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TRANSCRIPT
January 13, 2012
CMM 5001
L t Investigative MethodsLecture: Investigative Methodsin Pathology
Lecturer: Mercedes L Kuroski de Bold PhDLecturer: Mercedes L. Kuroski de Bold, PhDCardiosvacular Endocrinology LaboratoryLaboratoryDepartment of Pathology and Laboratory Medicine
Learning Objectives
1. Understand different types of microscopy and theapplications of each.
2. Understand the steps in tissue/cell, culture/pelletpreparation and discuss the importance of each.p p p
3. Understand the chemical interactions between tissuecomponents and stains for LM FM and EMcomponents and stains for LM, FM and EM.
4. Understand and discuss the procedure for localizingt i i ti / lt ll / ll t d th f tproteins in tissue/ culture cells/pellets and the factors
affecting the IHC procedure for LM, FM and EM.
Investigative Methods in Morphology
Histology:
Study of tissues
Interrelationships between cells, tissues, organs and organ systems
microanatomy
F tiFunctions
Histology
M l l ll ll tiMolecules organelles cells tissues organs organ systems organisms/bodies populations
Cardiac muscle longitudinal sectionc = capillaryc = capillaryID = intercalated diskf = fibroblast nucleusn = cardiac muscle nucleuswww.courseweb.uottawa.ca science.howstuffworks.com
Understanding histology is a vital link between anatomy and areas of physiology, pharmacology, molecular biology and pathology.
How do we proceed to examine tissuesHow do we proceed to examine tissuesvia microscopy?
Tissues have to be: fixed, processed adequately to avoid cell damage, embedded and sliced into very thin sections
Cells cultured/suspensions/pellets : need fixing for microscopic examination
Fresh tissues : need fixing freezing to provide a hardenedFresh tissues : need fixing-freezing to provide a hardenedmatrix for sectioning
Lead-Hematoxilin
SAGSAG
Portions of a rat auricle showing:Secretory atrial granules (SAG) containing atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) stained blue.
Source: de Bold, AJ, Cardiovascular Endocrinology Laboratory, UOHI
Light Microscopy
FATNucleus
Section of human fatty liver stained with oil red O in isopropanol and counterstained with Mayer's haemalum.Fat red nucleus blueFat red, nucleus blue
Source: http://home.primus.com.au
Fluorescent Microscopy
Embryonic Rat Thoracic Aorta Medial Layer Myoblast Cells (A-10 Line)
F actin AlexaHoechst 33258 F-actin- Alexa Fluor 488-phalloidin
Hoechst 33258
Alexa Fluor 568-goatanti mouse PDI
PDI= protein disulfide isomeraseSource:www.microscopyu.com/galleries/fluorescence/
Confocal Microscopy
C t l iDNA
Lysosomes
Cytoplasmic protein
DNA
NRK cell labeled for lysosomes with; an AlexaFluor-546 conjugate (green), a cytoplasmic protein with an AlexaFluor-350 conjugate (blue), and DNA with TOTO-3 dye (red). Imaged with laser lines 364 nms., 543 nms., and 633 nms., using a C-APO 40 x objective.
Prepared by Joe Goodhouse.Source: www.molbio.princeton.edu/facility/confocal
Scanning Electron Microscopy
Portion of a rat atrialcardiocyte showing:
Mitochondria (M),Mitochondria (M),
Secretory atrial granules(SAG)(SAG),
Golgi complexes (G) GM
G
Source: M Kuroski de Bold,Cardiovascular Endocrinology Laboratory,UOHI
Electron Microscopy
T
L
M t fib bl t ithi l ll ti ti iMature fibroblast within a loose, collagenous connective tissue in kidney. The collagen fibrils are shown in both longitudinal (L) and Transverse (T) section. Mag: 8,000
Source: http://home.primus.com.au
TISSUE PROCESSINGTISSUE PROCESSING
FIXATIONFIXATION
DEHYDRATION and CLEARING
EMBEDDING
SECTIONING
STAINING
COVERSLIPPING
PROCESSING OF TISSUESPROCESSING OF TISSUESFixation
Purpose: to preserve the tissues permanently as close as they were in vivo
Fixation should take place immediately after removal of th ti t t t l i ( t d d b ld dthe tissues to prevent autolysis (retarded by cold and accelerated at 300C)
A fixative must change the soluble content of the cell into insoluble so the they are not lost during the processing steps. This process is called denaturation
Denaturation: The protein molecule unfold and thei t l b d di t dinternal bonds are disrupted
Chemical: Uses fixative solutionsChemical: Uses fixative solutions
Physical: Uses heat (microwave), freeze drying,y ( ), y g,freezing
Additive fixation: The protein combines chemically withthe fixative molecule and its rendered insoluble
Non additive fixatives: Alcohol, acetone the fixative doesnot combined with the protein but preserve thesecondary structure of proteins
The selection the appropriate fixative is based on:
a) the structures and entities to be demonstrated
andand
b) the effects of short-term and long-term storage
TYPES of FIXATIVES
Aldehydes: formaldehyde glutaraldehyde
S o S
Aldehydes: formaldehyde, glutaraldehyde
Oxidizing agents: osmiun tetroxide, chromic acid
Protein denaturing agents: methyl alcohol, ethyl alcohol,agents: methyl alcohol, ethyl alcohol,
acetic acid
Combined reagents: BouinCombined reagents: Bouin
Microwave irradiation
FIXATIVES-Aldehydes:
methylene bridgeThe most commonly used is 10 % phosphate buffered Formalin.It t i d 37% f ld h d b i ht d llIt contains around 37% formaldehyde by weight and usually7% methanol to prevent formaldehyde polymerization.http://publish.uwo.ca/~jkiernan/FixAnti1.pdf
4-8% Formaldehyde prepared from paraformaldehydeyields a pure formaldehyde solution. It is the fixativeof choice for Electron Microscopy. Need heat and an slightlyalkaline solution for depolymerization. Penetrate very quickly butalkaline solution for depolymerization. Penetrate very quickly butfixes slowly because cross-linkage to proteins takes timeVery good for LM, EM, IHC & ICC
GlutaraldehydeO
HC - CH2 - CH2 - CH2 - C
H
O
H H
This is a dialdehyde acts as formaldehyde in cross-linking proteins but with only one of the aldehyde groups It penetratesproteins but with only one of the aldehyde groups. It penetrates tissue slowly and poorly. Tissue blocks should be very small or thin. Mostly used in EM at 2-4 % in various buffers at 40C.
