INTRODUCTION
I. ORGANIZATION OF THE HUMAN BODY
II. THE CELL COMPOSITION
THE CELL MEMBRANE
I. FUNCTIONS OF THE CELL MEMBRANE
II. ELECTRON MICROSCOPY OF THE CELL MEMBRANE
III. BASIC STRUCTURES OF THE CELL MEMBRANE
THE CYTOPLASM
I. COMPOSITION OF THE CYTOPLASM
II. CYTOPLASMIC ORGANELLES
III. FIBRILLAR ELEMENTS
IV. INCLUSIONS
NUCLEUS
I. FUNCTIONS OF THE NUCLEUS
II. COMPONENTS OF THE NUCLEUS
III. NUCLEAR ENVELOPE OF THE NUCLEUS
IV. THE CHROMOSOMES
V. PROTEINS
MOVEMENT OF SUBSTANCES ACROSS MEMBRANE
I. ENDOCYTOSIS
II. EXOCYTOSIS
HISTOLOGY 2: LABORATORY
INTRODUCTION TO HISTOLOGY
I. LIGHT MICROSCOPE
II. ELECTRON MISCROSCOPE
III. COMPARISION BETWEEN LIGHT AND ELECTRON MICROSCOPE
HISTOTECHNIQUES
I. CONCEPTS IN HISTOLOGY
II. IMPORTANT TERMINOLOGIES
III. STEPS IN MICROSCOPY (PREPARING SLIDES)
V. COMPARISON BETWEEN LIGHT AND ELECTRON MICROSCOPY
VI. DESCRIPTION OF STEPS
VII. STAININGS USED
HISTOCHEMISTRY
INTRODUCTION
I. ORGANIZATION OF THE HUMAN BODY
· Cell -basic morphological and functional unit of all living things
· Tissue -combination of cells with the same general function
· Organs -combination of tissues that form a more complex functional unit
· Systems -organs that have interrelated functions
II. THE CELL COMPOSITION
· Cell Membrane -delimits the cell from its surroundings
· Cytoplasm -enclosed in the cell membrane
· Nucleus -separated from the cytoplasm by a nuclear envelope
THE CELL MEMBRANE
I. FUNCTIONS OF THE CELL MEMBRANE
· Delimits cell from surroundings
· Protective cover
· Determines substances that move in and out of the cell
· Attachment for cytoskeleton
· Forms specialized junctions with membrane of other cells
· Receives and gives out stimuli
· Provides binding sites and receptors for enzymes and other substances
· Allows for cell recognition
II. ELECTRON MICROSCOPY OF THE CELL MEMBRANE
-8-10 nm thick (not seen in Light Microscope)
-Electron Lucent Line (formed by tails) between 2 Electron-Dense Lines (formed by heads)
III. BASIC STRUCTURES OF THE CELL MEMBRANE
A. Phospholipid Bilayer
-contains a head and two tails which forms a bilayer
-head is the polar and tail is non-polar
1. Head -Globular Polar and Hydrophilic
-Glycerol conjugated to a nitrogenous compound by a Phosphate Bridge
-Occupies the outer surface of bilayer
-forms the 2-electron dense lines
2. Tails -Non Polar / Hydrophobic
-Straight, Saturated Fatty Acids
-May contain unsaturated fatty acids (with slight Kinks)
-occupies the inner surface
-forms the electron-lucent tails
B. Proteins
-half of the mass of cell membranes
-has two types: Integral Proteins + Peripheral Proteins
1. Integral Proteins -forms part of the membrane
-also called Transmembrane proteins
-span the whole thickness of the membrane
-hydrophobic because it interacts with tails
2. Peripheral Proteins -inserted on / loosely bound to outer or inner surfaces
-hydrophilic / polar
C. Cholesterol
-forms part of all cell membranes
-functions for rigidity
-can be synthesized by body from other substances
D. Glycocalyx
-present in some cells
-thin layer of amorphous electron dense material on surface of cell (outer)
-functions for Cell Recognition, Cell to Cell Adhesion, Immunological Response
-consists of Glycoproteins and Glycolipids
§ Glycolipids = carbohydrates + lipids
§ Glycoprotein = carbohydrates + proteins
THE CYTOPLASM
I. COMPOSITION OF THE CYTOPLASM (MATRIX + FORMED ELEMENTS)
A. Matrix of the Cytoplasm
-Viscid, translucent, homogenous, colloidal substance
-amorphous but important complex dynamic part of the cell
-sites of many important biochemical processes
-provides milieu for the organelles to perform their functions
**Composition of the Cytoplasmic Matrix
§ Water (70% or more by volume)
§ Inorganic Ions
§ Organic Molecules: Proteins, Lipids, Carbo, Nucleic Acids, Enzymes, Product of Enzymatic Activity
B. Formed Elements
-includes Organelles + Fibrillar Structures + Inclusions (discussed in detail)
-Cytoplasmic Organelles: Mitochondria + Ribosomes + Endoplasmic Reticulum + Golgi Complex +
Lysosomes + Peroxisomes (Microbodies) + Centrosomes and Centrioles
-Fibrillar Structures: Microfilaments + Intermediate Filaments + Microtubules
