Histology: Microscopy Techniques

• Histology is the study of the microscopic structures of tissues and organs, also known as microscopic anatomy.
• Microscopes magnify images to reveal details unseen by the naked eye, essential for studying tissue microstructure.

Types of Microscopes

• Light Microscopes (LM) are commonly used in teaching labs.
• Electron Microscopes (EM) include Transmission EM and scanning EM.
• Final magnification of the microscope = magnification of an objective lens x magnification of ocular lens.
• LM magnifies objects up to 1000x, while EM can magnify up to 100,000x.
• Resolution is the ability to produce separate images of two adjacent objects.
• The resolution limit for the human eye is 0.2 mm, while for LM it is 0.2 micron and for TEM it is 0.1 nanometer.

Parts of LM

• A light source illuminates the specimen using daylight or a lamp.
• A condenser focuses the light beam onto the specimen.
• A stage holds the slide.
• Ocular lenses (eyepieces) can be single (uniocular) or double (binocular).
• Objective lenses gather light from the specimen, with magnification powers such as ×4, ×10, ×40, and ×100.
• Magnification increases the linear dimension of an object without optical defects.

Cell Structure

• Cells are the basic structural and functional units of the body.
• Cells and extracellular matrix collectively form tissues.
• Organelles, cells, tissues, organs, and systems constitute the human body.
• Eukaryotic cells are common in human bodies.
• Cell shapes vary based on function.
• Red blood cells are nearly 6 microns in diameter.
• Human oocytes may reach 100 microns in diameter.
• Most cells typically measure 8-10 microns in diameter.
• The two major components of the cell are the nucleus and the cytoplasm.

Cytoplasm

• Contains cytoplasmic organelles, cell inclusions (e.g., lipid droplets, pigments), cytoskeleton (microtubules, microfilaments, intermediate filaments), and cytoplasmic matrix (variable solutes of sodium and potassium ions).

Organelles

• They are metabolically active structures classified as membranous or non-membranous.
• Membranous organelles include the plasma membrane (PM), mitochondria, Golgi apparatus, endoplasmic reticulum (smooth and rough), lysosomes, and transport vesicles.

Plasma Membrane

• It is also called the plasmalemma or cell membrane.
• It is a dynamic structure involved in physiological and biochemical activities.
• In living cells, it exhibits a fluid mosaic appearance with proteins moving within a lipid bilayer.
• With EM, the thickness ranges from 7.5 to 10 nm, below the resolution limit of LM.
• EM reveals a trilaminar structure: inner and outer dark bands with a light band in between.
• Components include lipids (phospholipids, cholesterol), proteins, and carbohydrates.

Lipids in the Plasma Membrane

• Phospholipids form the bilayer, with hydrophilic parts facing outwards and hydrophobic parts facing inwards.
• Certain outer layer lipids attach to oligosaccharide chains, forming glycolipids.
• Glycolipids contribute to the glycocalyx of the cell.
• Cholesterol is distributed patchily and stabilizes the plasma membrane.

Proteins in the Plasma Membrane

• They are located between phospholipid bilayers, classified as integral or peripheral.
• Integral membrane proteins are embedded in the phospholipid bilayer, either passing once (one pass integral protein) or multiple times (multi pass integral protein).
• Peripheral membrane proteins are bound to the PM surfaces.
• Carbohydrate chains attach to proteins, forming glycoproteins, which, along with glycolipids, form the cell's glycocalyx (cell coat).

Functions of Plasma Membrane Proteins

• Pumps actively transport ions (e.g., sodium and potassium) using ATP.
• Channels passively allow the passage of small molecules, ions, and water.
• Receptors recognize and bind molecules like hormones and antibodies.
• Linker proteins anchor intracellular proteins to the extracellular matrix.
• Enzymes, like ATP synthase, are located in the inner mitochondrial membrane.

Endoplasmic Reticulum (ER)

• It is a tortuous network of tubules, small vesicles, and flattened cisternae, usually in parallel stacks.
• Separated from the cytoplasm by a membrane.
• Amount, type, and distribution vary by cell type and function.

Smooth Endoplasmic Reticulum (sER)

• Lacks polyribosomes, resulting in a smooth outer surface.
• Cells with abundant sER display acidophilic cytoplasm under LM due to protein content.
• Best observed with EM.
• Abundant in steroid-secreting cells like Leydig cells.
• Functions include lipid synthesis, drug detoxification, and calcium ion regulation in muscles.

