Histology- microscopic structure assoc. with function!Tissue: cells + extracellular matrix!Organs: Basic tissue types together to accomplish something!!Objectives!1) Optical properties!2) Light and electron microscopy!3) Tissue preparation!4) 2D and 3D images!!!1) Optical properties!Detection: being an able to tell that an object is there!•Resolution: being able to tell two object apart that are close together!•Resolving power: min. distance 2 objects can be apart and be seen as separate!◦eye: 250 microns, EM: 0.001micron!‣0.61x lamda (wavelength)/NA!‣NA = numerical aperture= ability of a lens to gather light!•wavelength= distance between 2 peaks!•Condenser lens focuses light rays!•Objective lens collects the light rays to make an image!•Magnification= size of an image compared to the size of the object!•Empty magnification= magnifying beyond the resolving power of the lens!◦Resolution increases, the magnification can increase, can see more details!•2) Light and electron microscopy!LM- use light to create an image!•Resolving power= 0.25 microns!◦Brightfield- stained!◦Phase contrast- unstained and live cells!◦Nomarkski/DIC- unstained and live cells!◦Fluorescense- localize proteins!◦Laser scanning confocal- get 3D views!◦EM- uses electrons to create an image!•Resolving pwoer= 0.001 microns!◦Transmission EM (TEM)!◦Scanning EM (SEM)!◦!3) Preparation of tissues!For LM:!•Fixation (aka crosslink proteins to preserve biological structure), Embedding in paraffin/plastic, ◦Sectioning into small pieces, Staining (detection method)!Contrast: image must contrast with surroundings so that we can see it!•Phase of light altered by passing through thick regions-> amplify change-> visible structures!◦Dyes: colored molecule that binds to a substrate. Can use these to get contrast. Dyes can bind ◦to cellular components. Red stain, red wavelength passes through, others don't, you can see it.!Hematoxylin- positively charged blue dye, tissue that stains with basic dyes= basophilic!‣Eosin- negatively charged red dye, tissues that stain with acidic dyes= acidophilic!‣H&E!‣Azan- collagen->blue!‣Verhoeff- elastin-> black!‣Fluorescent dyes: different excitation and emission wavelengths!‣!4) 2D and 3D images!Can use confocal microscope to eliminate out of focus light that would blur the image, uses a •pinhole aperture!3-D projections of optical sections, don't have to mechanically section them!◦!5) Detection method: depends on question!overall structure- stain!•specific type of molecule- stain, histochemistry (ex. Periodic Acid Schiff [PAS] causes chemical rxn-•> magenta stains when you have carbohydrates)!specific enzyme!•enzyme histochemistry= enzyme+ substrate-> product 1, product 1+ chromagen-> product 2!◦Chromagen= a color reagent that binds to your product!◦specfic protein- !•immuno-detection: primary antibody that binds the substrate, then add the secondary antibody ◦(which is bound to a visible marker) that binds the primary antibody. Can tag with enzymes (immuno-enzymehistochemistry), gold (-electron microscopy), or fluor. molecules (-fluorescense)!GFP-tagged proteins!◦Can visualize it in living cells!‣GFP fusion protein-> cellular locations of the protein will fluoresce!‣cells where a gene is transcribed-!•GFP-reporter= reporter construct, GFP is joined to the promoter of interest, cells where ◦promoter is transcribed will express GFP and fluoresce !In situ hybridization!◦Autoradiography= radioactivity is added to whatever probe you're using, incubate the tissues ◦with the radioactive probe. Tissues with probe attached will be radioactive.!Detect and localize specific mRNAs using cRNA from radio-labeled nucleotides (incubate, binds ◦to complementary sequence, detect)!specific DNA sequence- !•In situ hybridization!◦FISH= Fluorescense In Situ Hybridization!‣Fluor. dyes conjugated to nucleotide probes, can visualize many targets ex. HIV infection
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