Stabilizing Agents
RL BALATBAT MSPH 2
Importance
Prevents autolysis
Prevents putrefaction
RL BALATBAT MSPH 3
Should disable intrinsic biomolecules
RL BALATBAT MSPH 4
Denatures proteins by coagulation
RL BALATBAT MSPH 5
Denatures proteins by forming additive
compounds
RL BALATBAT MSPH 6
A. Heat Fixation
Routinely used with bacteria and archaea
Adv: Preserves overall morphology
Disadv: Damages internal structures
RL BALATBAT MSPH 7
B. Perfusion
Fixation via blood flow
Fixative is injected into the heart with the
injection volume matching cardiac output
Adv: Preserves perfect morphology
Disadv: Expensive
RL BALATBAT MSPH 8
C. Immersion
Tissue is immersed in fixative (20:1)
RL BALATBAT MSPH 9
A. Phosphate buffered formalin
10ml 10% formalin (40%) + 900ml DW
NaH2PO4H2O
Na2HPO4
pH: 6.8
Fixation time: 12-24h
RL BALATBAT MSPH 10
3 hrs vs 8 hrs
RL BALATBAT MSPH 11
RL BALATBAT MSPH 12
B. Formal calcium
100ml 40% formalin + 900ml DW
CaCl
RL BALATBAT MSPH 13
C. Formal saline
100ml 40% formalin + 900ml DW
NaCl
RL BALATBAT MSPH 14
3 hrs vs 8 hrs
RL BALATBAT MSPH 15
D. Zinc formalin
ZnSO4
Deionized H2O
100ml 40% formalin
RL BALATBAT MSPH 16
RL BALATBAT MSPH 17
E. Zenkers fixative
HgCl2 + DW
KCr2O2
Hac
RL BALATBAT MSPH 18
RL BALATBAT MSPH 19
F. Hellys fixative
KCr2O2
NaSO4
HgCl
50 ml 40% formalin + 1000ml DW
RL BALATBAT MSPH 20
RL BALATBAT MSPH 21
g. B-5 fixative
C2H3NaO2
HgCl
2ml 40% formalin + 200ml DW
RL BALATBAT MSPH 22
RL BALATBAT MSPH 23
H. Bouins solution
Picric cid
Hac
Hg
250ml 40% formalin
RL BALATBAT MSPH 24
RL BALATBAT MSPH 25
I. Hollandes solution
Copper acetate
Picric acid
Hac
100ml 40% formalin
RL BALATBAT MSPH 28
L. Carnoys solution
100% ETOH
Hac
Chloroform
RL BALATBAT MSPH 29
M. Methacarn
100% METOH
Hac
Chloroform
RL BALATBAT MSPH 30
N. Alcoholic formalin
100ml 40% formalin
900ml 95% ETOH
Ca acetate
RL BALATBAT MSPH 31
O. Formol acetic alcohol
10ml 40% formalin
85ml 100% ETOH
HAc
RL BALATBAT MSPH 32
RL BALATBAT MSPH 1
Gr. Mikros + temnein
RL BALATBAT MSPH 2
Blade
Base
Block Holder
RL BALATBAT MSPH 3
RL BALATBAT MSPH 4
RL BALATBAT MSPH 5
RL BALATBAT MSPH 6
A. Blades
Steel- for light microscopy
Glass- for light and electron microscopy
Industrial Grade Diamond- used for
bones, teeth and hard plant tissues
Gem Quality Diamond- for thin sections
for electron microscopy
RL BALATBAT MSPH 7
A.Blades
Edge
Planoconcave edge: for sledge and some
rotary types
Planoconcave: for celloidin blocks
Plane wedge: used for all types of
microtomes
Biconcave: for rocking and sledge types
Tool/Chisel edge: for hard tissues
RL BALATBAT MSPH 8
B.Base
Platform that secures the blade
RL BALATBAT MSPH 9
C.Block Holder
Holds the specimen in place
RL BALATBAT MSPH 10
1. Rotary
2. Rocking
3. Base-sledge
4. Sliding
5. Freezing
6. Vibrating
7. Saw
8. Cryostat
9. Ultra-Microtome
RL BALATBAT MSPH 11
AKA Minot microtome
Features:
Knife is stationary
Block is moved vertically by the rotary
action of the hand wheel
Maybe manually operated or automated
Thickness = 0.5 60m
RL BALATBAT MSPH 12
Advantages:
Ideal for cutting serial sections
More stable
Knife is heavier, so less vibrations
Cutting angle of knife is
adjustable
Disadvantage
Can not cut large or hard tissues
RL BALATBAT MSPH 13
RL BALATBAT MSPH 14
1. Rotary
2. Rocking
3. Base-sledge
4. Sliding
5. Freezing
6. Vibrating
