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Transmission Electron Microscopy (TEM) - Advances in 透射电子显微镜（TEM）研究进展
Fluorescence Fluorochromes are excited by specific wavelengths of light and emit specific wavelengths of a lower energy (longer wavelength) Filter Cubes for Fluorescence Filter cubes generally have an excitation filter, a dichroic element, and an emission filter The elements of a cube are selected for the excitation and fluorescence detection desired Choose Fluorochrome/Filter Combos Laser Scanning Confocal Microscopy Fluorescence technique Uses laser light for excitation Improves image resolution over conventional fluorescence techniques Optically removes out-of-focus light and detects only signal from focal plane Can construct an in-focus image of considerable depth from a stack of images taken from different focal planes of a thick specimen Can then make a 3-D image that can be tilted, rotated, and sliced Principal Light Pathway in Confocal Microscopy Laser light is scanned pixel by pixel across the sample through the objective lens Fluorescent light is reflected back through the objective and filters (dichroic mirrors) Adjustable pinhole apertures for PMTs eliminate out-of-focus flare Image is detected by photomultiplier(s) and digitized on computer TEM Transmission Electron Microscope Illumination source is beam of electrons from tungsten wire Electromagnetic lenses perform same function as glass lenses in LM Higher resolution and higher magnification of thin specimens Specimen Preparation for TEM Chemical fixation with buffered glutaraldehyde Or 4% paraformaldehyde with 1% glutaraldehyde Postfixation with osmium tetroxide Or not, or with subsequent removal from sections Dehydration and infiltration with liquid epoxy or acrylic resin Polymerization of hard blocks by heat or UV Ultramicrotomy – 60-80nm sections Labeling and/or staining View with TEM High pressure freezing: Plant tissue is flash frozen in a pressure bomb -197 C Water in the tissue is replaced with acetone over 5 day period Acetone saturated tissue is embedded in resin Resin is cut in th
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