-Oxidizing agents
Osmium Tetroxide (OsO4)
f f fNormally used for EM after primary aldehyde fixation to seecell membranes. Vapors are used to darken PAP reaction product in IHCp
It combined with lipids making them insoluble thus thePhospholipids (component of cell membranes) becamePhospholipids (component of cell membranes) became electron-dense.
P t t l f ll l th i t bPenetrates only a few cell layers so the specimen must beextremely small (2-3 mm3). It is expensive, hazardous (vaporizes quickly)( p q y)
MUST BE USED IN FUMEHOOD
Secretory atrial granules
O i t fi dOsmium post-fixed
No osmium ICCNo osmium ICC
ANF: 5nm gold
BNP: 10 nm gold
Source: M Kuroski de Bold,Cardiovascular Endocrinology Laboratory,UOHI
Chromic acid (chromium trioxide)
Strong oxidizer never used aloneStrong oxidizer never used aloneForm complexes with H2O and combine with reactive groups(-COOH and –OH) adjacent to protein chains to form
li k i il t f li U d t id tif ti ithcross-linkage similar to formalin. Used to identify tissues witharomatic amines e.g. adrenal medullary tumors forming a brownor black precipitateSpecimens need to be washed thoroughly to remove the oxideformed
Potassium dichromate (K2Cr2O7)
Rarely used alone The mode of action is the same as aboveRarely used alone. The mode of action is the same as abovePreserve lipids component in mitochondria’s membranesReadily dissolves DNABoth highly toxic by both inhalation and ingestion
Protein denaturing agents These are coagulants fixatives Affect terciary structure ofThese are coagulants fixatives. Affect terciary structure ofproteins but preserve secondary structure
M th l l h l d f bl dMethyl alcohol used for blood smears
Ethyl alcohol to preserve water soluble components e.g. Glycogen and urate crystals deposited in gout (acute arthritis )It causes distortion of nuclear detail and shrinkage of cytoplasm. Prolong fixation extract DNA RNA and remove histonesProlong fixation extract DNA, RNA and remove histones
AcetoneFixation and dehydration occurs at the same time and rapidly atFixation and dehydration occurs at the same time and rapidly at4oC. Used for brain tissue e.g. rabies diagnosis and acid and alkaline phosphatase. Used to fix frozen tissue sections forIHC. Causes shrinkage, distortion and extract lipids
Carnoy Solution (Metharcan)(Metharcan)
A 6:3:1 a mixture of absolute ethyl alcohol, chloroform andglacial acetic acid, rapid acting, preserves glycogen and helicalproteins in myofibrils and collagen but lyses erythrocytes.Fixative of choice to demonstrate intermediary filaments byFixative of choice to demonstrate intermediary filaments by immunohistochemical techniques.Fixation should not be prolong (4 h maximum)
Acetic acid
Always used in combination with other fixatives.Penetrates thoroughly and rapidly but extract proteins and lyseserythrocytese yt ocytes
Combined reagentsThe disadvantages of one component is counterbalanced by theThe disadvantages of one component is counterbalanced by theadvantage or disadvantage of another
Bouin: picric acid, formalin and glacial acetic acid.Excellent fixative for gastrointestinal and endocrine specimens.Good antigenic preservation.
Zenker-Formol: mercuric chloride, K2Cr2O7 , water and formalinZenker Formol: mercuric chloride, K2Cr2O7 , water and formalinGood nuclear fixative, used for needle biopsy specimens ofbone marrow.Recommended for Mallory phosphotungstic acid hematoxylinRecommended for Mallory phosphotungstic acid-hematoxylinstain. Hazardous fixative due to the presence of mercury.
Microwave (MV) irradiation
Heat denaturation. Exposure of dipolar molecules like water and polar side chains of proteins to alternatingand polar side chains of proteins to alternating electromagnetic fields generates heat. MV (2.5 GHz) penetrate several cm into the specimen. The heat produced and duration of exposure can be controlledand duration of exposure can be controlled.