7. Saw
8. Cryostat
9. Ultra-Microtome
RL BALATBAT MSPH 15
Oldest type
Features:
Knife is stationary
Block is moved in an arc to strike the
knife
RL BALATBAT MSPH 16
Advantages:
Mechanism is simple
Machine can last for hundreds of years
Disadvantages:
Size of blocks that can be cut is limited
Cutting produces vibrations
Cutting angle cannot be adjusted
Serial section is not possible
RL BALATBAT MSPH 17
RL BALATBAT MSPH 18
1. Rotary
2. Rocking
3. Base-sledge
4. Sliding
5. Freezing
6. Vibrating
7. Saw
8. Cryostat
9. Ultra-Microtome
RL BALATBAT MSPH 19
Originally designed for cutting very large
blocks
Commonly used in neuropathology and
ophthalmic pathology
Features:
Block is fixed
Blade is moved horizontally against the block
Thickness = < 60m
RL BALATBAT MSPH 20
RL BALATBAT MSPH 21
Advantages:
Stable
No vibrations
Knife angle is adjustable
24 cm knife
Disadvantages
Heavy blade
Slower in use
RL BALATBAT MSPH 22
1. Rotary
2. Rocking
3. Base-sledge
4. Sliding
5. Freezing
6. Vibrating
7. Saw
8. Cryostat
9. Ultra-Microtome
RL BALATBAT MSPH 23
Features:
Block is fixed
Blade is moved
horizontally against the
block
RL BALATBAT MSPH 24
Advantages:
Adjustable angle
Ideal for hard and thick specimen
Designed for paraffin and celloiden blockes
Disavantages:
Similar with Base-sledge
RL BALATBAT MSPH 25
1. Rotary
2. Rocking
3. Base-sledge
4. Sliding
5. Freezing
6. Vibrating
7. Saw
8. Cryostat
9. Ultra-Microtome
RL BALATBAT MSPH 26
Freeze the tissue thru CO2 blast or Peltier
module (-10C to -20C)
Tissue must be fixed with formalin
RL BALATBAT MSPH 27
Features:
Similar with sliding microtome
RL BALATBAT MSPH 28
Advantages:
Does not require extensive processing
Processing time is reduced
Disadvantages:
Formalin may overharden the tissue
Sections may melt and crumple
RL BALATBAT MSPH 29
RL BALATBAT MSPH 30
1. Rotary
2. Rocking
3. Base-sledge
4. Sliding
5. Freezing
6. Vibrating
7. Saw
8. Cryostat
9. Ultra-Microtome
RL BALATBAT MSPH 31
Designed to cut fresh unfixed tissue
Cutting power of the blade is from high
speed vibration
Amplitude if vibration can be adjusted
by altering the electrical voltage applied
to the blade
RL BALATBAT MSPH 32
Features:
Block is fixed
Knife moves horizontally against the block
Thickness = 10 30m
RL BALATBAT MSPH 33
Advantages:
Ideal for diagnosis
Does not alter the macromolecular
component of the tissue
Sections can be used in histochemistry and
serology
RL BALATBAT MSPH 34
1. Rotary
2. Rocking
3. Base-sledge
4. Sliding
5. Freezing
6. Vibrating
7. Saw
8. Cryostat
9. Ultra-Microtome
RL BALATBAT MSPH 35
Designed for cutting extremely hard and
brittle industrial materials
Features:
Samples embedded in resin are moved
extremely slowly against the rotating saw
(600rpm)
RL BALATBAT MSPH 36
Advantage:
Can cut hard and brittle specimen
Disadvantage:
Very thin sections are not possible (>30m)
RL BALATBAT MSPH 37
1. Rotary
2. Rocking
3. Base-sledge
4. Sliding
5. Freezing
6. Vibrating
7. Saw
8. Cryostat
9. Ultra-Microtome
RL BALATBAT MSPH 38
Used in medicine to cut histo slides
Uses liquid helium to maintain low
temperature (-20C to -185C)
Features:
Blade is stationary
Block is moved against the blade
RL BALATBAT MSPH 39
RL BALATBAT MSPH 40
Advantages
Can cut up to 1m thick
Reduce processing time from prep to
examination (10-20 mins.)
