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COURSE GOALS: Use appropriate descriptive language and terms. * Understand the appropriate use of techniques to study material at the histological level. * Identify tissues. (Epithelial, Nervous, Muscle, Connective Tissue) Identify composite of tissues as an organ. *We begin to address these goals TODAY! Exercise: Learn the Language of Histology Examine your image and write a description that can be used to identify it. - Form groups of four--> same letter/numbers 1-4 examples: group 1: A1, A2, A3, A4 group 2: E1, E2, E3, E4 - Mix up your group’s images and descriptions and exchange them with another group. Yours 1-4 Theirs 1-4 - Work as a group to match the description with the image. - Check results on slide that is coming up Learn the Language of Histology BEFORE: “-looks like an abstract painting…..Looks like it was colored with colored pencils.” “-looks like a bunch of worms on a pink background…..3 white areas- bigger one to the left of picture.” “-tree bark with blue bugs crawling on it.” “-looks like a yellow river with some kind of fish swimming upstream and the edges of the river are made up of big hunks of ice.” “-the center has light thin dashes/stripes. One side of the slide has rectangular blocks. The other border is light.” Learn the Language of Histology After: Cross section of nonkeratinized stratified squamous epithelium facing a lumen. Undifferentiated cells at basal end of epithelium are smaller with darker nuclei and are positioned on a loose connective lamina propria; likely esophagus. ORGANISM IMAGE TISSUE PREPARATION 1) Fixation 2) Embedding 3) Sectioning 4) Staining 5) Imaging Tissue Preparation 1) Fixation: halts cell metabolism, preserves cell/tissue structure • Different fixatives- different degrees of protein denaturing • Choice of fixative depends on level of analysis – Light microscopy: formaldehyde, glutaraldehyde – Electron Microscopy: glutaraldehyde, osmium Tissue Preparation 1) Fixation Mode of action: - cross link proteins: glutaraldehyde/formalin - precipitate proteins: methanol* - react with membrane lipids: osmium tetroxide - membranes become permeable Produce different levels of tissue preservation * Methanol often solubilizes membranes Tissue Preparation 2) Embedding: infiltrate water-filled spaces with embedding medium Series of soluble replacements H2O/fix alcohol xylene embedding medium • Dehydration: replace with ethanol, acetone • Clearing: replace with xylene • Embedding: replace with paraffin wax, plastic resin Tissue Preparation 3) Sectioning 3 dimensions --> 2 dimensions Orientation: Planes of Section - whole mount (unsectioned) cross section longitudinal section random Planes of Section Kidney Tubules KIDNEY CORTEX Box #17, slide 51 (B), 52 (T) Nicole Monteiro – Wed, 03/25/2009 Tissue Preparation 3) Sectioning Section thickness depends on imaging method. -Microtome (Light microscopy) ~ 1-10 um -Cryostat - frozen tissues (Light microscopy) ~ 1-30um -Ultramicrotome (Electron Microscopy) ~ 0.1 um HistoTip: For sharper images, cut thinner sections. Tissue Preparation 4) Staining* • Nonspecific: general • Specific: identified molecules * To be discussed in detail in a few days Tissue Preparation 4) Imaging ----> Microscopy • Compound light microscope - light • Confocal microscopy - coherent light • Electron microscopy- electron beam Microscopy Imaging Resources Websites: links are on course website- Review materials NIKON-- recommended for clarity http://www.microscopyu.com/articles/optics/ ZEISS http://zeiss-campus.magnet.fsu.edu/ OLYMPUS http://www.olympusmicro.com/primer/virtual/virtual.html Compound microscope Optical Components - Light source - Diaphragm - Condenser - Lenses - objectives - oculars Nikon E200 2 Sets of Conjugate Focal Planes: 1) Image-forming (field planes) 2) Illuminating (aperture planes) The sets of focal planes are in focus and superimposed in properly aligned microscope http://www.microscopyu.com/articles/formulas/formulasconjugate.html Conjugate Planes: 1) Focused at 1, focused at all (pointers etc.) 2) Planes alternate in succession: illumination / image-form 3) Poor image quality: dirt, dust, poor alignment Magnification: Objective lens - gathers light from specimen - projects a magnified, real image up into body tube. Ocular lens - produces a secondarily enlarged real image projected by the objective. - can be fitted with scales, markers or crosshairs whose images can be superimposed on the image of the specimen. Compound microscope MAGNIFICATION Magnifying power of Ocular lens (Mocular) Magnifying power of Objective lens (Mobjective) Visual Magnification = Mocular X Mobjective Compound Microscope Resolution= Resolving Power -the smallest distance (d) at which two objects can be successfully distinguished. Resolution (d): d = (0.61 x )/ NA = wave length NA= numerical aperture Quick Question: How can you make d smaller? Numerical Aperture (NA): measure of objective’s ability to collect light from specimen NA= n sin n = refractive index of medium = one half of angular aperture http://www.microscopyu.com/tutorials/java/imageformation/airyna/index.html Resolution: d = 0.61 x NA NA=0.22 NA=1.0 NA= n sin Refractive index (η) of different media Air=1.0003 Water=1.33 Immersion Oil=1.515 Resolution versus Wavelength Resolution: d= 0.61 x NA Wavelength (nanometers) 360 400 450 500 550 600 650 700 Resolution (micrometers) .19 .21 .24 .26 .29 .32 .34 .37 Resolving Distance (d) Human eye Light Microscope Scanning Electron Microscope Transmission Electron Microscope 0.2 mm 0.2 um 2.5 nm 1.0 nm Resolution: d= (0.61 x )/ NA HistoTip: Avoid confusion when discussing resolution. Increased resolution or resolving power usually means a SMALLER value of d (distance). PROBLEM: Objective lens A: Magnification = 40X N.A. = 0.45 Objective lens B: Magnification = 40X N.A. = 0.80 -->Which objective lens would give the sharper image and why? PROBLEM: You photograph some liquid crystalline DNA using objective D and objective E. You then enlarge the images to the same size using Photoshop in the manner described below. Image D : 20X objective, NA= 0.40, enlarged 10X Image E : 4X objective, NA= 0.10, enlarged 50X Which image would be sharper and why? Empty Magnification: an image is enlarged, but no additional detail is resolved. A : 20X objective, NA= 0.40, enlarged 10X. Magnified 200 B : 4X objective, NA= 0.10, enlarged 50X. Magnified 200 HistoTip: Maximum useful magnification=1000 X N.A. Empty Magnification: an image is enlarged, but no additional detail is resolved. A : 20X objective, NA= 0.40, enlarged 10X. B : 4X objective, NA= 0.10, enlarged 50X. HistoTip: Maximum useful magnification=1000 X N.A.