Rough Endoplasmic Reticulum (rER)

• Rough outer surface due to ribosome attachment.
• Cells with abundant rER show basophilic cytoplasm because of ribosomes.
• Made of sac-like parallel cisternae with attached ribosomes.
• Cisternae are continuous with the outer nuclear envelope.
• Involved in protein synthesis, prolific in protein-secreting cells.

Golgi Complex (Apparatus)

• Golgi body does not stain by Hx&E. In large cells, Golgi appears as a clear area surrounded by cytoplasm.
• Can be visualized by LM with special stains such as silver stain.
• Commonly located near the nucleus.
• Number and size vary based on cell activity.
• Active secretory cells have numerous well-developed Golgi complexes.
• EM shows a complex of flattened, curved cisternae with vesicles and vacuoles.

Distinct Functional Faces

• Golgi receiving (forming) region: convex, near rough ER, receives transport vesicles with synthesized materials.
• Trans face (maturing face): concave, generates secretory vesicles carrying protein products.
• Medial compartment: contains flattened cisternae between the two compartments.

Golgi Functions

• Condensation and packaging of proteins.
• Protein modification via chemical group addition.
• Hydrolytic lysosomal enzyme packaging.

Mitochondria

• Membrane-bound organelles with enzymes for aerobic respiration and ATP production.
• The powerhouse of the cell.
• It can divide because it contains its own DNA.
• Highly acidophilic under LM.
• Can be stained with iron Hematoxylin and Janus green B.
• Appear as elongated filaments, spherical rods, or pleomorphic shapes.

Mitochondrial Components

• Outer mitochondrial membrane.
• Inner mitochondrial membrane.
• Intermembranous space.
• Matrix.

Outer Mitochondrial Membrane

• Smooth and sieve-like with porins for molecule transport.
• Contains enzymes and receptors.

Inner Mitochondrial Membrane

• Features cristae (folds) projecting into the matrix for increased surface area.
• Cristae number, shape, and size depend on cell activity.
• Tubular cristae are present in steroid-secreting cells.
• Enzymes for the respiratory chain are attached to the inner membrane; their heads appear as elementary particles.

Intermembranous Space

• Located between the inner and outer membranes.
• It contains enzymes for ATP generation.

Matrix

• Surrounded by the inner mitochondrial membrane.
• Contains Krebs cycle enzymes and enzymes for β-oxidation of fatty acids.
• Stores calcium using dense matrix granules and contains mitochondrial DNA, ribosomes, and tRNA.

Mitochondrial Functions

• Energy production.
• Calcium homeostasis.
• Initiation of programmed cell death (apoptosis).

Lysosomes

• The site of intracellular digestion and turnover of cellular components.
• Contains more than 40 hydrolytic enzymes.
• Found in large numbers in phagocytic cells like macrophages and neutrophils.
• Lysosomal membrane is resistant to degradation by lysosomal enzymes, containing special phospholipids.
• Originates from Golgi bodies or the rough ER.
• Visible by histochemical stains (acid phosphatase test) under LM and appear as membrane-bound vesicles under EM.

Types of Lysosomes

• Primary lysosome: Contains homogenous granular content without digestive material (EM).
• Secondary lysosome: Larger and more heterogeneous, formed by the fusion of primary lysosomes with foreign bodies.

Lysosome Functions

• Defense against infection by engulfing viruses and bacteria.
• Removal of worn-out organelles via autophagy.
• Self-destruction of cells via autolysis after death.

Centrioles

• Cylindrical structures with walls of nine longitudinally arranged microtubule bundles each containing three microtubules (triplet).
• The centrosome consists of two perpendicular centrioles surrounded by a pericentriolar matrix, usually near the nucleus.
• Responsible for the formation of mitotic spindles during cell division.

Ribosomes

• Small spherical macromolecules that synthesize proteins.
• Uniform in size, ranging from 15-20 nm in diameter, mainly composed of rRNA and proteins.
• Synthesized in the nucleolus and transported to the cytoplasm via nuclear pores.