Can be used in serology
Disadvantage:
Quality of section is poor compared to wax
mounted types
RL BALATBAT MSPH 41
1. Rotary
2. Rocking
3. Base-sledge
4. Sliding
5. Freezing
6. Vibrating
7. Saw
8. Cryostat
9. Ultra-Microtome
RL BALATBAT MSPH 42
Exclusive for Electron Microscopy
Knives made from glass, diamond or sapphire
Advantage:
Section thickness = 10nm
Disadvantage:
Expensive ($10K)
RL BALATBAT MSPH 43
RL BALATBAT MSPH 44
RL BALATBAT MSPH
MICROSCOPE
SLIDES
1
TYPES
Permanent
Temporary
RL BALATBAT MSPH
Wet Mounts
Dry Mounts
Heat Fixed
Smear
Squash
2
I. PERMANENT/PREPARED MOUNT
Long-term
Use mounting medium
RL BALATBAT MSPH
3
II. TEMPORARY
For short time observations
RL BALATBAT MSPH
4
III. WET MOUNTS
Use liquid mountant:
water,
RL BALATBAT MSPH
nss,
immersion oil- for nonpolar organic
samples
Glycerine- high refractive index
Mostly temporary slides
For aquatic samples
RL BALATBAT MSPH
For hair, feathers, airborne
particles (dust, pollens), insect
parts
Adv: most simple type of slide
6
V. HEAT FIXED
No cover glass is used
Heat fixing makes the cells stick to the slide
RL BALATBAT MSPH
7
VI. SMEAR
For body fluids
Angle of 2nd slide determines the length of the smear
RL BALATBAT MSPH
>45: shorter
<45: 1.5
8
RL BALATBAT MSPH
9
VII. SQUASH
For soft samples
RL BALATBAT MSPH
10
RL BALATBAT MSPH 1
Terrestrial Invertebrates
5-10% Isoflurane
Sevoflurane
5-10% Halothane
10-20% CO2
RL BALATBAT MSPH 2
RL BALATBAT MSPH 3
Isoflurane
RL BALATBAT MSPH 4
Aquatic Species
MS-222 (Tricaine Methanesulfonate)
Benzocaine dissolved in acetone
100mg of anesthesia/L of water
RL BALATBAT MSPH 5
Aquatic Species
Chloral Hydrate/Methanol- annelids, mollusks,
tunicates, bryozoan, turbellarians
sprinkled on water surface
RL BALATBAT MSPH 6
Vertebrates Isoflurane
CO2
Chloretone
Ethanol
Chloroform
MgCl
Isobutanol
Lidocaine
RL BALATBAT MSPH 7
RL BALATBAT MSPH 8
Shaudins for protozoa
Aceto-orcein- for chromosomes
Bouins- GI tract, soft tissues, invertebrates
Gendre Solution- preserves glycogen and
other carbohydrates
Hollande Solution- stabilizes RBC
membranes and granules of Eosiophil and
endocrine cells
RL BALATBAT MSPH 9
Gomoris- with Hg content