-Inclusions: Endogenous Inclusions + Exogenous Inclusions
II. CYTOPLASMIC ORGANELLES
-consists of Mitochondria, Ribosomes, ER, GC, Lysosomes, Peroxisomes, Centrosome
-all organelles have Deliminating Membrane Ribosomes and Centrosomes
-organelles are more or less permanent structures which perform functions in the cell
A. The Mitochondria
**Size and Shape
-0.5-1.0 micra diameter; 10 micra long
-hot dog shaped
**Properties of Mitochondria
§ present in all cells except RBC
§ not visible in H&E; visible in EM, special staining, Phase Contrast Microscopy
§ motile and can change shape
§ synthesizes all its enzymes and can self replicate
**Structure of the Mitochondria
| STRUCTURE | DESCRIPTION |
| Wall of Mitochondria | *Portions invaginate into intercristal space & form cisternae *Double Walled: Outer Membrane + Inner Membrane |
| Intercristal Space | *Cavity enclosed by wall filled with matrix |
| Intermembranous Space | *Space between the two walls |
| Intracristal Space | *Projections into Cristae Mitochondriales |
**Content of the Mitochondrial Matrix
§ granules rich in Magnesium and Calcium
§ enzymes of the Kreb’s Tricarboxylic Acid Cycle
§ strand of DNA
§ ribosomes (RNA containing granules)
§ messenger and transfer RNAs
**Functions of the Mitochondria
§ Powerhouse of the cell (source of energy)
§ Generate ATP (principal source of energy of various metabolic processes)
B. The Ribosomes
**Size / Shape / Properties
-small granules: 15-30 nm in size only seen in EM
-Basophilic; numerous phosphate groups in their RNA
-previously known as Ergastoplasm or Basophilic Bodies
-occur singly or in clusters (Polysomes or Polyribosomes)
-Polyribosomes are clusters of ribosomes connected to each other by mRNA
-ribosomes and polyribosomes occer free in cytoplasm or attached to RER
**Functions of Ribosomes:
1. Free Ribosomes -synthesis of proteins for cytoplasm only (internal)
1. Free Ribosomes -synthesis of proteins for cytoplasm only (internal)
-synthesis of proteins (assembly of amino acids)
-synthesis of proteins of structures that are renewed
2. Attached Ribosomes -synthesis of proteins that are for export by cell
-also synthesize proteins for internal use
**Composition of Ribosomes
-Ribosomes = Large Subunit + Small Subunit
-both subunits are dense globular structures w/ rRNA & associated proteins
-large and small units (not together yet) are transported to cytoplasm via nuclear pores; in the
cytoplasm, Union of Large and Small Sub-unit to form Ribosomes
**Ribosomes Consist of:
§ rRNA Molecules
§ Proteins that are linked to rRNA
C. Endoplasmic Reticulum (ER)
-Composed of Interconnecting: Tubules, Vesicles, Cisternae (flattened sacs)
-they are supporting structure for the cytoplasm and involved in the production of certain substances
**Properties of the ER
-present in all cells but seen only in EM and special preparations
-most extensive membranous structure in cytoplasm
-dynamic organelle capable of remodeling, disassembly/assembly & interacts w/ other organelles
**Membranes of the ER
-thinner than the plasmelemma (cell membrane)
-continuous w/ nuclear membrane; cavity continuous w/ space bet outer&inner layers of nuclear membrane
**Regions of the ER (Smooth and Rough)
1. Rough Endoplasmic Reticulum
-with ribosomes (and polyribosomes)
-involved in synthesis and transport of proteins
2. Smooth Endoplasmic Reticulum (no ribosomes)
-site for synthesis of cholesterol and phospholipids
-involved in transport of fatty acids and other lipids
-in most cells, its less developed than rER but well developed in liver cells
-in striated muscles, it is modified into Sarcoplasmic Reticulum
D. Golgi Complex
-also called the Golgi Apparatus or Golgi Body
-Function: involved in protein synthesis (see below)
**Properties of Golgi Apparatus
-all cells have one; some more
-not seen in H & E, but location is marked by a pale region (negative Golgi Image)
-seen in EM and cells impregnated with Silver salts or Osmium
**Golgi Complex is a Dynamic Organ:
§ at Forming Surface, membrane is added (transfer vesicle)
§ at Maturing Face, membrane removed (secretory vesicle)
**Faces of Golgi Complex:
1. Trans / Maturing Face -related (faces) to the nucleus
