COURSE GOALS: Use appropriate descriptive language and terms. * Understand the appropriate use of...
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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!
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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 1-4examples: 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
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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.”
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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.
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ORGANISM
IMAGE
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TISSUE PREPARATION
1) Fixation
2) Embedding
3) Sectioning
4) Staining
5) Imaging
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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
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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
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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
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Tissue Preparation
3) Sectioning
3 dimensions --> 2 dimensions
Orientation: Planes of Section
- whole mount (unsectioned)- cross section- longitudinal section- random
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Planes of Section
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KIDNEY CORTEXBox #17, slide 51 (B), 52 (T)Nicole Monteiro – Wed, 03/25/2009
Kidney Tubules
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Tissue Preparation3) 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.
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Tissue Preparation
4) Staining*
• Nonspecific: general
• Specific: identified molecules
* To be discussed in detail in a few days
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Tissue Preparation
4) Imaging ----> Microscopy
• Compound light microscope - light
• Confocal microscopy - coherent light
• Electron microscopy- electron beam
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Microscopy
Imaging Resources Websites: links are on course website- Review materials
NIKON-- recommended for clarityhttp://www.microscopyu.com/articles/optics/
ZEISS http://zeiss-campus.magnet.fsu.edu/ OLYMPUShttp://www.olympusmicro.com/primer/virtual/virtual.html
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Compound microscope
Nikon E200
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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
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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
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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.
Magnification:
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MAGNIFICATION
Magnifying power of Ocular lens (Mocular)
Magnifying power of Objective lens (Mobjective)
Visual Magnification = Mocular X Mobjective
Compound microscope
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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
Compound Microscope
Quick Question: How can you make d smaller?
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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
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NA=0.22 NA=1.0
Resolution: d = 0.61 x NA
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Refractive index (η) of different media Air=1.0003 Water=1.33 Immersion Oil=1.515
NA= n sin
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Resolution versus Wavelength
Resolution: d= 0.61 x NA
Wavelength (nanometers) Resolution (micrometers)360 .19400 .21450 .24500 .26550 .29600 .32650 .34700 .37
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Resolving Distance (d)
Human eye 0.2 mmLight Microscope 0.2 umScanning Electron Microscope 2.5 nmTransmission Electron Microscope 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).
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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?
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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?
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Empty Magnification: an image is enlarged, but no additional detail is resolved. A : 20X objective, NA= 0.40, enlarged 10X. Magnified 200B : 4X objective, NA= 0.10, enlarged 50X. Magnified 200HistoTip: Maximum useful magnification=1000 X N.A.
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HistoTip: Maximum useful magnification=1000 X N.A.