Download Tissue Preparation

ANNOUNCEMENTS
Lab starts this week!
ORGANISM
- Tuesday or Wednesday 1:25 ISB 264
-  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)
IMAGE
Tissue Preparation
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
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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
Tissue Preparation
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
Planes of Section
3) Sectioning
3 dimensions --> 2 dimensions
Orientation: Planes of Section
- whole mount (unsectioned)
- cross section
- longitudinal section
- random
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Tissue Preparation
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
Tissue Preparation
4)  Staining*
•  Nonspecific: general
•  Specific: identified molecules
* To be discussed in detail in a few days
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Microscopy
Tissue Preparation
4) Imaging ----> Microscopy
Imaging Resources Websites: links are on
course website- Review materials
•  Compound light microscope - light
NIKON-- recommended for clarity
http://www.microscopyu.com/articles/optics/
•  Confocal microscopy - coherent light
ZEISS
http://zeiss-campus.magnet.fsu.edu/
•  Electron microscopy- electron beam
Compound microscope
Optical Components
- Light source
- Diaphragm
- Condenser
- Lenses
- objectives
- oculars
Nikon E200
OLYMPUS
http://www.olympusmicro.com/primer/virtual/virtual.html
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
http://www.microscopyu.com/articles/formulas/formulasconjugate.html
Magnification:
Objective lens
3) Poor image quality:
dirt, dust, poor
alignment
Compound microscope
- gathers light from specimen
MAGNIFICATION
-  projects a magnified, real
image up into body tube.
Magnifying power of Ocular lens (Mocular)
Ocular lens
Magnifying power of Objective lens (Mobjective)
- 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.
Visual Magnification = Mocular X Mobjective
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Compound Microscope
Numerical Aperture (NA): measure of objective’s
ability to collect light from specimen
NA= n sin α
Resolution= Resolving Power
-the smallest distance (d) at which two
objects can be successfully distinguished.
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?
http://www.microscopyu.com/tutorials/java/imageformation/airyna/index.html
Resolution: d = 0.61 x λ
NA
NA=0.22
NA= n sin α
Refractive index (η) of different media
Air=1.0003
Water=1.33
Immersion Oil=1.515
NA=1.0
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Resolving Distance
(d)
Resolution versus Wavelength
Resolution: d= 0.61 x λ
NA
Wavelength (nanometers)
360
400
450
500
550
600
650
700
Resolution (micrometers)
.19
.21
.24
.26
.29
.32
.34
.37
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?
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 λ)/ NA
HistoTip: Avoid confusion when discussing
resolution. Increased resolution or resolving power
usually means a SMALLER value of d (distance).
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 200
B : 4X objective, NA= 0.10, enlarged 50X. Magnified 200
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.
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:
NAa<NAb<NAc
http://www.microscopy.fsu.edu/primer/anatomy/numaperture.html
Resolution determined by overlap of Airy disks.
http://www.microscopy.fsu.edu/primer/anatomy/numaperture.html
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Point sources
of light appear
as Airy
diffraction
patterns
(disks) in the
microscope.
“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.”
http://www.microscopyu.com/articles/formulas/formulasresolution.html
Condenser Aperture Setting and Image Quality
Contrast increases as less light passes through condenser
(a) 90% (b) 60 % (c) 20%
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