-concave side
2. Cis / Forming Face -convex side
**Protein Synthesis
Lysosomes
Ribosomes à rER à Golgi Complex à Secretory Vesicles Integral Proteins Export outside Cell
· Proteins are synthesized in attached ribosomes
· Fully formed polypeptide chains traverse rER membrane and enter the lumen
· While in transit in rER, proteins are processed (new chemicals are added)
· Polypeptide chains are brought to the Golgi Complex in form of Transfer Vesicles (Because there is no continuity between ER and Golgi)
· At Cis Face (Forming face / Convex), transfer vesicles coalesce w/ membrane of Golgi Complex
· While in transit in Golgi Complex, proteins are processed, concentrated, sorted, packaged and labeled
· At Trans Face (Maturing / Concave) of Golgi Complex, Secretory Vesicles (Condensing Vacuoules Bud-off
**Secretory Vesicles -some are incorporated into developing lysosomes
-some incorporated into integral proteins
-some concentraded further and become secretory granules,
later released at apical region of cell by Exocytosis
E. The Lysosomes
**Properties of Lysosomes
-heterogenous group of structures
-vary in shape and size (usually ovoid: 0.05 –0.8 micra)
-not seen in H & E
-seen by histochemical methods that identify enzymes
-constitute an Intracellular Digestive System: can digest and degrade nearly all organic substances
found in the cells
**Similarities of Lysosomes
-membrane bound
-contain hydrolytic enzymes (hydrolases): more than 40 identified today
**Lysosomal Enzymes
-synthesized in rER
-modified and packaged in Golgi Complex
-secretory vesicles pinch of and fuse with developing lysosomes
**Functions of Lysosomes
-Heterophagy -digestion of substances foreign to the cell (ex. Bacteria)
-Autophagy -digestion of unneeded cell organelles
**Lysosomes and Phagocytosis
-lysosomes are numerous in phagocytes (such as Neutrophils)
1. Phagocytosis -bacteria engulfed & brought to cytoplasm in membrane bound structures
(phagosomes or phagocytic vacuole)
2. Phagosomes -attacked by primary lysosomes
**Fate of Engulfed Material
1. Digested Nutrients -diffuse from lysosome and recycled
2. Undigested Materials -also called Residual Bodies
-kept within lysosome
-coalesce to form Lipochrome / Lipofuschin pigments (ex. Macrophages)
**Types of Lysosomes:
1. Primary Lysosomes -lysosomes which have not digested anything yet
-surrounds phagosome, fuse membrane with phagosome, and release
hydrolytic enzymes
2. Secondary Lysosomes -refers to primary lysosome and digested material
-also called as Phagolysosome
**Lysosomes in Bone Resorption
-Osteoclasts release hydrolytic enzymes extracellularly
-digest bone matrix
F. Peroxisomes (Microbodies)
-found in all cells
-simillar to lysosomes morphologically
**Properties of Peroxisomes
-cant be distinguished in LM and EM
-0.5 – 1.2 Micra
-distinguished by using Histochemical Techniques
-contain oxidases and catalases instead of hydrolytic enzymes
**Functions of Peroxisomes
-Enzymes (more than 40 identified) catalyze many metabolic reactions
G. Centrosomes and Centrioles
1. Centrosome -dense spherical area usually near nucleus
-contains the centrioles
2. Centrioles -pair of tubular organelles, collectively referred as Diplosome
-lie perpendicular to each other
-composed of electron-dense wall that surrounds an electron luscent (hollow) space under EM
**Wall -Formed by 9 groups of Microtubules
-each group (Triplet) consists of three Microtubules
-Triplet Obliquely set
-innermost microtubule of triplet is connected to the outermost microtubule of
adjacent triplet by fine filament
**Functions of Centrioles
o sources of mitotic spindles that appear during mitosis
o produce cilia of ciliated cells and flagellum (sperm cells)
**Replication of Centrioles
o prior to mitosis, bud (Procentriole) grows out of lateral surface
o bud elongates perpendicular to mother centriole
o daughter centriole separates from mother centriole
o daughter and mother centrioles move to one pole of cell
o other daughter & mother centrioles move to opposite poles
III. FIBRILLAR ELEMENTS
-forms the structural framework or Skeleton of the Cell (Cytoskeleton)
-seen only in EM or using special histological techniques