Used for:Used for:-Fixation
To accelerate fixation-To accelerate fixation
-To accelerate histochemical staining for LM & EM
- Antigen retrieval in IHC
Immersion: small pieces (5 mm3) for 1-48 h at 4oC depending on purpose of p g p pfixation. Morphological preservationdepends on the rate of penetrationof the fixative
Fixation
of the fixative
Perfusion: retrograde through aorta or(pump or transcardiac
it f d)gravity-fed)
ADVANTAGES of Perfusion Fixation:excellent morphology, good antigen’s preservation
Factors affecting immersion fixation:
1.-Temperature
2 Penetration2.- Penetration
3.- pH and buffers
4.- Osmolality: hypertonic fixatives = to cell shrinkage
(300mOsm). Isotonic (long term) and hypotonic
fixatives = cell swelling and poor fixation
5.- Time: prolong fixation = cell shrinkage anp g g
hardening of tissue, loss of peptide
6 - Choice and concentration of fixative6.- Choice and concentration of fixative
7.-Volume ratio: ideal 1 (tissue vol): 20 (fixative vol)
A very important pitfall arising from fixation by immersion of a normal tissue sampleby immersion of a normal tissue sample
GOOD POORFIXATION FIXATION
(misinterpreted as pathological)
All i ht t th
1 mm3 tissue sample
All you might see at theEM
1 mm3 tissue sampleEmphasizes the need to study your tissue section with the light microscope before trimming the block to cut for TEM
Factors affecting perfusion fixation:
-Perfusion pressure-Flow rate of fixative: Initial 200 ml at 150 mmHg (rat) or
100 120 mmHg until the desire volume100-120 mmHg until the desire volumehas been perfused ~500-1000 ml for rat or70-150 ml for a mouse. By gravity, 2bottles 5 feet above animals (rinse andfixative)
-Cannula size: For retrograde cannula should fit snugly inCannula size: For retrograde cannula should fit snugly inthe aorta.If perfused through needles inserted into LV backflow and suboptimal fixation willLV backflow and suboptimal fixation willresult
-Choice of fixative
Toluidine BlueStaining A section of a rat
Islet of Langerhans
→ IC
Islet of Langerhans
* PC
X 720
PCIC→
X 720
IC→
Mercedes L. Kuroski de Bold PhD
X 720
Toluidine Blue A section of rat liverToluidine BlueStaining
→ PCS→ Vacuoles
X 720
X 720
→ Lipid droplets
X 720Mercedes L. Kuroski de Bold PhD
A portion of arat Islet ofStain: Uranyl
* PC
rat Islet of Langerhansand lead
PC
* IC
MV
X 53 600
→ Mv ICS
X 7200
X 53,600
Mercedes L. Kuroski de Bold PhD
A portion of aStain: Uranyld l d rat Islet of
Langerhansand lead
X 7200
Mercedes L. Kuroski de Bold PhD
CryotechniquesUses: rapid intraoperative diagnosisUses: rapid intraoperative diagnosis
lipids’s demonstrationpreservation of antigens for immunohistochemistrypreservation of enzymes or diffusible compounds for histochemical demonstrationdemonstration of neurological elementsg
Factors affecting morphology:Ice crystal formation during cooling
Prevention:-Prevention:Cryoprotectants reduce the rate of ice crystal nucleation e.g. PVP, dextran, sucrose, DMSO, glycerol
C-Cryogens:Liquid nitrogen-isopentane (-150oC)
-Embedding compound: OCTg p
Other cryotechniquesOther cryotechniques
F b tit tiFreeze substitution:involves immersing frozen tissue in adehydrating agent at low temperature
Freeze drying: removes water from frozen tissue byFreeze drying: removes water from frozen tissue by sublimation at low temperature undervacuum. It can be infiltrated with resin t l t tat low temperature
TISSUE PROCESSINGTISSUE PROCESSING
FIXATIONFIXATION
DEHYDRATION and CLEARING
EMBEDDING
SECTIONING
STAINING
COVERSLIPPING
•DEHYDRATION and CLEARING
DEHYDRATION R l f t i l i i f-DEHYDRATION: Removal of aqueous material in specimen for embedding in non-aqueous mediume.g. paraffin, plastic.g p pDone by immersing specimens through agraded series of alcohols (70%, 95%, 100%). Inadequate water removal = artifactsInadequate water removal = artifacts
-CLEARING: Use in processing and stainingPurpose: Removal of alcohol and make specimen
receptive to infiltration media. If dehydration isreceptive to infiltration media. If dehydration isincomplete clearing agent will not work = mushy blocks. In staining it renders a clear tissue section Xylene most widely used (hightissue section. Xylene most widely used (high index of refraction)
•EMBEDDING Specimen orientation is the most critical step. Infiltration greatlySpecimen orientation is the most critical step. Infiltration greatly aided by vacuum. -Paraffin Celloidin (Parlodion)-Celloidin (Parlodion)
-Plastics: Glycol metacrylate, epoxy resins y y , p yAraldite, Epon and Spurr
-Sucrose 30% : Cryo-protectant for frozen specimens
•SECTIONING-Vibratome: Thick slices, unfixed tissue for IHC steel
bl d-Microtome: Production of 5-10 μm sections for LM -Ultramicrotome: 50-100 nm for EM. Diamond or glass
blade
-Artifacts: Wrinkles, scratches, chatter
Picking up Paraffinti fsections from
warm water
Mounted unstainedtisection
Frozen sectionsFrozen sections
Coverslipping
http://www-medlib.med.utah.edu/WebPath/HISTHTML/HISTO.html#
Interpretation of Results in Microscopy:Plane of sections
Longitudinal
Oblique
Cross
Cross
Tangential
Compound Light Microscope
Diaphragm
www.southwestschools.org
EyepieceTube
Nosepiece
Aperture
Objectives
Stage clips
Stage
Light source
CoarseAdjustmentknob
Fine
Condenser
Compound: more than one lens
Light source FineAdjustmentknob
On-OffswitchDiaphragm
(Iris)
Compound: more than one lensMicro: smallScope: view