Hellys- has Hg, preserves cytoplasm
Sinhas- destains hematoxylin
Smiths- fixes yolks, reacts w/ formalin
RL BALATBAT MSPH 11
Alcohol
Can be used in storing specimen
Used at RT
RL BALATBAT MSPH 12
Xylene
Benzene- has low boiling point
Toluene
RL BALATBAT MSPH 13
Paraffin
Soft: for 5 7 m sections
Melting point: 50-55C
Hard: for <5 m sections
Melting point: 56-68C
RL BALATBAT MSPH 14
Nitrocellulose
Water waxes
Gelatin
Agar
Ice
RL BALATBAT MSPH 15
Natural Dyes
Hematoxylene- from the Logwood tree
(Hematoxylon campechianum)
RL BALATBAT MSPH 16
RL BALATBAT MSPH 17
Natural Dyes
Indigo- from Indigofera
RL BALATBAT MSPH 18
RL BALATBAT MSPH 19
Natural Dyes
Orcein and Litmus- from Lecanora and Rocella
RL BALATBAT MSPH 20
RL BALATBAT MSPH 21
Natural Dyes
Saffron- from Crocus
RL BALATBAT MSPH 22
RL BALATBAT MSPH 23
Natural Dyes
Berberine- from barberry
RL BALATBAT MSPH 24
RL BALATBAT MSPH 25
Natural Dyes
Brazilin- from brazilwood (Caesalpinia)
RL BALATBAT MSPH 26
Natural Dyes
Cochineal and carmine- from Coccus cacti
RL BALATBAT MSPH 27
RL BALATBAT MSPH 28
Synthetic Dyes Janus green B
Methylene blue Alizarine red
Azure A and B Brilliant Cresyl blue
Eosin
Toluidine blue
Methyl violet
Fast green
Congo red
RL BALATBAT MSPH 29
RL BALATBAT MSPH 30
RL BALATBAT MSPH 31
RL BALATBAT MSPH 32
RL BALATBAT MSPH 33
RL BALATBAT MSPH 34
Resinous
Natural or Synthetic
Most frequently used
Provide firm adhesion of the coverslip
Slow hardening
Uses Xylene as solvent
Premature darkening
Yellows with age
RL BALATBAT MSPH 35
Resinous
Canada balsam- from the bark of Abies balsamea
Phenol balsam
Dammar balsam
Euparal
RL BALATBAT MSPH 36
RL BALATBAT MSPH 37
Aqueous
cheaper, safer, little prep time
Needs anti-fungal agents
Types
Syrups
Gelatin
Gum Arabic
RL BALATBAT MSPH 38
Aqueous
Syrups
Apathys medium
RL BALATBAT MSPH 39
Aqueous
Gelatin
Glycerine jelly
Glycerine-glycerol
RL BALATBAT MSPH 40
Aqueous
Gum Arabic
Farrants medium
RL BALATBAT MSPH 41
RL BALATBAT MSPH 42
Cooper JE. 2011. Anesthesia, Analgesia and
Euthanasia of Invertebrates. ILAR J. 52(2):
196-204.
http://www.acuc.berkeley.edu/guidelines/ane
sthesia
http://journal.plastination.org/archive/jp_vol.
