Multi-modality Laser Scanning Biological Microscopy

• Main Authors: Tsung-Han Tsai, 蔡宗涵
• Other Authors: Chi-Kuang Sun
• Format: Others
• Language: en_US
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/25872190518230747833

碩士 === 國立臺灣大學 === 光電工程學研究所 === 93 === In this thesis, a multi-modality laser scanning microscopy system is build based on a modified optical scanning microscope and a Cr:forsterite laser. Reflection confocal, second harmonic generation (SHG), third harmonic generation (THG), and 2-photon fluorescence (2PF) have been used as imaging modalities. Both confocal techniques and nonlinear natures can achieve the optical sectioning property desirable for microscopy applications. Combined with the near-infrared femtosecond source working in the biological penetration window and a high numerical aperture (NA) objective, multi-modality laser scanning microscopy can offer >1-mm penetration depth and sub-micron spatial resolution in biological samples with much-reduced photodamage and phototoxicity. Multi-modality laser scanning microscopy can thus provide sufficient information deep inside a live biological specimen for biological studies less invasively than Ti:sapphire based techniques. Such a microscopy system would “enlarge” the field of view on histology and developmental biology researches, as presented in the following chapter. As a well-developed optical biopsy tool, reflection confocal images of normal skin correlate very well with images from conventional histology. Combining reflection confocal imaging modality with SHG and THG imaging modalities, this microscopic tool can provide much more information than traditional confocal laser scanning microscopy with better spatial resolution and multiple imaging modalities that are very useful for dermatology studies. The images in human skin taken from reflection confocal and THG microscopy provided very helpful evidences that the light scattering from cells is dominated by Rayleigh scattering when the volume fraction of organelles increases, which confirms the bright reflection confocal and THG signals from cytoplasm that containing multiple organelles such as mitochondria. The signal intensity decay in deeper parts of human skin was found to be mainly caused by the focal plane broadening which was also the reason of the resolution degradation of each imaging modality. With the aid of transgenic techniques, specific tissues tagged by fluorescent protein have become a powerful tool in developmental biology studies because the spatial expression of that fluorescent protein enables to encapsulate the expression pattern of endogenous genes. Based on a 2PF microscope, images high 3-dimensional (3D) resolution can be obtained due to its nonlinear nature. We have combined 2PF microscopy with higher-harmonic optical microscopy based on the femtosceond Cr:forsterite laser with the aid of new transgenic lines tagged with HC-red fluorescent protein to obtain molecular, morphological, and structural protein information in biological tissues. With its optical sectioning property, high penetration depth, and much-reduced photodamages, this multi-modal method provides superb imaging capability for dynamic developmental studies of vertebrate embryos in the future.