LIGHT MICROSCOPY APPLICATIONSLIGHT MICROSCOPY APPLICATIONS
1.- Phenotypic characterization of cells andsubcellular organelles
2.- To monitor cell fractionation as a supplement tobiochemical studies
3.- Ultrastructural changes correlates of experimentally-induced disease
4.- Human pathology diagnosis
Types of MicroscopesBRIGHT FIELD MICROSCOPYBRIGHT FIELD MICROSCOPY
Specimens need to be stained
PHASE CONTRAST (PC)( )For live unstained specimens, uses an annular ring in condenser. As light passes through object it isAs light passes through object it isdeviated = phase retarded.PC is the artificial transfer of thesePh diff i t b i htPhases differences into brightness
www.microscopy-uk.org.uk
DARK FIELD MICROSCOPYDARK FIELD MICROSCOPY
Unstained living cells. Creates contrastBetween object and surrounding fieldBetween object and surrounding field.Uses an annular stop so only light scattered by part of the object entersthe objective lens resulting in high contrast of specimen
Amoeba Proteus
NORMANSKI DIFFERENTIALINTERFERENCE CONTRAST (DIC)
Unstained living cells. Uses polarized Light, a DIC prism, a DIC slider and ananalyzer
www.microscopy-uk.org.uk
KÖELLER ILLUMINATION:Proper alignment of incident light for LM. The intensity and wavelength spectrum of light emitted by the illumination source iswavelength spectrum of light emitted by the illumination source isof significant importance
Condenser lens not aligned Close diaphragm
Focus edge diaphragm byadjusting condenser height
Open field diaphragmwww.aecom.yu.edu
Image Quality
B i ht B i ht i l t d t th ill i ti t d bBrightness - Brightness is related to the illumination system and can bechanged by changing the voltage to the lamp (rheostat) and adjusting thecondenser and diaphragm/pinhole apertures. It is also related to thenumerical aperture of the objective lens the larger the numerical aperturenumerical aperture of the objective lens, the larger the numerical aperture,the brighter the image
Image of pollen grain
Focus Is the image blurry or well defined? Focus is related to focal lengthFocus - Is the image blurry or well-defined? Focus is related to focal lengthand can be controlled with the focus knobs. The thickness of the cover glasson the specimen slide can also affect your ability to focus the image -- it can betoo thick for the objective lens The correct cover-glass thickness is written ontoo thick for the objective lens. The correct cover glass thickness is written on the side of the objective lens.
R l ti H l t i t i th i b b f thImage of pollen grain
Resolution - How close can two points in the image be before they are no longer seen as two separate points? Resolution is related to the numerical aperture of the objective lens (the higher the numerical aperture, the betterThe resolution) and the wavelength of light passing through the lens (theThe resolution) and the wavelength of light passing through the lens (the shorter the wavelength, the better the resolution).
Contrast - What is the difference in lighting between adjacent areas of thespecimen? Contrast is related to the illumination system and can be adjusted
Image of pollen grain
p y jby changing the intensity of the light and the diaphragm/pinhole aperture. Also, chemical stains applied to the specimen can enhance contrast.
Basic Concepts and Terms in Microscopy
Depth of field: Is the thickness of the specimen which isDepth of field: Is the thickness of the specimen which isacceptable sharp at a given focus
Depth of Focus: Refers to the range over which the image plane can be moved while an acceptableamount of sharpness is maintainedamount of sharpness is maintained
Field of View: Is the area of the specimen that can be seenField of View: Is the area of the specimen that can be seenthrough the microscope with a given objective
Basic Concepts and Terms in Microscopy
Focal Length: Distance required for a lens to bringFocal Length: Distance required for a lens to bring the light to a focus (measured inmicrons)
Numerical Aperture: Measure of the light-collecting abilityof the lens
Magnification: The product of the magnifying powers of the objective and eyepowers of the objective and eyepieces
Fluorescence Microscopy
The basic function of the fluorescence microscope is to irradiate the specimenwith a desired and specific band of wavelengths.Fluorescent molecules absorb light at oneFluorescent molecules absorb light at one wavelength and emit at another, longer wavelength.
Light source:Halogen or Hg lamps
Filters: Exciter filter, between light source andobject to select the excitation wavelengthBarrier filter, in the objective to allow visible rays to pass
www.jic.ac.uk/microscopy
PRINCIPLE OF FLUORESCENCE
1. Energy is absorbed by the atom which becomes excited.
2. The electron jumps to a higher energy level.2. The electron jumps to a higher energy level.
3. Soon, the electron drops back to the ground state, emitting aphoton (or a packet of light) - the atom is fluorescing.p ( p g ) g
http://nobelprize.org/educational_games/physics/microscopes/fluorescence
Multiphoton Fluorescence MicroscopyMPFM: Combines the advanced optical techniques of laser scanning p q gMicroscopy with long wavelength multiphoton fluorescence excitation to captureHigh-resolution, three-dimensional images of specimens tagged with highly Specific fluorophores.