10/jp_vol.10_27-30.pdf
RL BALATBAT MSPH 43
1
RL BALATBAT MSPH
History
85-150 AD- Ptolemy described a stick
appearing to bend in a pool of water
2
3
History
1st Century- glass was invented
Romans experimented with the
magnifying property of glasses
4
5
6
7
History
1st Century- Seneca (c. 4 BC-65 AD)
described actual magnification by a
globe of water
8
History
13th Century- Spectacles were invented
1268- Roger Bacon made the earliest
recorded comment on the use of
lenses for optical purposes
9
10
History
1590- the first compound microscope was
invented
Hans and Zacharias Jansen mounted 2
lenses in a tube, making the 1st
compound microscope
3 10 X magnification
11
12
History
1609- Galileo Galilei developed a
compound microscope with a convex
and concave lens
13
14
History
1665- Robert Hooke coined the word
cellula
Published Micrographia where he
documented a wide rage of
observations through the
microscope
15
16
17
18
19
20
21
22
History
1674- Anton van Leeuwenhoek
observed unicells under the
microscope (200X)
23
24
25
26
27
28
29
30
History
1826- Joseph Jackson Lister created
the achromatic lens
31
Lister combined lenses that improved
the image resolution by eliminating
chromatic aberrations
32
Listers notes 33
History
1872- Ernst Abbe (fr Zeiss Optical
Works) wrote a mathematical formula
that provided calculations for maximum
resolution
34
35
Abbe Condenser
History
1903- Richard Zsigmondy
developed the Ultramicroscope
36
History
Ultramicroscope- system is based on
light scattering and not light reflection
37
38
History
1932- Frits Zernike invented the phase-
contrast microscope
Allowed the study of colorless and
transparent biological materials
39
40
41
History
1938- Ernst Ruska developed the
electron microscope
42
History
Electron microscope depends on
electrons rather than light to view an
object
43
44
1953
45
History
1981- Gerd Binnig and Heinrich Rohrer
invented the Scanning Tunneling
Microscope
46
History
SEM- gives 3D images of objects down
to the atoms
47
48
49
50
END
51
TYPES
A. Optical D. Scanning Helium Ion
1. Compound (SHIM/HeIM)
2. Stereo
3. Confocal Laser E. Neutron
Scanning (CLSM)
4. Fluorescent F. Electron
1. Transmission (TEM)
B. X-ray 2. Scanning (SEM)
52
A. Optical
Use visible/UV light
Allow viewing of live
specimen
Magnification: 1X-1000X
Cheaper
Easy to maintain
53
TYPES
A. Optical D. Scanning Helium Ion
1. Compound (SHIM/HeIM)
2. Stereo
3. Confocal Laser E. Neutron
Scanning (CLSM)
4. Fluorescent F. Electron
1. Transmission (TEM)
B. X-ray 2. Scanning (SEM)
54
A1. Compound
Use 2 lens systems
Ocular (5X, 10X, 12X)
Objectives
Scanner (4X)
LPO (10X)
HPO (40X)
OIO (100X)
55
56
TYPES
A. Optical D. Scanning Helium Ion
1. Compound (SHIM/HeIM)
2. Stereo
3. Confocal Laser E. Neutron
Scanning (CLSM)
4. Fluorescent F. Electron
1. Transmission (TEM)
B. X-ray 2. Scanning (SEM)
57
A2. Stereo
Use 2 lens systems
Ocular (5X - 30X)
Objectives
2- 4 X
Useful in observing
opaque specimen
Supply a 3D view of the
specimen 58
A2. Stereo
59
60
TYPES
A. Optical D. Scanning Helium Ion
1. Compound (SHIM/HeIM)
2. Stereo
3. Confocal Laser E. Neutron
Scanning (CLSM)
4. Fluorescent F. Electron
1. Transmission (TEM)
B. X-ray 2. Scanning (SEM)
61
A3. Confocal Laser