Types of Ribosomes

• Free ribosomes (polysomes): scattered in the cytoplasm and have a rosette shape; consist of ribosomes along mRNA; synthesize proteins used to form organelles.
• Attached ribosomes: attached to the rough ER or outer nuclear membrane by docking proteins; synthesize proteins for secretion.
• Ribosomes located below LM resolution limits are stained by Hematoxylin (basophilic).

Function

• Translation of mRNA into amino acids forming polypeptide chains (proteins).

Cell Inclusions

• Metabolically inactive structures, including stored food (glycogen in liver and muscle, stained by PAS) and lipids (adipose tissue, stained by Sudan III or Sudan black).
• Pigments can be endogenous (melanin, hemoglobin, lipofuscin) or exogenous (tattooing).

Nucleus

• Largest structure in the cell responsible for DNA replication and RNA synthesis.
• Carries genetic material (DNA).
• Shape and size depend on cell type (rounded, elongated, flattened, multilobed, or bilobed).
• Usually a single nucleus (mononuclear cell). Some cells are binucleated (liver cells, transitional epithelial cells) or multinucleated (osteoclasts, skeletal muscle, giant cells). Anucleated cells have no nucleus (red blood cells).

Nuclear Components

• Nuclear envelope
• Chromatin
• Nucleolus

Nuclear Envelope

• Forms a selective permeable barrier between nuclear and cytoplasmic components.
• Consists of two membrane units separated by a narrow perinuclear space (EM).
• The outer membrane and perinuclear space are continuous with the rough ER.
• Many ribosomes attach to the outer surface of the outer nuclear membrane.
• Nuclear pores perforate the nuclear membrane, guarded by nuclear pore complexes that regulate molecule passage.

Chromatin

• Composed of DNA, histone proteins, non-histone proteins, and RNA transcribed from DNA.
• Responsible for basophilia in the nucleus and condenses into chromosomes during mitosis (46 in human cells).

Types of Chromatin

• Heterochromatin: Densely stained, transcriptionally inactive, located at the periphery of the nucleus, and abundant in metabolically inactive cells (small lymphocytes).
• Euchromatin: lightly stained, transcriptionally active, prevalent in nuclei of metabolically active cells (nerve cells, liver cells).

Nucleolus

• Dense, discrete, ovoid, non-membranous area inside the nucleus, responsible for ribosomal RNA (rRNA) synthesis and ribosome production.
• Nuclei may have more than one nucleolus, especially in active protein-secreting cells.

Types

• Vesicular nucleus: seen in active cells (liver cells) with more euchromatin than heterochromatin (open face nucleus).
• Dense nucleus: seen in inactive cells (circulating lymphocyte), with most chromatin as heterochromatin (condensed nucleus).

Basic Tissues

• Epithelium (epithelial tissue)
• Connective tissue
• Muscle tissue
• Nervous tissue

Organized to Perform Functions

• Tissues are groups of cells organized to perform one or more specific functions.

Epithelium Characteristics

• Features high cellularity, with cells closely adhered to one another by specialized cell-to-cell junctions.
• Resting on a basal lamina (basement membrane).
• Avascular (no blood vessels between epithelial cells).
• Nutrition depends on diffusion from underlying connective tissue.
• Rapid regeneration.

Functions

• Epithelium covers body surfaces and lines body cavities. Types vary according to function.

Classifications

• Classified according to the number of cell layers (simple or stratified) and the shape of the surface cells (squamous, cuboidal, or columnar).

Simple Epithelia

• Consists of one cell layer.
• Simple squamous epithelium is formed of one layer of squamous cells, lining Bowman's capsule, blood vessels (endothelium), and lung alveoli.
• Simple cuboidal epithelium is formed of one layer of cuboidal cells. Site collects renal tubules and follicles of thyroid gland and has collected renal tubules.
• Simple columnar epithelium is formed of one layer of columnar cells and lines the stomach, intestine, and gall bladder.
• Mainly involved in absorption or secretion.
• Pseudostratified columnar ciliated epithelium appears stratified due to nuclei at different levels but is actually simple and found in the trachea, bronchi (with goblet cells), and epididymis.