Combination 2 photon (red and green) and 3 photon (blue) image of C elegans embryoCombination 2-photon (red and green) and 3-photon (blue) image of C.elegans embryo
Uses:To study dynamic processes in living cells and tissues without inducing significant damage to the specimensignificant damage to the specimen
Disadvantages: Slightly lower resolution with a given fluorophore when compared to confocal imagingcompared to confocal imaging. Thermal damage can occur in a specimen if it contains chromophores that absorb the excitation wavelengths, such as the pigment melanin.Only works with fluorescence imaging.y g gCurrently rather expensive.
Fluorescence Resonance Energy Transfer (FRET)Transfer (FRET)
Is a process by which radiationless transfer of energy occurs from an excited state fluorophore to a second chromophore in close proximity. Because the
hi h th t f t k l i li it d t i t lrange over which the energy transfer can take place is limited to approximately10 nm (100 Å), and the efficiency of transfer is extremely sensitive to the separation distance between fluorophores, Resonance Energy Transfer measurements can be a valuable tool for probing molecular interactionsmeasurements can be a valuable tool for probing molecular interactions.Uses: protein-protein interactions, protein-DNA interactions, and protein conformational changes.
Blue:donor
Green:acceptordonor
fluorochromeacceptor emission
www.olympusmicro.com/
Confocal MicroscopyConfocal laser scanning microscopy (CLSM ) is a valuable tool forobtaining high resolution images and 3-D reconstructions. The key feature is its ability to obtain serial optical sections and to produce blur-free images of thi k i t i d ththick specimens at various depths.
1-A point light source forIllumination
2-A point light focus within the Specimen
3-A pinhole at theimage detecting plane
www.olympusfluoview.com
Human Rabbit sunflower medulla muscle pollen grain
plane
http://www.hi.helsinki.fi/amu/AMU%20Cf_tut/cf_tut_part1-4tm
Transmission Electron Microscopy (TEM)
Electron gun: An electrified tungsten filament that emit electrons
Transmission: Specimen very thin and either transmit electronsproducing electron-lucent areas in the imageproducing electron lucent areas in the image
or
deflect electrons producing electron dense areas in the image.
USE: study ultrastructure of cells and relationships between cells. Expensive and need a skill operator
Resolution: 3.5 Å (0.0004 μm)
TEM
TRANSMITION ELECTRON MICROSCOPY APPLICATIONS
1 - Phenotypic characterization of cells and1.- Phenotypic characterization of cells andsubcellular organelles
2 T it ll f ti ti l t t2.- To monitor cell fractionation as a supplement tobiochemical studies
3.- Ultrastructural changes correlates of experimentally-induced disease
4.- Human pathology diagnosis
Electron Microscopy
Portion of a rat atrial cardiocyte showing:cardiocyte showing:
Mitochondria (M)
Secretory atrial granules (SAG) N(deflect electrons)
A portion of its nuclei MN
Source: M. Kuroski de BoldCardiovascular Endocrinology Laboratory,UOHI
Trimming of Specimen Block for EM
Color Thickness for EM Sections
TEM
Sectioning
Ribbon of sectionsRibbon of sections in trough
Comparison of the light and electron microscope
Light Microscope Electron MicroscopeCheap to purchase Expensive to buy
Cheap to operate. Expensive to produce electron beam.
S ll d t bl L d i i lSmall and portable. Large and requires special rooms.
Simple and easy sample preparation. Lengthy and complex sample prep.
Material rarely distorted by P ti di t t t i ly ypreparation. Preparation distorts material.
Vacuum is might be required. Vacuum is required.Natural color of sample maintained. All images in black and white.Natural color of sample maintained. All images in black and white.Magnifies objects only up to 2000
times Magnifies over 500 000 times.
Specimens are dead as they must beSpecimens can be living or dead Specimens are dead, as they must be fixed in plastic and viewed in a vacuum
Stains are often needed to make theThe electron beam can damage
specimens; must be stained with anStains are often needed to make the cells visible
specimens; must be stained with an electron-dense chemical ( osmium,
lead or gold).http://www.biologymad.com
Scanning Electron MicroscopyElectrons are reflected off the surface of the specimen which has been previously
t d ith h t l U T bt i 3 D i f f R l ti lcoated with heavy metals. Use: To obtain 3-D images of surfaces. Resolution lower than TEM
Portion of a rat atrialcardiocyte showing:
Mitochondria (M),Mitochondria (M),
Secretory atrial granules(SAG)(SAG),
Golgi complexes (G) GM
G
Source: M Kuroski de Bold,Cardiovascular Endocrinology Laboratory,UOHI
X-Ray DiffractionDiffraction occur when electromagnetic radiation interacts with a periodic g pstructure whose repeat distance is about the same as the wavelength of the radiation.