Scanning (CLSM)
Used in research
Can acquire in-focus
images from selected
depths (i.e. optical
sectioning)
A computer is attached to
reconstruct the 3D image
62
A3. Confocal Laser
Scanning (CLSM)
63
64
TYPES
A. Optical D. Scanning Helium Ion
1. Compound (SHIM/HeIM)
2. Stereo
3. Confocal Laser E. Neutron
Scanning (CLSM)
4. Fluorescent F. Electron
1. Transmission (TEM)
B. X-ray 2. Scanning (SEM)
65
A4. Fluorescent
Instead of visible light, FM
uses light source w/
higher intensity
Ex. Mercury-vapor
lamps
Xenon lamps
lasers
66
67
A4. Fluorescent
fluorophores emit light
with longer wavelength
that results to magnified
image
68
69
Cryptosporidium oocyst
70
TYPES
A. Optical D. Scanning Helium Ion
1. Compound (SHIM/HeIM)
2. Stereo
3. Confocal Laser E. Neutron
Scanning (CLSM)
4. Fluorescent F. Electron
1. Transmission (TEM)
B. X-ray 2. Scanning (SEM)
71
B. X-ray
Use X-ray beam to create
an image
Higher resolution than
optical microscopes
72
B. X-ray
73
74
TYPES
A. Optical D. Scanning Helium Ion
1. Compound (SHIM/HeIM)
2. Stereo
3. Confocal Laser E. Neutron
Scanning (CLSM)
4. Fluorescent F. Electron
1. Transmission (TEM)
B. X-ray 2. Scanning (SEM)
75
C. Scanning Acoustic
Use focused sound waves
to generate an image
Physics- detects small
cracks and tensions
Biology- uncover stress,
tensions, elasticity in
tissues/organs
76
C. Scanning Acoustic
77
78
TYPES
A. Optical D. Scanning Helium Ion
1. Compound (SHIM/HeIM)
2. Stereo
3. Confocal Laser E. Neutron
Scanning (CLSM)
4. Fluorescent F. Electron
1. Transmission (TEM)
B. X-ray 2. Scanning (SEM)
79
D. Scanning Helium Ion
80
81
Al on Si
82
TYPES
A. Optical D. Scanning Helium Ion
1. Compound (SHIM/HeIM)
2. Stereo
3. Confocal Laser E. Neutron
Scanning (CLSM)
4. Fluorescent F. Electron
1. Transmission (TEM)
B. X-ray 2. Scanning (SEM)
83
E. Neutron
Still in experimental stage
(2013)
Higher resolution than EM
84
85
86
TYPES
A. Optical D. Scanning Helium Ion
1. Compound (SHIM/HeIM)
2. Stereo
3. Confocal Laser E. Neutron
Scanning (CLSM)
4. Fluorescent F. Electron
1. Transmission (TEM)
B. X-ray 2. Scanning (SEM)
87
F. Electron
Can magnify up to 200M
times
Specimen has to be
coated with metal (e.g.
gold)
89
TYPES
A. Optical D. Scanning Helium Ion
1. Compound (SHIM/HeIM)
2. Stereo
3. Confocal Laser E. Neutron
Scanning (CLSM)
4. Fluorescent F. Electron
1. Transmission (TEM)
B. X-ray 2. Scanning (SEM)
90
F1. Transmission
The electron beam is
passed through the
sample
Result in 2D image
Resolution- up to 2
Angstroms
Magnification- 50M X 91
92
93
94
95
TYPES
A. Optical D. Scanning Helium Ion
1. Compound (SHIM/HeIM)
2. Stereo
3. Confocal Laser E. Neutron
Scanning (CLSM)
4. Fluorescent F. Electron
1. Transmission (TEM)
B. X-ray 2. Scanning (SEM)
96
F2. Scanning
The electron beam is
projected on the sample
Result in 3D image
Magnification- 200M X
97
98
99
100
TYPES
A. Optical D. Scanning Helium Ion
1. Compound (SHIM/HeIM)
2. Stereo
3. Confocal Laser E. Neutron
Scanning (CLSM)
4. Fluorescent F. Electron
1. Transmission (TEM)
B. X-ray 2. Scanning (SEM)
101
G. Scanning Probe
Specimen is viewed at the
nanometer level
Has an electrically
charged probe that traces
the specimens surface
102
103
Sb dopant on Si
104
END
105
106
107