Stratified Epithelia

• Stratified squamous epithelium consists of many cell layers.
• The basal layer is cuboidal or columnar, with polyhedral middle layers and squamous superficial layers. It can be keratinized (epidermis of skin) or non-keratinized (esophagus, oral cavity, anal canal). Stratified cuboidal epithelium contains many layers with a superficial layer of cuboidal cells, lining the duct of the sweat gland.
• Stratified columnar epithelium contains many layers with a superficial layer of columnar cells, present in conjunctiva of the eye.
• Transitional epithelium has many layers with rounded or dome-shaped superficial cells. This epithelium is found in the ureter & urinary bladder. Transformed from rounded cells when empty to transitional when full hence the name. Protection.

Glandular Epithelium

• Epithelial cells produce and secrete macromolecules.
• Pancreatic acini cells secrete enzymes and sebaceous glands secrete lipids.

Classification of Glandular Epithelium

• Endocrine glands: secrete products directly into blood capillaries, such as the pituitary, thyroid, and adrenal glands which are called hormones..
• Exocrine glands: secrete products through ducts, such as salivary glands and pancreatic acini (exocrine part of the pancreas). The secretory portion of exocrine glands is called acinus, and made of Epithelial resting on basement membrane.
• Serous glands secrete a watery, protein-rich substance, that are high pyramidal in shape and have developed RER. Includes the Golgi complex. Parotid and pancreatic acini.
• Mucous glands secrete viscous mucus which are are low in column and they have flattened basal nucleus with foamy cytoplasm. Example is the Esophageal and Brunner's.
• Mixed glands formed of mucus, serous acini, and submandibular and sublingual salivary glands.
• A highly specialized type of epithelium that functions in perception of stimuli located in taste buds of oral cavity and the organ of Corti in the inner ear.

Connective Tissues

• Connective tissue consists of cells, fibers, and ground substance. Fibers and ground substance are is described as extracellular matrix.
• Functions include binding structures, supporting and protecting, serving as a framework for organs, forming organ capsules, synthesizing and storing fat, repairing wounds, and acting as a site of immune cell action.
• Classified as fixed (resident) or free (wondering), are classified as fixed or free with undifferentiated cells, fibroblast, mast cells with cells that react to stimuli.

UMSC

• A stem cell responsible for: connective tissue, cartilage, bone, and smooth muscles with minimal cytoplasm.

Fixed (Resident)

• Fibroblast (active cell) is the most common connective tissue cell. The cells have basophilic cytoplasm, abundant cytoplasm and reticula that produce most of the extracellular matrix.

Quiescent Cells

• Fibrocytes are smaller than fibroblasts, with few processes, a nucleus cell that contains few organelles.

Macrophages

• Derived from blood monocytes. Shape and size vary due to activity with irregular outer surface and lysosome cytoplasm that phagositizes tissue debris and foreign invaders during phagocytosis.

Adipocytes (fat cells)

• Common in connective tissues with synthesizing and storing fat: unilocular (white)
• Features rounded nucleus, cytoplasm in, smooth complex, a single large fat droplet with signet appearance and cytoplasm. stained by Sudan III or osmic.
• Multilocular (brown) have a rounded nucleus, small fat droplets. It also contains a smooth reticulum.

Mast Cells

• Has central nuclei. They are characterized by histamine a mediator of inflammation in which the basic dyes are changed by the granules. Heparin is found.

Plasma Cell

• Arise from activated B lymphocytes displaying cytoplasmic protein and spherical nuclei with cartwheel appearance that produce antibodies.

Baophils

• They have irregular lobes. Have cells with basophilic granules that change dye color that contain histamine and heparin chemicals and share features.
• Esinophils are involved with parasitic problems and allergic reactions and are created in the bloodstream.
• Esinphils are large with granules with multiple bilobed nucleuses.

Neutrophils

• Neutrophils are a granular leucocyte. First responders to infection Nucleus has multiple lobes linked by fine chromatin threads. Neutrophil cytoplasm contains two types of granules. Lysosomal Their numbers increase in bacterial infection.

Lymphocyte

• Lymphocytes are classified according to function into antibody mediated or cell regulated.

Fibers

• Fibers are produced by fibroblasts.
• Types: collagen, reticular, elastic.

Collagen Fibers

• The most components of the connective tissue appearing not branched. They can be stained by stain or Eosin.

Reticular

• III collagen and extensive network Visualize PAS

Elastic

• Stained in brown.
• A complex of macromolecule that fills cells and fibers.