Energy emitted = photons have a wavelength between 10-6 to 10 -10 cm theEnergy emitted = photons have a wavelength between 10 6 to 10 10 cm, the energy = X-rays.Energy emitted ~wave moving at the speed of light, c (~3 x 10-10 cm/s in a vacuum), and having associated with it a wavelength, λ, and a frequency, v, such that the relationship c = λ.v is satisfied.
Diffraction patterns constitute evidence for the periodicallyrepeating arrangement of atoms in crystals. The symmetry of the diffraction patterns corresponds to theThe symmetry of the diffraction patterns corresponds to the symmetry of the atomic packing.
Atomic Force MicroscopyWhat is Atomic Force Microscopy?What is Atomic Force Microscopy?
The atomic force microscope (AFM) is one of the family of scanningis one of the family of scanning probe microscopes, and is widely used in biological applications. The optical lever principle is used which means that a smallThe optical lever principle is used, which means that a smallchange in the bending angle of the cantilever is converted to ameasurably large deflection in the position of the reflected spot.Living cells can be imaged as well as single molecules such asLiving cells can be imaged, as well as single molecules such asproteins or DNA. The force contrast gives 3-dimensionaltopography information. www.jpk.com
MALDI Imaging for Pathology Proteomic StudiesCurr Pharm Des, 2007,13(32):3317
Tissue sections from fresh organ or biopsy are laid out on the MALDI target sections first covered with a specific matrix. For peptides/proteins, very intense signals are obtained with α-cyano-4-hydroxycinnamic acid (α-CHCA) as a matrix. S ti d ith CHCA ( th t i ) d th i t d d i th MALDI TOF f l iSections are covered with α-CHCA (or another matrix) and are then introduced in the MALDI-TOF for analysis.Next, MALDI laser is used to scan each point of the surface area and the mass spectra representative of the peptides/proteins or lipids present in each point are analyzed. Automated analysis of the complete tissue is obtained by performing mass spectra every 10-50μm, providing representative information of selected ions (each ion is a specific bio-molecule). Analysis is obtained within 2-6 hours and images are reconstructed using Flex-imaging software.
Chemistry of Staining
LM: Cationic dyes: Stain basophilic structures e.g., nuclei,ribosomes
Anionic dyes: Stain acidophilic structures e.g., mitochondria,llcollagen
Purpose of Staining
A) Different colors
B) Coloring to different intensities
Categories of Stains
Acid-Base: Hematoxylin- EosinBasic dye; stains nucleous blueAcidic dye; stains cytoplasm and intercellular
components pink
Trichrome Methods3 colors allows differentiation between
muscle & keratin
3 colors, allows differentiation betweencytoplasmic & intercellular components
collagenHuman skin
nuclei (blue-black)
Specific StainsSpecific StainsIron hematoxylin: cytological details e.g.; subcellular organelles
striations
Categories of Stains
Specific Stains:
Periodic-Acid Schiff: stains glycogen, muco & glycoproteins, glycosaminocansred
Silver Impregnation: outlines reticularSilver Impregnation: outlines reticularfibers
Orcein Resorcin Fuchsin: elastic fibersOrcein, Resorcin-Fuchsin: elastic fibers
Sudan Black B: stains fat
TYPES of BONDS
Ionic:tissue protein-NH2 + H+ tissue protein -NH3+ + Eosin-
tissue protein -NH3 Eosin
tissue proteoglycan —OSO3 + H+p g y
tissue proteoglycan —OSO3 - + Methylene Blue+
tissue proteoglycan —OSO3 Methylene Blue
www.scionpublishing.com
Hydrogen bonds van der Waal’s bonds
www.google.ca www.scionpublishing.com
Metachromatic Dyes and Metachromasia
Metachromasia: Dye stains tissue different colour to the dye solution. Needs water between dye molecules, water mount. No dehydration and clearing.y , y g
M t ll l blMast cell: nucleous: bluegranules: pink
Mordants:non dying compound to improve binding of the dyenon-dying compound to improve binding of the dye.
DYE
TISSUE DYELAKELAKE
MORDANT
e.g., Haemalum, Iron hematoxylin
Light Microscopy
Section of a lymph node H & E and reticular silver stain. The latter is specific for type III collagen
::
Source: http://www.upei.ca/~morph/webct/Modules/Light_Microscopy/stain.html
EM staining of tissues
EM: Post-embedding stainingElectron dense heavy metals salts e g ;Electron dense heavy metals salts e.g.;Lead citrate: Binds to RNA-containing structures and hydroxyl groups of carbohydrates;U l t t St i b dUranyl acetate: Stains membranes and nucleic acids.
