging depths, and focus limited photodamage enable multiphoton microscopy to be especially useful for in vivo bioimaging applications. For multiphoton microscopy to be widely applicable in for in vivo biopsy applications, characterization of the tissues of interest must first be performed ex vivo. This presentation is an overview of the microscopic and spectroscopic characterization of the tissue types being pursued by my group. Both multiphoton images and spectroscopic information of a diverse range of normal, damaged, or diseased tissues will be presented and the implications of our results discussed.