-Types: Microfilaments, Intermediate Filaments, Microtubules
A. Microfilaments (Thinnest) 5-7 nm
-made up of Actin Sub-units (F-Actin and G-Actin)
-abundant in peripheral areas of the cell
-present in all cells (except RBC)
-undergo frequent assembly and disassembly to accommodate changes in cell shape & movement of cell
**Actin (Monomer)
-basic Monomer of Microfilaments
-comprises 10-15% of total proteins in the cell
-can bind with ATP and other proteins
1. F-Actin -Filamentous form (50% of actin)
-formed as needed
-formed by two strands of globular G-Actin
2. G-Actin -Soluble form
**Functions of Microfilaments:
§ provide internal support
§ plays a role in shape changes and locomotion of the cell
§ involved in movement of organelles
B. Intermediate Filaments
- not present on all cells
-size between microfilaments and microtubule in size (10-12nm)
-unlike Actin filaments, assemble and disassembly is not frequent
-has five major types: Keratin, Desmin, Vimentin, Neurofilament, Glial Filament
| TYPE | DESCRIPTION |
| Keratin | *only in epithelial cells, numerous in keratinocytes, involved in cell to cell attachment |
| Desmin (Skeletin) | *only in muscle cells (more numerous in smooth than striated) |
| Vimentin | *present in all cells from Mesenchyme (ex. Fibroblasts) *scattered all over cytoplasm |
| Neurofilament | *found in Neurons for neural and internal support *in cell body and processes |
| Glial Filament | *Glial Fibrillary Acidic Protein (GFA) *found in Neuroglial Cells for internal support |
C. Microtubules (Thickest: 25nm in diameter)
-Polymers of Tubulin
-hollow tubules
-assembled and disassembled as needed (like microfilaments)
**Tubulin (Monomer)
-monomer of Micotubules
-formed by Polymerization of 2 sub-units: Alpha-Tubulin & Beta-Tubulin
**Structure of Microtubules
-formed by 13 Tubulin Molecules arranged around a Lumen
-stabilized by Microtubule-Associated Proteins (MAPs)
1 Centriole = 9 Microtubules
1 Microtubule = 13 Tubulin Molecules
**Functions of Microtubules
§ comprise the mitotic spindles and cilia of ciliated cells
§ play a role in movement of organelles
§ lend internal support for the cell
IV. INCLUSIONS
-generally temporary and inert structures
-most not enclosed by membranes
-vary in size, shape and content
-some are useful, some harmful to cell
-two classifications: Endogenous and Exogenous
A. Endogenous Inclusions
-arise from within cell
-ex) Lipid Droplets, Glycogen, Zymogen Granules, Pigment Granules, and Crystals
1. Lipid Droplets -in routine preparations, lipid extracted by reagents; clear areas appear
-in tissue fixed with Gluteraldehyde and Osmic Acid – gray or black globules
-in adipose cells -huge blob
2. Glycogen -storage form of carbohydrates
-seen only in special preparations like PAS method
-sizes: Large Alpha Particles (90nm diameter)
Small Beta Particles (20-30nm diameter)
-EM: Electron Dense; not enclosed in membrane
3. Zymogen Granules -in secretory epithelial cells, often in apical portion of cell
-membrane bound
-secretory products of precursors
4. Pigment Granules àMelanin -in keratinocytes of skin (synthesized by melanocytes)
-cells of substantia nigra in brain
-cells in pigment epithelium of retina
àHemosiderin -brown pigment
-seen by selectively staining iron
-product of lysosomal digestion of hemoglobin
5. Crystals -in few cells (interstitial cells; sertoli cells of testis)
-not membrane bound
-chemical composition is unknown
B. Exogenous Inclusions
-originate from outside the cell
-ex) Liposome (Lipofuschin) Pigments, Dust Particles
1. Lipochrome Pigments-Lipofuschin
-most common; membrane bound
-undigested remnants of Lysosomal digestion (Residual Bodies)
2. Dust Particles -in Macrophages or Phagocytic Cells in lungs
NUCLEUS
-largest structure inside the cell (3-10 micra in diameter) which often round or spherical (but occurs in other shapes)
-present in all cells, except RBC
-vital structure: removal of nucleus leads to death of cell
I. FUNCTIONS OF THE NUCLEUS
· Data Bank of the cell
· Genes in its Chromosomes contain information needed for synthesis of proteins and nucleic Acids
II. COMPONENTS OF THE NUCLEUS (MATRIX + CHROMATIN + NUCLEOLUS):