Special Connective includes

• Loose
• irregular
• Mesenchyme

Functions of CT

• Vessels can be stained. Blood includes erythrocytes and volume accounts for 5 to six.

Erythorcytes

• Most are in circulation that 99% do but the rest lose it. RBC are and contain with a blood film biconcave. A certain shape provides large gas exchange but adhere too
• Rouleaux can be removed. WBC also contains granules of leukoctryes and non cells and they are also motile. Normal is 4000 -110000

Neutrophils

• Account for 60 to 70 nucleolus. They have 5 segments and their nucleus has strands with the function to first kill invaders.

Granules contain

• Small membrane but stins with dyes
• Azuro stain

Eosinphills

• Is around 1 to 15 percentage, with a 12 to 15 diameters that also include 2 granules and are acidiophilic
• The eosinophils are is abundance and in tract. They complex and parasite infections.

Basophilis

• They are the least. Some include are that make them stain intensely. They have allergic which they involve and increase

Lymphoctyes

• Account for the mass amount or leukocyte also with small mitochondria Differentiation happens in the bone marrow and the thymus. They can be separated.

Platelets

• They have little cytoplasm with megakaryocytes. Normal is 400 x 10. They also have different muscles.

Muscle Tissue

• Striated muscles include cardiac and skeletal.

Elongated Fivers

• The plasmic membrane is sarcolemma. Each has two proteins for contractile parts: myosin and action Skeletal can include that a the diameter can vary.

Skeletal Organization

• Is made of fibers and delicate parts. A long section shows alternating parts with banding. The filaments is parallel.

Myofibrils

• Banging dark a a light band and each part by light is Z.

Functional unit

• Sarcomere the zone to z.
• thick myosin is present and the center is also length wise a band.
• thin activity from line to both sides running Iband. the line is formed. the center is H

Skeletal Muscle

• Skeletal non cylindrical and has parts inside it The sarcolemma. the reticulum is triaed that aids the the releasing the calcium

Cardiac

• It is also involuntary. Is with acidophilic and less branched. They contain no trials and complex mitochondrion.

Vascular parts

• Non involuntary in walls but include the ureter And present with the end.

Vessels

• Transports and lympth. It contains the heart blood and vessels with a layer: tunics.

Endothelium

• Specialized a membrane barrier with semi that fluid the endothelium presents a layer.

Layers

• The middle layer has circumferentially. it increases. Elastin are also intervened.

Outer

• Has long collagen and fiber that supply vascular
• Capillaries has single tubes and pericytes. These are divided because tight a continuous with a high amount of molecular fluid and a membrane.

Sinuses

• Have high amount for cellular. fluid levels. In the veins they vascular has the intima.

Vessels

• And an increased blood levels that include. Lymphocytes respond that provides immunity with leukoctyes and has many organs.

Organization

• Nodes and lymphoid in blood. Are a certain surface that blood enter. The cortex. the nodes and cells perform. The medulla tissue is in.

Medullary

• Are mass Sinuses also connect that are used for the drain. The paracole is in area.

Spleen

• It can be the body location. filtration of blood. In tissue and cells in a mesh part with tissue that can contract. Pass. It can have.

White

• The white consists while cells are in form and branching of the artery. The cells

Lymphodials

• Are primary . In section stained are apparent. Puld for the number.

Red

• From a large and large cells. Made of cell cords and billsroth The connect or.

Dendric

• The system are that are cell system in many and a cells. The gland or cell. The in most tissue. Endocrine

Thyroid

• Under is with connected. with to. with vascular with follicle. synthesis the metalblim. with calcitonin and calcium

The

• The capsule connect with the cells. This is well. The is the thyroid. million and follicle

Cell Types

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Parathyroid

• Has capsules. Cells.

GI Consists

• The system small intestines and gland. and

Functions

• Ingestion movement hormone action digestion Features that forms. The linings.

The Inner

• Lumen the tissue smooth movement

Muscleras and Externa

• Composed of muscle and. with With

GI

• The tracts for in called or covered by the and. that is. the. the of

LIVER

• is about. three. proteins. with. the the side where by the from what the from and. connected

Hepatocytes

• And.

Structures

• Also, the and.

Cells

• derived recognize or. in with. and that They.

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• The is describes the system. the and. contains with with what.