Cell Histology

Histology

Study of tissues of the body and how these tissues are arranged to constitute organs

Histology levels of organization smallest to most complex

Molecules, Cells, Tissues, Organs, Organ Systems

Light microscope can see how much better than the human eye?

1000X

Preparation of tissue for microscopy steps (9)

Fixation, Dehydration, Embedding, Trimming/Sectioning, Removal of paraffin, Rehydration, Staining, Mounting sections of glass slide, Viewing the tissue (LM)

Fixation

Step 1: Small pieces of tissue are placed in solutions of chemicals that preserve by cross-linking proteins and inactivating degradative enzymes

Dehydration

Step 2: In alcohols, removes all water

Embedding

Step 3: Paraffin-infiltrated tissue is placed in a small mold with melted paraffin and allowed to harden

Trimming/Sectioning

Step 4: Paraffin block is trimmed to expose the tissue for sectioning (slicing) on a microtome

Staining

Step 6

Tissues

Cells that carry out the same general function

Four basic tissues

Epithelia, Connective tissue, Muscle, Nerve

Epethelia

Cover and/or line internal and external body surfaces

Form secretory glands and ducts

Connective tissue

Packing, support, connecting

Muscle

Contractility

Nerve

Irritability, conduction

Plane of section

Cutting the specimen, crucial to how specimen is viewed. Improper plane of section can lead to misdiagnosis

Different plane of sections

Longitudinal section, Cross section, Oblique section

Basic dyes

Bond with acidic molecules. Toluidine blue, methylene blue, Hematoxylin. BLUE

Acidic dyes

Bond with basic molecules. Eosin, fuchsia. RED

Hematoxylin will react with what molecules to stain them blue?

Acidic molecules in the nucleus

Basophilic

Anionic components (acidic components) stain more readily with basic dyes

Acidophilic

Cationic components (basic molecules) stain more readily with acidic dyes

Cytoplasm will stain what color?

Red/pink due to cationic proteins, acidophilic

Nucleus will stain what color?

Blue due to anionic nuclei acids, basophilic

Luxol fast blue stain (LFBS) / Luxol blue and hematoxylin

Shows myelin

Hematoxylin and Eosin stain (H&E)

Red=basic molecules (Eosin)

Blue=acidic molecules (Hematoxylin)

Dissolves fat (adipose tissue) and mucous during preparation process. Appears as white circle (empty )

Trichrome stain

3 colors, shows connective tissue (collagen, fibrin) in green/blue color

Golgi stain

Potassium dichromate and silver nitrate

Gold & Luxol blue & Cresyl violet

Used to demonstrate the Nissl substance in the neurons and cell nuclei

Periodic Acid Scheff Reaction stain (PAS)

Shows mucous, microvilli, basement membrane and glycogen molecules

Distortions and artifacts

Result due to problems in interpretation of tissue sections due to either

A) Shrinkage because of fixation, dehydration and embedding

B) Loss of molecules that were not retained after fixation or removed during dehydration (glycogen and lipids)

Two types of electron microscopy

1) Transmission Election Microscopy (TEM)

2) Scanning Electron Microscopy (SEM)

Electron Microscopy

Electron beam passed through a very thin section of tissue

Transmission Electron Microscopy (TEM)

Permits high resolution

Allows magnification of up to 400,000 times

Looks at small portion of the cell

Can view organelles

Utilizes osmium tetroxide

Osmium tetroxide

Reacts with phospholipids and imparts electron density to cell and tissue structures because it is a heavy metal to enhance image formation in TEM

Electron dense

Dark areas in TEM

Electron lucent

Light areas in TEM

Scanning Electron Microscope (SEM)

Shows only surface views

10 nm resolution

Can view large depth of field

Inside of organs are viewed by freezing and fracturing tissues to expose internal surfaces

Membranous organelles

Contain plasma membranes that separate internal environment of organelle from the cytoplasm (cytosol + organelles)

RER, SER, Golgi, Mitochondria, Lysosomes, Peroxisomes

Non-membranous organelles

Lack a plasma membrane

Ribosomes, cytoskeleton and inclusions

Nucleus functions

1) Cellular regulation - houses genetic material which directs all cellular activities and regulates cell structure

2) Produces ribosomal subunits in nucleolus and exports them into cytoplasm for assembly into ribosomes

Outer nuclear membrane

Faces the cytoplasm, contains ribosomes and is continuous as certain sites with RER

Inner nuclear membrane

Faces the nuclear material and is supported on its inner surface by the nuclear laminate

Functions: stability to the nucleus, chromosomes attach

Nuclear envelope

Perinuclear space between outer and inner membranes. Function: Allows molecules in/out of the nucleus.

Molecules >9nm transported by active process mediated by receptors and utilize energy.

Molecules <9 nm (ions and smaller water-soluble molecules) cross via simple diffusion

Nucleosome

DNA sequence wrapped around a core of histone proteins. The basic repeating subunit of chromatin packed inside the cell’s nucleus.

Heterochromatin

Dense staining (dark), highly condensed chromatin (closed). No active transcription

Eurochromatin

Light staining, open conformation chromatin. Active gene transcription occurs

Nucleolus function

1) Site of ribosomal RNA (rRNA) synthesis

2) Initial assembly, once assembled transported to cell cytoplasm via nuclear pores to serve as sites for protein synthesis

Ribosomes functions

1) Site of translation’s

2) Protein synthesis

AKA reads messenger RNA (mRNA) and translates into amino acids to form proteins

Found in cytoplasm or bound to RER

RER functions

Protein synthesis

Post translational modifications of proteins (N-linked glycolysation)

Glycolysation

Modification process in which a carbohydrate covalently attaches to a target macromolecule, typically lipids (glycolipids) and proteins (glycoproteins)

N-linked glycolysation

Begins in RER

Co-translational mechanism

O-linked glycolysation

Occurs in Golgi after protein is translated, occurs post-translationally

SER functions

1) Synthesis and break down of glycogen

2) Detoxification of drugs, metabolic wastes, etc.

3) