Tissue Preparation ORGANISM IMAGE TISSUE PREPARATION. 1) Fixation: halts cell metabolism, preserves cell/tissue structure
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1 Lab starts this week! ANNOUNCEMENTS - Tuesday or Wednesday 1:25 ISB Read Lab 1: Microscopy and Imaging (see Web Page) - Getting started on Lab Group project - Organ for investigation - Lab project team members (3 in all) ORGANISM IMAGE TISSUE PREPARATION 1) Fixation 2) Embedding 3) Sectioning 4) Staining 5) Imaging 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 1
2 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 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 Planes of Section 3) Sectioning 3 dimensions --> 2 dimensions Orientation: Planes of Section - whole mount (unsectioned) - cross section - longitudinal section - random 2
3 Kidney Tubules 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. KIDNEY CORTEX Box #17, slide 51 (B), 52 (T) Nicole Monteiro Wed, 03/25/2009 4) Staining* Nonspecific: general Specific: identified molecules * To be discussed in detail in a few days 3
4 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 ZEISS OLYMPUS Optical Components Compound microscope - Light source - Diaphragm - Condenser - Lenses - objectives - oculars 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 Nikon E
5 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 (M ocular ) Magnifying power of Objective lens (M objective ) Visual Magnification = M ocular X M objective 5
6 Compound Microscope Resolution= Resolving Power -the smallest distance (d) at which two objects can be successfully distinguished. Numerical Aperture (NA): measure of objective s ability to collect light from specimen NA= n sin α n = refractive index of medium α = one half of angular aperture Resolution (d): d = (0.61 x λ)/ NA λ= wave length NA= numerical aperture Quick Question: How can you make d smaller? Resolution: d = 0.61 x λ NA NA= n sin α Refractive index (η) of different media Air= Water=1.33 Immersion Oil=1.515 NA=0.22 NA=1.0 6
7 Resolution versus Wavelength Resolution: d= 0.61 x λ NA Wavelength (nanometers) Resolution (micrometers) Human eye Light Microscope Scanning Electron Microscope Transmission Electron Microscope Resolving Distance (d) Resolution: d= (0.61 x λ)/ NA 0.2 mm 0.2 um 2.5 nm 1.0 nm 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. = >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? 7
8 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. Image of specimen: - made of points appearing as Airy patterns with center disk. - result of light diffracted as it passes through specimen. - size influenced by NA: NA a <NA b <NA c Resolution determined by overlap of Airy disks. 8
9 Criterion for resolution: the central ring in the diffraction pattern of one image should fall on the first dark interval between the Airy disk of the other and its first diffraction ring. Point sources of light appear as Airy diffraction patterns (disks) in the microscope. Condenser Aperture Setting and Image Quality Contrast increases as less light passes through condenser (a) 90% (b) 60 % (c) 20% 9
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