Ping Chin Cheng

Multi-Photon Fluorescence Microscopy: Behavior of Biological Specimens Under High Intensity Illumination

Recent development in multi-photon fluorescence microscopy, second and third harmonic generation microscopy (SHG and THG) and CARS open new dimensions in biological studies. Not only the technologies allow probing the biological specimen both functionally and structurally with increasing spatial and temporal resolution, but also raise the interest in how biological specimens respond to high intensity illumination commonly used in these types of microscopy. We have used maize leaf protoplast as a model system to evaluate the photo-induced response of living sample under high intensity illumination. It was found that cells can be seriously damaged by high intensity NIR irradiation even the linear absorption coefficient in low in these wavelengths. Micro-spectroscopy of single chloroplast also allows us to gain insight on the possible photo-damage mechanism. In addition to fluorescence emission, second harmonic generation was observed in the maize protoplasts.

Multiphoton fluorescence spectroscopy of flourescent bioprobes and biomolecules

Multi-photon fluorescence spectra of a number of commonly used biological probes were measured in this study. Significant spectral variation has been detected between single and multi- photon excitation. The result is important for the proper selection of spectral setting/dichroic beam splitter in the set- up of a multi-photon fluorescence microscope. The information can also be useful in the detection of multi-photon fluorescence in bio-chip technology. In addition, we have investigated a few highly fluorescent bio-molecules commonly found in plant cells.

Nonlinear multimodality spectromicroscopy: multiphoton fluorescence, SHG, and THG of biological specimen

The non-linear nature of multi-photon fluorescence excitation, SHG and THG restricts the signal detecting volume to the vicinity of the focal point. As a result, the technology has intrinsic optical sectioning capability. The use of multi-photon fluorescence excitation also allows micro-fluorometry at high spatial resolution. Under high intensity illumination, biological specimen not only emits fluorescence, but also generates harmonic emissions. Conventional ultra-fast Ti-sapphire laser allows efficient excitation of most biologically important fluorescent probes and SHG in the deep blue range. In contrast, the use of ultra-fast Cr-forsterite laser makes possible simultaneous detection of two- and three-photon fluorescence, SHG and THG

Two-photon optical-beam-induced current microscopy of indium gallium nitride light-emitting diodes

In this study, epilayers of packaged indium gallium nitride light emitting diodes (LEDs) are characterized by optical beam induced current (OBIC) and photoluminescence laser scanning microscopy through two-photon excitation. OBIC reveals spatial and electrical characteristics of LEDs which can not be distinguished by photoluminescence. When compared with single- photon OBIC, two-photon OBIC imaging not only exhibits superior image quality but also reveals more clearly the characteristics of the epilayers that are being focused on. The uniformity of these LEDs OBIC images can also be related to their light emitting efficiency.

Second-harmonic generation microscopy of tooth

In this study, we have developed a high performance microscopic system to perform second-harmonic (SH)imaging on a tooth. The high sensitivity of the system allows an acquisition rate of 300 seconds/frame with a resolution at 512x512 pixels. The surface SH signal generated from the tooth is also carefully verified through micro-spectroscopy, polarization rotation, and wavelength tuning. In this way, we can ensure the authenticity of the signal. The enamel that encapsulates the dentine is known to possess highly ordered structures. The anisotrophy of the structure is revealed in the microscopic SH images of the tooth sample.

Biological photonic crystals revealed by multimodality nonlinear microscopy

A novel multi-modality nonlinear microscopy reveals highly optically-active biophotonic crystal structures in living cells. Numerous biological structures, including stacked membranes and arranged protein structures are highly organized in optical scale and are found to exhibit strong optical activities through second-harmonic-generation (SHG) interactions, behaving similar to man-made photonic crystals. The microscopic technology developed is based on a combination of imaging modalities including not only SHG, but also third-harmonic-generation and multi-photonfluorescence. With no energy deposition during harmonic generation processes, the demonstrated highly-penetrative yet non-invasive microscopy is useful for investigating the dynamics of structure-function relationship at the molecular and subcellular levels and is ideal for studying living cells that require minimal or no preparation.

Multiphoton microspectroscopy of biological specimens

The non-linear nature of multi-photon fluorescence excitation restricts the fluorescing volume to the vicinity of the focal point. As a result, the technology has the capacity for micro- spectroscopy of biological specimen at high spatial resolution. Chloroplasts in mesophyll protoplast of Arabidopsis thaliana and maize stem sections were used to demonstrate the feasibility of multi-photon fluorescence micro-spectroscopy at subcellular compartments. Time-lapse spectral recording provides a means for studying the response of cell organelles to high intensity illumination.

Multi-photon confocal microscopy by using a femtosecond Cr forsterite laser

Confocal laser scanning microscopy provides a significant improvement in axial resolution over conventional epi-fluorescence microscopy by eliminating out of focus fluorescence using a spatial filter in the form of a confocal aperture. In this presentation, we will present our recent studies on two-photon confocal scanning microscopy by using a femtosecond Cr:forsterite laser with output wavelength centered at 1220-1240 nm.

Multiphoton fluorescence microspectroscopy

The intrinsic confined photo-interacting volume in multi- photon fluorescence microscopy provides the possibility of obtaining fluorescence spectrum from specific cellular structure in a tissue. In this article, we demonstrated that it is feasible to obtain useful two-photon pumped fluorescence spectrum from cell wall and single chloroplast. The difference in fluorescence spectra obtained with single- and two-photon excitation indicates that a significant shift in fluorescence maximum may occur due to the non-linear nature of excitation. Therefore, in order to properly interpret two-photon fluorescence micrographs, it is important to characterize the fluorescence spectrum of the specimen and the commonly used fluorescence probes. The fluorescence spectra will in turn be useful in the selection of filter sets in multi-photon fluorescence microscopy.