LtLecture: Investigative MethodsInvestigative Methods in ......The disadvantages of one component is counterbalanced by theThe disadvantages of one component is counterbalanced by

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


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


A portion of aStain: Uranyld l d rat Islet of

Langerhansand lead

X 7200


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



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


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



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

LtLecture: Investigative MethodsInvestigative Methods in ......The disadvantages of one component is counterbalanced by theThe disadvantages of one component is counterbalanced by

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