NUCLEUSNUCLEUS
MYOFIBRILSMYOFIBRILS
STORAGE STORAGE GRANULESGRANULESNUCLEUSNUCLEUS
MYOFIBRILSMYOFIBRILS
STORAGE STORAGE GRANULESGRANULES
MYOFIBRILSMYOFIBRILSMYOFIBRILSMYOFIBRILS
Pre-Embedding for EM: Ruthenium Red
Figs 6 and 7. Rat pars intermedia, ruthenium red stainingde Bold AJ et al Cell Tissue Res 207(3):347, 1980
Pre-Embedding for EMLanthanum, Uranyl en block, Horseradish peroxidaseIntracellular space
Fig. 11. Rat pars intermedia, HRPde Bold, AJ et al ., Cell Tissue Res, 207(3):347,1980
I hi t h i tImmunohistochemistryDirect
ImmunohistochemistryImmunohistochemistryIndirect
FACTORS AFFECTING ICC STAINING
Choice of fix or frozen sections; pre or post embeddingChoice of subbed material for slidesChoice of subbed material for slidesChoice of antibodies monoclonals or polyclonalEstablishing a specific binding and appropriate antibody titerChoice of secondary antibodies and appropriate titerAntigen retrieval reagents break the protein-cross-links formedBy formalin fixation uncovering the antigenic sites e.g., y g g g ,Citrate buffer pH 6.0, TRIS-EDTA pH 9, Proteinase KUse of detergents facilitate antibody penetration e.g.,Triton X 100 not indicated for use of membrane antigensTriton X-100 not indicated for use of membrane antigensUnmasking antigens for EM sodium borohydryde in 1 %Phosphate buffer is recommended for glutaraldehyde fixed
tissueIt reduces the glutaraldehyde linkages.
CONTROLSMust be run to: Test the protocol and specificity of antibody for
LM and EM
Positive control: To test protocol and procedure. If negativestaining is obtained, the protocol need to becheckedchecked
Negative control: To test the specificity of the antibody used.
1.- Replacement of primary antibody with normal serum (from the same species as primary)p a y)
2.- Adsoption of the primary antibody with purified antigen prior to staining. The staining must be inhibited
Avidin-Biotin method multiple Flurorecentantigen labelingantigen labeling
www.vectorlabs.com
IHC stainingmethodsmethods(a) Tissue microarrayer device.
(b) Overview of tissuei bl kmicroarray block.
(c) Overview of H&E–stained tissuestained tissuemicroarray slide.
Modern Pathology (2004) 17, 790–797,www.modernpathology.org
HL-1 cells: ANF,Texas red; Goα, Fluorescein green; Nucleous,DAPI blue. ( Mercedes L. Kuroski de Bold, PhD, 2007)
ICC Staining Methods for EM
ASG
Electron microscopic level staining by immunogold of ANF (small particles)and BNP (large particles) in atrial secretory granules.
Mercedes L. Kuroski de Bold
TRANSMITION ELECTRON MICROSCOPY APPLICATIONS
1 - Phenotypic characterization of cells and1.- Phenotypic characterization of cells andsubcellular organelles
2 T it ll f ti ti l t t2.- To monitor cell fractionation as a supplement tobiochemical studies
3.- Ultrastructural changes correlates of experimentally-induced disease
4.- Human pathology diagnosis
1.- Phenotypic characterization of cells andsubcellular organelles
EXAMPLE OF GOOD
FIXATION
VERY HIGHPOWERPOWER (x70,000)
2.- To monitor cell fractionation as a supplement tobiochemical studies
DIFFERENTIAL CENTRIFUGATION FRACTIONS
MITOCHONDRIAL CRUDE GRANULAR MICROSOMAL
3.- Ultrastructural changes correlates with experimentally-induced disease
Cardiac Tissue-Restricted Deletion of Plakoglobin Results in ProgressiveCardiomyopathy and Activation of -Catenin Signaling. Jifen Li, et al, Mol Cell Biol, Mar. 2011, p. 1134–1144
Mutations in the plakoglobin (JUP) gene have been identified in arrhythmogenic right ventricularMutations in the plakoglobin (JUP) gene have been identified in arrhythmogenic right ventricular
cardiomyopathy (ARVC) patients. However, the mechanisms underlying plakoglobin dysfunction
involved in the pathogenesis of ARVC remain poorly understood. Plakoglobin is a component of both
desmosomes and adherens junctions located at the intercalated disc (ICD) of cardiomyocytes where itdesmosomes and adherens junctions located at the intercalated disc (ICD) of cardiomyocytes, where it
functions to link cadherins to the cytoskeleton. In addition, plakoglobin functions as a signaling protein
via its ability to modulate the Wnt/-catenin signaling pathway. To investigate the role of plakoglobin in
ARVC we generated an inducible cardiorestricted knockout (CKO) of the plakoglobin gene in miceARVC, we generated an inducible cardiorestricted knockout (CKO) of the plakoglobin gene in mice.
Plakoglobin CKO mice exhibited progressive loss of cardiac myocytes, extensive inflammatory
infiltration, fibrous tissue replacement, and cardiac dysfunction similar to those of ARVC patients.
Desmosomal proteins from the ICD were decreased, consistent with altered desmosome ultrastructureDesmosomal proteins from the ICD were decreased, consistent with altered desmosome ultrastructure
in plakoglobin CKO hearts. Despite gap junction remodeling, plakoglobin CKO hearts were refractory to
induced arrhythmias. Ablation of plakoglobin caused increase -catenin stabilization associated with
activated AKT and inhibition of glycogen synthase kinase 3. Finally, -catenin/TCF transcriptional activityactivated AKT and inhibition of glycogen synthase kinase 3. Finally, catenin/TCF transcriptional activity
may contribute to the cardiac hypertrophy response in plakoglobin CKO mice. This novel model of
ARVC demonstrates for the first time how plakoglobin affects-catenin activity in the heart and its
implications for disease pathogenesis.