A. Nuclear Matrix (Amorphous Substance + Nuclear Scaffolf)
1. Amorphous Substance = water, proteins, metabolites, ion
2. Nuclear Scaffold (Nuclear Skeleton; Nuclear Matrix)
-filamentous protein network seen in Interphase Nucleus
-anchored on inner surface of nuclear membrane
-supporting framework that maintains overall size and shape of nucleus
B. Chromatin (Chromatin Material; Chromatin Threads)
-chromosomes at interphase which are fine threads that are entangled with each other
-before cell division, they condense to form basophilic rod-like structures (chromosomes)
**Dispersal Patterns of Chromatin:
1. Heterochromatin -collective term for condensed areas of chromatins which take up stains
-clump or granules (Chromatin Granules)
-portions of chromosomes NOT actively producing RNA
2. Euchromatin -extended areas of chromatin which DONT take up stains
-composed of portions of chromosomes producing RNA
C. Nucleoli (one or more)
-spherical, highly basophilic, usually eccentrically located in nucleus
-no deliminating membrane
-present only in Interphase (disappear during early mitosis / reappear during late telophase)
-Function: synthesize Ribosomal Sub-units
**Number of Nucleoli
-usually one per nucleus but may be more than 1 and larger in cells that actively secrete protein
-absent in cells that don’t synthesize (or little) proteins (muscle cells)
**Three Regions of Nucleolus (Under EM)
1. Nuclear Organizing Region
-several per nucleolus
-circular pale area surrounded by Pars Fibrosa
-area where chromosomes w/ Nucleolar Organizers get together to transcribe rRNA
-resulting rRNA form Pars Fibrosa
*Nuclear Organizers -sequence of bases that code for rRNA
-in humans, 5 pairs of chromosomes are known to have these
2. Pars Fibrosa
-Electron-Dense area surrounding nucleolar region
-rRNA molecules formed in Nucleolar Organizing Region
3. Pars Granulosa
-granular appearance
-accumulation of Ribonucleoproteins (ribosomal sub-units)
III. NUCLEAR ENVELOPE OF THE NUCLEUS
-two membranes (outer and inner nuclear membrane)
-both membranes are similar and thinner than Cell Membrane
-7-8nm thick (cell membrane is 8-10nm)
-seen in EM, not in LM
-can be regarded as a specialized portion of endoplasmic reticulum
o Perinuclear space is continuous with cavity of ER
o Outer nuclear membrane is continuous with membrane of ER
o also has attached Ribosomes
A. Perinuclear Space
-also called Perinuclear Cisterna or Intermembranous Space
-separates the two membranes and continuous with cavity of ER
-size: 10-30nm
B. Nuclear Pores
-perforates the Nuclear Envelope
-circular openings (70-75nm diameter); hundreds to thousands
-in pores, inner and outer membranes are continuous with each other
-channel for exchange of substances between cytoplasm & nucleus
-stabilized by Fibrous Lamina
1. Fibrous Lamina -associated with inner nuclear membrane
-stabilizes nuclear pore; fibrilar protein (30-100nm thick)
-clumps of Chromatin attached, forming outline of n. envelope
2. Nuclear Pore Complex -electron dense structure surrounding the pore
3. Pore Diaphragm -thin diaphragm that covers the pore w/ electron-dense granule in the center
IV. THE CHROMOSOMES
-23 Pairs (22 Somatic Chromosomes + 1 Pair of Sex Chromosome)
-seen during cell division
-consist of Nucleoproteins + DNA Molecules
A. Nucleoproteins
-attached proteins
-has 2 major types: Histones + Non-Histones
B. DNA Molecule
-twisted ladder w/ two strands wrapped around each other
-rungs connect strands with H-bonds
· Strands = Sugar and Phosphate molecules
· Rungs = Nitrogen containing bases (Bases: Adenine, Thymine, Guanine, Cytosine)
**DNA Bases -DNA sequence refers to order of bases along double strand
-there are 3 billion nitrogen containing bases in chromosomes
-it accounts for uniqueness of each protein
**Genes -a segment of DNA strand of variable length
-contains the code for production of specific proteins
-numerous per chromosomes
-humans have between 30,000-40,000 genes
-Human Genome is the collective term for all DNA in all chromosomes (database with all
needed instructions for synthesis of all proteins)
-Nucleus à Chromosomes à DNA à Genes
V. PROTEINS
