BMB 251 1st Edition Lecture 10Outline of Last Lecture I. Optical diffraction effectsII. ResolutionIII. Light Microscopya. Dark Fieldb. Bright FieldIV. Fluorescent MicroscopyV. AntibodiesOutline of Current Lecture VI. PhotobleachingVII. FRABVIII. Electron Microscopesa. Immunogoldb. SEMc. TEMIX. Cryo-Electron Microscopya. Single particle reconstructionCurrent Lecture- Indirect immunocyto chemistry: using an unlabeled primary antibody and then detecting it with a group of labeled secondary antibodies gives a stronger chemical signal- Genes can code for proteins that are inherently fluorescent; genetic engineering enables creation of lines of cells or organisms that make their own visible tags and labels, without the introduction of foreign moleculeso Green fluorescent protein (GFP) is used for this; it was isolated from a jellyfish Can be fused with a protein which will remain functional- Photobleachingo Uses a higher laser to bleach the cell; the cell will never be fluorescent again on its owno The laser is put right onto the desired part of the cell and some of it gets hit with the intense beam selectively bleaches only one part of the cello The bleaching causes a dark spot within the cell that is easily visible- Fluorescence Recovery After Bleaching (FRAB)o After bleaching, the fluorescence starts to recover where the dark spot is within the cello Fluorescent molecules are not recovering form the bleaching, but rather more fluorescent molecules are diffusing back into the cellThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.o How long it takes for the proteins in Golgi to turnover (come back): half-life is about one hour- Deconvolution: way in which the out-of-focus fluorescent images can be “cleaned up” using mathematics- Confocal microscopy: way in which the out-of-focus fluorescent images can be “cleaned up” using physics- Electron microscopes allow higher resolutions can now see actual macromoleculeso Wavelength of electron decreases as its velocity increaseso Problems: Resolution could be 0.002 nm, but really only shows about 0.1 nm because of lens aberrations (numerical aperture able to be used is very tiny) Specimen preparation Contrast Radiation damageo Specimen must be placed in a vacuum because electrons are scattered by collisions with air moleculeso Electrons then accelerated and pass through a tiny hole to create a beamo Magnetic coils placed at different intervals focus electron beamo **Contrast in the electron microscope depends on atomic number of atoms in the specimen Higher atomic number =more electrons scattered = higher contrast- Immunogold electron microscopy: analogous to antibodies with fluorescent microscopes, but is used with the electron microscopeo Incubate thin section with primary antibodyo Attach colloidal gold particle to secondary antibody (this is seen as a black dot in electron microscope)- Scanning electron microscope (SEM): directly produces image of 3D structure of the surface of a specimen (smaller, simpler, cheaper than electron microscope)o Uses electrons scattered or emitted from specimen’s surface o Can only examine surface features whole cell and tissues rather than subcellular organelles are used with this microscopeo Much thinner samples needed than with the light microscope- Transmission electron microscope (TEM): uses heavy metals (such as uranium, lead, platinum, osmium, or gold) containing many electrons to create a 2D image of the cell; otherwise, it would be transparent and biologists would not be able to see molecular images (such as ribosomes)o Uses electrons that have passed through the specimen to create an imageo Can also examine surface features, but at higher resolutions than SEM and can depict macromoleculeso Radiation beams are electrons with very short wavelengthso Lenses are magnets which can focus the electron beam, unlike the glass lenses in light microscopyo Samples must be extremely thin (between about 50-100nm)o Samples are both fixed and dead- Negative staining: used to see finer details (small macromolecular assemblies) o Smallest metal particle limits the resolution- Since the negative staining has limited resolution, cryoelectron microscope is usedo Rapid freezing to form vitreous ice in order to see interior 3D structures or viruses/organelles at high resolutionso Does not need to be fixed, stained or dried- Single-particle reconstruction: obtain thousands of identical images and combine them to produce an averaged image (gets rid of the background
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