Desmosome Adherent junction
users.rcn.com/jkimball.ma.ultranet/BiologyPages/J/Junctions.htmlutm.utoronto.ca
Adherent junction hold cardiac muscle cells tightly together as the heart expands and contracts
Localization in muscle
Fig 2. LM Fig 3. EM
Fig 2, A B,C and E: Hematoxiln/EosinArrows in E: loss of cardiomyocytesArrowheads in F: fibrosis,Masson’s trichrome
Fig 3, Black and white arrows: desmosomesDoble-headed arrows: sarcomeresArro heads Z linesMasson’s trichrome
WT: wild type CKO: knockoutArrowheads: Z lines
Mol Cell Biol, Mar. 2011, p. 1134–1144
Mol Cell Biol, Mar. 2011, p. 1134–1144
4 H th l di i4. Human pathology diagnosis
Negatively stained Rhabdovirus as seen Ribonucleoprotein - notice the abundant through an electron microscope. Notice thebullet shape of the virus (A). See the"bee hive" like striations of the RNP (B).Notice the glycoprotein spikes in the outer
strands of coiled RNP (almost everything in the image is RNP).
Notice the glycoprotein spikes in the outer member bilayer (C).
USCDC 2
Fig7 EM appearance of Helicobacter pylori infected gastric surface mucous cells. (A) H pylori were also demonstrated on the apical plasma membrane and/or between the lateral plasma membranes of the surface mucous cells. Detaching cells also had H pylori attached to their surface. H pylori located in the reticular pattern layer (arrows) frequently ran parallel to the mucosal surface or to the mucins of the thick band like patternthe thick band like pattern(bar=1 μm). (B) Higher magnification of H pylori identified with arrows in the surfacemucous gel layer in (A) (bar=0.5 μm). Gut 2001, 49:474-480
Gut 2001, 49:474-480
Fig 8 EM appearance of Helicobacter pylori within the surface mucous gel layer. H pylori were trapped by frayed thin threads of mucins of the thick b d lik ttband like pattern(bar=0.5 μm). Gut 2001, 49:474-480
Helicobacter Pilory EM
Wikipedia.org
Summary-Microscopy different kinds
Fixation different types-Fixation different types
-Frozen specimens
-Processing tissue, cells, pellets specimens
-Embedding specimens
Sectioning LM and EM specimens-Sectioning LM and EM specimens
-Staining LM, EM, IHC
CMM 5001 P th l i l B i f DiJanuary 13, 2012 Professor: Mercedes Kuroski de Bold, PhD
CMM 5001 Pathological Basis of Disease
Investigative Methods in Pathologyg gy
Essay Guidelines
Title Page
IntroductionIntroduction
Material and Methods
Di iDiscussion
References
References
WEBSITES
http://dept.kent.edu/projects/cell/FLUORO.HTMp p p j
http://www.fluorescense.com/tutorial/fm-optic.htm
http://micro.magnet.fsu.edu
http://www.protocol-online.org/prot/Histology
http://www.jpk.com
www.dako.com
www.millipore.com
www.scionpublishing.com
ih / b kwww.ihc.com/_books
www.olympusmicro.com
BOOKS
Th d P ti f Hi t l i l T h i Ed B ft JD dTheory and Practice of Histological Techniques; Eds: Bancroft, JD andGamble, M. 5th Edition, Churchill Livingstone, Publishers, 2002(Available at the Heart Institute Library)
Basic Techniques for Transmission Electron Microscopy, Eds: Hayat, M.A., AP Harcourt Brace Jovanovich, Publishers, 1986
Electron Microscopy Methods and Protocols, Ed; Hajbagheri, M. A. Nasser , IN: Methods in Molecular Biology, John M Walker, Series Editor, Humana Press, Totowa, New Jersey, 1999
Functional Ultrastructure, Atlas of Tissue Biology and Pathology. Eds: Pavelka, M and Roth, J.Springer-Verlag/Wien, 2005 (Available at the Heart Institute Library)
Histotechnology. A Self-instructional Text, Ed: Carson L. Freida,2 d di i ASCP P A i S i f Cli i l P h l P bli h2nd edition, ASCP Press, American Society for Clinical Pathology Publishers,Chicago 1997 (Available at the Heart Institute Library)
Introduction to Diagnostic Electron Microscopy Ed: Mackay BIntroduction to Diagnostic Electron Microscopy, Ed: Mackay, B., Appleton-Century-Crofts/New York, 1981
Immunocytochemical Technology, Eds: Childs, G.V., Alan R. Liss Inc., New y gy, , , ,York Publishers, 1986
In situ Hybridization, Principles and Practice, Eds: Polak, J. M. and McGee, J O’D., Oxford University Press, New York, 1990
Selected Histochemical and Histopathological Methods. Eds: Thompson S W Ch l C Th P bli h S i fi ld Illi i U S A 1966S.W, Charles C. Thomas Publishers, Springfield, Illinois, U.S.A., 1966
Using Antibodies. A laboratory Manual, Ed: Harlow, E and Lau, D. Cold Spring Harbour Press CSH New York 1999Spring Harbour Press, CSH, New York, 1999
E-BooksMetastasis Research Protocols Vol 1: Analysis of Cells and Tissues Eds:Metastasis Research Protocols, Vol., 1: Analysis of Cells and Tissues, Eds: Brooks, SA & Schumacher, U, Humana Press, P42-48, 2001.http://site.ebrary.com/lib/oculottawa/Doc