-nucleus contains the code for their production
-synthesis occurs in the cytoplasm
-code for a particular protein is first transcribed from DNA of gene to an mRNA (carries code to cytoplasm)
-aside from mRNA, two other RNAs also transcribed in Nucleus & brought to the cytoplasm:
· transfer RNA (tRNA)
· ribosomal RNA (rRNA)
· messenger RNA (mRNA)
-all proteins in nucleus are imported from cytoplasm
-all RNA produced in nucleus destined for cytoplasm
MOVEMENT OF SUBSTANCES ACROSS MEMBRANE
I. ENDOCYTOSIS
-from extracellular space into the cell
-phagocytosis and pinocytosis
A. Phagocytosis
-if substance is solid like bacteria or dust
-uses receptors
-ingestion of phagocytes of: Bacteria, Exogenous particulate matter
-ex) Neutrophils; Macrophages
**Stages of Phagocytosis:
§ Receptors of Phagocyte start to bind particulate material
§ Pseudopodia start to form (cell processes)
§ Pseudopodia encircles particulate material and fuse
§ Membrane bound particulate is now a Phagocytic Vacuole
§ Phagocytic Vacuole is attacked by Lysosomes
B. Pinocytosis
-if substance is liquid
-common to all cells
-no receptors necessary
-cell membrane invaginates to enclose fluid
**Pinocytic Vesicle -resulting membrane bound structure that has budded off
-attacked by lysosomes
**Trancytosis -transport of pinocytic vesicle across cell
**Forms of Pinocytosis:
a. Macropinocytosis: large amounts of liquid
b. Micropinocytosis: minute amounts of liquid
II. EXOCYTOSIS
-from inside the cell to extracellular space (mostly secretory products)
-membrane of secretory vesicle fuses with plasmalemma
-secretion flows out into extracellular area
-excess cell membrane is generated by removed when plasmalemma invaginates to form vesicle
that is pinched off and brought to Golgi Complex
**Secretory Vesicle -secretory product and its membrane
-often appears as a granule (Secretory Granule)
**Constitutive Secretion -exocytosis where secretory vesicles are not visible as granules (very small)
HISTOLOGY 2: LABORATORY
INTRODUCTION TO HISTOLOGY
-study of tissues which are composed of cells that carry the same of similar function(s)
-usually with the aid of microscopes
-Objective: To identify the function of the tissue
**Cytology -study of cells and cellular parts that make up the tissue
-Objective: To identify the function of the cell through the parts present
-Two Types of Microscope: Light Microscope and Electron Microscope
I. LIGHT MICROSCOPE
A. Components of the Light Microscope
-Light Source = Bulb or direct sunlight
-Microscope Lenses:
a. Condenser Lens -between light source and specimen
-collects light from source; projects it through specimen
b. Objective Lens -one or more ion-rotating turret located between specimen and ocular lens
-enlarges & resolves specimen’s image and projects image to ocular lens
c. Ocular Lens -further enlarges image and projects onto the observer’s retina
B. Types of Light Microscopes
1. Compound Bright Field -uses a series of lens
-entire field id illuminated by light through a condenser
-specimen translucent and needs to be stained
2. Dark-Field Microscope -needs special condenser for contrast
-specimen unstained
3. Phase-Contrast Microscope -with special lens system
-based on the differences in the light speed retardation by different structures
in specimen giving diff. light intensity
-used for live specimens
4. Polarizing Microscope -allows for visualization of repetitive/ crystalline structures (birefringent)
such as collagen fibers or myofibrils
-stain not necessary
5. Fluorescence Microscopes -allowed localization of substances labeled with fluorescing compounds
such as Fluorescein or Rhodamine
6. Interference Microscopes -combined features of Phase Contrast + Polarizing
-provides contrast on unstained material
-can be used to calculate mass of cellular components
7. Confocal Scanning Microscope -allows visualization of 3-D structures w/o cutting sections
-uses laser optics and computerized imaging light is focused at
specific depth and specimen is scanner point by point
II. ELECTRON MISCROSCOPE
-electrons deflected or absorbed do not reach the screen
-electrons pass through the specimen reach the screen
