BME 100 Optical Microscopy

BME 100 Optical Microscopy - Optical Microscopy...

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Unformatted text preview: Optical Microscopy Fluorescence http://www.olympusmicro.com/primer/techniques/fluorescence/fluorhome.html Fluorescence microscopy The inner works of a Fluorescence Microscope Visualizing cells This mitotic spindle is stained to show the spindle microtubules (green), the DNA in the chromosomes (blue), and the centromeres (red). (Courtesy of Kevin F. Sullivan.) antibodies Indirect immunocytochemistry Immunofluorescence (A) A transmission electron micrograph of the periphery of a cultured epithelial cell showing the distribution of microtubules and other filaments. (B) The same area stained with fluorescent antibodies against tubulin, the protein subunit of microtubules, using the technique of indirect immunocytochemistry (see Figure 9-16). Arrows indicate individual microtubules that are readily recognizable in both images. Note that, because of diffraction effects, the microtubules in the light microscope appear 0.2 µm wide rather than their true width of 0.025 µm. (From M. Osborn, R. Webster, and K. Weber, J. Cell Biol. 77:R27–R34, 1978. © The Rockefeller University Press.) green fluorescent protein (GFP) The structure of GFP, shown here schematically, highlights the eleven β strands that form the staves of a barrel. Buried within the barrel is the active chromaphore (dark green) that is formed post-translationally from the protruding side chains of three amino acid residues. (Adapted from M. Ormö et al., Science 273:1392–1395, 1995.) GFP tagging A) The upper surface of the leaves of Arabidopsis plants are covered with huge branched single-cell hairs that rise up from the surface of the epidermis. These hairs, or trichomes, can be imaged in the SEM. (B) If an Arabidopsis plant is transformed with a DNA sequence coding for talin (an actin-binding protein), fused to a DNA sequence coding for GFP, the fluorescent talin protein produced binds to actin filaments in all the living cells of the transgenic plant. Confocal microscopy can reveal the dynamics of the entire actin cytoskeleton of the trichome (green). The red fluorescence arises from chlorophyl in cells within the leaf below the epidermis. (A, courtesy of Paul Linstead; B, courtesy of Jaideep Mathur.) Quantum dots Application to biology 1 hour later Injection of 2 Billion QD into one cell In vivo cancer targeting and imaging Gao X. et al., Nature Biotechnology 22(8),969-976,2004 Figure 4. Spectral imaging of QD-PSMA Ab conjugates in live animals harboring C4-2 tumor xenografts. Orange-red fluorescence signals indicate a prostate tumor growing in a live mouse (right). Control studies using a healthy mouse (no tumor) and the same amount of QD injection showed no localized fluorescence signals (left). (a) Original image; (b) unmixed autofluorescence image; (c) unmixed QD image; and (d) superimposed image. After in vivo imaging, histological and immunocytochemical examinations confirmed that the QD signals came from an underlying tumor. Note that QDs in deep organs such as liver and spleen were not detected because of the limited penetration depth of visible light. single particle tracking Image Deconvolution A) A light micrograph of the large polytene chromosomes from Drosophila, stained with a fluorescent DNA-binding dye. (B) The same field of view after image deconvolution clearly reveals the banding pattern on the chromosomes. Each band is about 0.25 µm thick, approaching the resolution limit of the light microscope. (Courtesy of the John Sedat Laboratory.) Confocal microscopy Comparison Conventional Confocal These two micrographs are of the same intact gastrula-stage Drosophila embryo that has been stained with a fluorescent probe for actin filaments. (A) The conventional, unprocessed image is blurred by the presence of fluorescent structures above and below the plane of focus. (B) In the confocal image, this out-of-focus information is removed, resulting in a crisp optical section of the cells in the embryo. (Courtesy of Richard Warn and Peter Shaw.) Challenges in bioseparations DNA sequencing: currently up to 1500 base pair resolution Post-genomic era: Single cell content analysis DNA fingerprinting Protein analysis Gel Electrophoresis Fiber radius 3.0 nm Pore radius 100 nm Slab gel electrophoresis DNA in Cavity Arrays ! 1µm M13mp18 (7250bp), L=3.4µm, Rg=0.26µm Lambda (48502bp), L=22µm, Rg=0.73µm Current directions Center for functional Nanomaterials at BNL Quantum dot end-labels DNA length = 17µm Quantum dot diameter = 100Å Resolution? Resolution Single molecule resolution blur ...
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