Po-Sheng Hu

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.

Characterizing the morphologic changes in collagen crosslinked–treated corneas by Fourier transform–second harmonic generation imaging

Purpose To evaluate the efficacy of using forward second harmonic generation (SHG) and 2‐dimensional fast Fourier‐transform (2D‐FFT) analysis for the label‐free characterization and quantification of morphologic changes in the corneal stroma after collagen crosslinking (CXL). Setting Department of Physics, National Taiwan University, Taipei, Taiwan. Design Experimental study. Methods En face forward SHG imaging and 2D‐FFT analysis were performed on ex vivo porcine corneas at the depths of 100, 200, 400, and 800 &mgr;m. Morphologic changes in stromal collagen fiber in control, ultraviolet‐A (UVA), riboflavin, and riboflavin–UVA treated porcine corneas were assessed. Hematoxylin–eosin staining and Sirius red staining were performed for comparison. Results Corneas after CXL treatment tended to have collagen fibers that were wavy compared with the linear pattern in normal corneas. Quantitative 2D‐FFT analysis of forward SHG images also showed an increase in the standard deviations of the distribution of stromal collagen fiber orientations, which is indicative of the changed pattern of crosslinked stromal collagen fibers. Conclusions Second harmonic generation imaging showed the morphologic changes in stromal collagen after CXL treatment. The linear collagen fibers in normal corneal stroma became wavy after treatment. With the introduction of 2D‐FFT analysis, the morphologic changes can be quantified. Financial Disclosure No author has a financial or proprietary interest in any material or method mentioned.

Elucidation of the mechanisms of optical clearing in collagen tissue with multiphoton imaging

Abstract. Optical clearing (OC) is a promising method to overcome limitations in biomedical depth-resolved optical studies. Mechanisms of OC in purified bovine Achilles tendon, chicken skin, and chicken tendon were studied using time-lapsed, three-dimensional second harmonic generation (SHG) and two-photon fluorescence microscopic imaging. Quantified nonlinear optical measurements allowed temporal separation of two processes in collagen OC with glycerol. The first one is a fast process of tissue dehydration accompanied with collagen shrinkage and the second relatively slow process is glycerol penetration into the interfibrillar space of collagen alongside with CF swelling. The use of 50% glycerol induced less-expressed OC via partial substitution of water molecules with glycerol molecules. We also found that phosphate-buffered saline- and glycerol-treatments were reversible, and fiber morphology and SHG signal intensity were recovered after the removal of immersion agents. It was shown that tissue OC was a dynamic process and elucidation of its physical mechanisms may help choose optimal diagnostic, treatment, and modification regimes for collagen-based as well as other types of biomaterials.

Spatial orientation mapping of fibers using polarization‐sensitive second harmonic generation microscopy

In this work, we present a non-invasive approach to determine azimuth and elevation angles of collagen fibers capable of generating second harmonic signal. The azimuth angle was determined using the minimum of second harmonic generation (SHG) signal while rotating the plane of polarization of excitation light. The elevation angle was estimated from the ratio of the minimal SHG intensity to the intensity when laser polarization and fiber directions were parallel to each other using experimentally determined calibration curve. Pixel-resolution images of collagen fiber spatial orientation in tendon from bovine leg, chicken leg, and chicken skin were acquired using our approach of SHG polarization-resolved microscopy.

Spatial and temporal analysis of skin glycation by the use of multiphoton microscopy and spectroscopy

BACKGROUND Tissue glycation, the main cause of many diabetes-related complications, results in the accumulation of advanced glycation endproducts (AGE). OBJECTIVES These AGEs are endogenous fluorophores that can serve as a viable pathological indicator for disease diagnostics. Here we explore the capabilities of multiphoton microscopy to non-invasively localize and quantify the skin glycation. METHODS In our study, multiphoton microscopy and spectroscopy were used to investigate glycation events-induced changes in the intensities of autofluorescence and second harmonic generation on ex vivo human skin. RESULTS Temporal and spatial dependence of degrees of glycation of the epidermis, collagen and elastin fibers of dermis were evaluated for their relevance to the changes in amplitudes of autofluorescence signals. We found that glycation drastically and linearly increases multiphoton autofluorescence intensity of epidermis and dermal collagen whereas changes in dermal elastin are moderate. We also found decrease in the level of second harmonic generation signal. CONCLUSION Our study suggests that due to intrinsically weak autofluorescence the dermal collagen is the most sensitive skin tissue to be used for detecting changes in tissue glycation.

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.

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.

Oleic acid-enhanced transdermal delivery pathways of fluorescent nanoparticles

Transdermal delivery of nanocarriers provides an alternative pathway to transport therapeutic agents, alleviating pain, improving compliance of patients, and increasing overall effectiveness of delivery. In this work, enhancement of transdermal delivery of fluorescent nanoparticles and sulforhodamine B with assistance of oleic acid was visualized utilizing multiphoton microscopy (MPM) and analyzed quantitatively using multi-photon excitation-induced fluorescent signals. Results of MPM imaging and MPM intensity-based spatial depth-dependent analysis showed that oleic acid is effective in facilitating transdermal delivery of nanoparticles.

Collagen fiber spatial orientation mapping using polarization-sensitive SHG microscopy

Collagen, the main structural protein in vertebrates, possesses different structural organization that is responsible for specific functions of the tissues. Polarization dependence of the second harmonic generation (SHG) signal on spatial orientation of optically nonlinear materials, such as collagen, provides information on characteristic organization and architecture not available from intensity measurements alone. Here we describe a simple approach for determining both the azimuth and elevation angles of collagen fiber orientation in biological tissues. Azimuth angle of the fiber orientation is determined as an orthogonal angle to the laser polarization direction, when laser-induced total SHG signal is minimal, whereas the elevation angle is estimated from the ratio of the minimal SHG intensity to the intensity when laser polarization and fiber directions are parallel to each other. Using this approach pixel-resolved mapping of the spatial orientation of collagen fibers in tendon and cornea is demonstrated. The new approach may be used for analyzing of biological tissues in vivo. Spatial orientation mapping method provides additional information concerning fiber organization, and may be incorporated into nonlinear optical imaging systems.