-electrons travel through vacuum
A. Components of Electron Microscopes
1. Cathode (Negative)
-metallic film
-emits electrons when intensely heated in a vacuum with electric current
2. Anode (Positive)
-positively charged metal plate with small hole in the center
-voltage difference between Cathode & Anode (60-100kV) accelerates the passage of electrons
towards the anode
-Electrons pass through the hole to form the electron beam
3. Condenser Electromagnet
-deflects and focuses the cone of bean towards the specimen
4. Objective Electromagnet
-deflects the electron beam that passed through the specimen
-magnifies the image
5. Projector Electromagnet
-further enlarges image and projects to fluorescent screen or photographic emulsion
6. Fluorescent Screen
-Plate coated with materials that Fluorescence as electrons strike it
B. Types of Electron Microscopes
1. Transmission Electron Microscopes (TEM)
-allows visualization of the internal ultrastructure of cells and tissues and minute structures inside
the cell or intercellular spaces
-resolution 0.2nm
2. Scanning Electron Microscopes (SEM)
-visualization of the Surface Structures only
-allows 3-D representation of the specimen
-resolution 2nm
III. COMPARISION BETWEEN LIGHT AND ELECTRON MICROSCOPE
| LIGHT MICROSCOPE | ELECTRON MICROSCOPE |
| Uses Light picked up by mirror | Uses Electrons emmited from a metallic filament (Cathode) |
| Focused by condenser to specimen | Electrons pass through positively charged metal plate (Anode) with small hole in the center |
| Condenser: Lens | Condenser: Electromagnet |
| Medium is Air | With Projector Electromagnet |
| Visualized directly through ocular lens to retina | Uses fluorescent screen or photographic emulsion; No ocular lens |
| Specimen: 3-8 um | Specimen: 0.08 - 0.1 um |
HISTOTECHNIQUES
-involves a series of steps for tissue preparation for subsequent viewing under the microscope
-different from Histochemistry
-Objective: To visualize morphology of tissues and cells
I. CONCEPTS IN HISTOLOGY
**Units of Measure:
àMillimeter mm 10 m
àMicrometer um 10 m
àNanometer nm 10 m
**Importance of Histotechnique:
· Needed in preparation of tissues for microscopic viewing
· To understand rationale behind the steps in preparation of slides and how they affect the outcome of tissues on the slide
II. IMPORTANT TERMINOLOGIES
A. Magnification
-increases apparent size of the specimen (makes image appear larger)
-function of the objective and ocular lenses
-Magnification = (Power of Objective) X (Power of Ocular Lens)
B. Resolution
-measures how close distance between 2 points are where these 2 points are still considered as separate
-the smaller the value, the greater the resolution
-independent of magnification
-dependent on the Numerical Aperture of Objective Lens and the wavelength of illumination
-Numerical Aperture: width of lens opening of objective
III. STEPS IN MICROSCOPY (PREPARING SLIDES)
 |
LIGHT MICROPSCOPY
Fixation Dehydration Clearing Infiltration Embedding  |
Microscopy Sectioning |
Staining Rehydration Paraffin Removal Mounting 
 |
ELECTRON MICROSCOPY
(NO Paraffin Removal and Rehydration)
Fixation Dehydration Clearing Infiltration Embedding
Microscopy
 |
Staining Mounting Sectioning |
V. COMPARISON BETWEEN LIGHT AND ELECTRON MICROSCOPY
| STEP | LM | TEM | SEM |
| Fixation | YES | YES | YES |
| Dehydration | YES | YES | YES |
| Clearing | YES | YES | XXX |
| Infiltration | YES | YES | XXX |
| Embedding | YES | YES | XXX |
| Sectioning | YES | YES | XXX |
| Mounting | YES | YES | YES |
| Removal of Paraffin | YES | XXX | XXX |
| Rehydration | YES | XXX | XXX |
| Staining | YES | YES | Sputter Coating |
VI. DESCRIPTION OF STEPS (IN DETAIL)
| STEPS | RATIONALE | SUBSTANCE USED |
| 1. Fixation | *Preservation of structural organization *Prevents Bacterial and Enzmyatic Digestion *Insolubilizes tissue components to prevent Diffusion *Protects damage from subsequent steps in tissue processing *Acts as mordant | LM: Formalin EM: Glutaraldehyde |
| 2. Dehydration | *Eases penetration of tissue by clearing agent *Prepares fixed tissue for infiltration with embedding medium *Fixed tissue immersed in alcohol concentration to replace water | Ethanol |
| 3. Clearing | *Prepares tissue for infiltration *Dehydrating agent replaced with Clearing agent | LM: Xylene EM: Propyleneoxide |
| 4. Infiltration | *Prepares cleared tissue for embedding *Tissue immersed in a series of clearing agent embedding medium | LM: Xyleneparaffin EM: Propylene Oxide/ Plastic |
| 5. Embedding | *Prepares tissue for sectioning *Makes tissue firm (for sectioning) *Allows thin sectioning *For EM: tissue block needs to be hard to allow very thin sections | LM: Paraffin EM: Plastic |
| 6. Sectioning | *Thin sections allow light or electrons to penetrate specimen and form image: àLM size: 3-8 um àEM size: 0.08-0.1um | Equipment: *Rotary Microtome *Ultra Microtome |
| 7. Mounting | *For ease of handling and to prevent damage | LM: Glass Slide EM: Copper Grid |
| 8. Paraffin Removal (LM only) | *Preparation for staining *Paraffin is dissolved | Warm Water Bath |
| 9. Rehydration (LM only) | *H & E are water soluble | Increasingly Dilute Alcohol |
| 10. Staining | *Tissue structures cannot be distinguished w/o stain | H & E Lead Citrate |
**Limitations and Associated Artifacts in the Steps:
1. Fixation -induces change in chemical composition and may produce staining artifacts
2. Dehydration -alcohol may denature proteins
-water loss causes uneven shrinkage of components with diff water content
-unnatural spaces between cells and tissue layers
3. Clearing -may denature proteins and cause uneven shrinkage of tissue components
4. Infiltration -heat may denature protein of interest
-bubbles may be left by poor infiltration
5. Sectioning -dull knife can crush of pinch tissue
-vibration can lead to varying thickness in tissue
6. Mounting -tissue section may develop folds
7, Staining -multiple staining may be needed to characterize structures
VII. STAININGS USED
A. Staining for Light Microscopy
1. Hematoxylin -blue basic dye: Basophilic Structures (BLUE)
-attracts acidic components of cell (RNA, DNA) which appear blue on the slide
2. Eosin -acidic red dye: Eosinophilic or Acidophilic (RED)
-attracts basic components of cell such as basic component Protein or Zymogen
Granules, Cytoplasm
3. Silver Stain -for Collagen III, Reticular Fibers
4. Resorcin-Fuschin -for Elastic Fibers
5. Periodic Acid Schiff -for Carbohydrates
**Lipids are usually dissolved during preparation involving Paraffin Method
| TYPES | STAINS | AFFINITY |
| Basic Dyes | Hematoxylin Toluidin Blue Alcian Blue | Basophilic Tissue Ex. DNA, RNA, Ribosomes Sulfated Glycosaminoglycans |
| Acidic Dyes | Eosin Orang G Acid Fuschin | Acidophilic Tissue Ex. Basic Proteins |
| Lipid-Soluble Dyes | Oil Red Sudan Black | Long-chain Hydrocarbons (Fats, oils, waxes) |
| Multicomponent Histochemical Reaction | PAS Feulgen’s Reaction | Complex Carbohydrates (Glycogen, Glycosaminoglycans) Nuclear Chromatin |
B. Staining for Electron Microscopy (Heavy Metal: Electron Dense)
1. Uranyl Acetate, Lead Citrate -Non-specific; adsorb to surfaces and enhance contrast
2. Osmium Tetroxide -fixative that binds to phosphate groups of membrane
phospholipids, enhancing contrast
3. Ruthenium Red -Polyanions; complex carbohydrates
-ex) Oligosaccharides of Glycocalyx and GAG
HISTOCHEMISTRY
-used for detecting ions, lipids, nucleic acids, proteins and amino acids, carbohydrates,
catecholamines, enzymes, antibodies, and antigens
-Objective: To reveal the chemical composition of tissues and cells beyond the acid-base distribution
shown by standard staining methods
A. Ions
-using chemical reactions to identify
-ex. Iron containing tissues is incubated w/ potassium ferrocyanide and HCl
B. Lipids
-osmium tetroxide reacts with lipid to form a precipitate
C. Nucleic Acid
-Feulgen reaction for DNA
-Acridine Orange Fluorescence (yellow green for DNA and red-orange for RNA)
-Toluidine Blue stains for RNA blue
D. Proteins
-Sakaguchi reaction for Arginine
-Million-reaction for Tyrosine
E. Carbohydrate
-Periodic Acid-Schiff for polysaccharides like glycogen
-Alcian blue for Glycoseaminoglycan
F. Cathecholamines
-through Fluorescence in presence of dry formaldehyde vapors
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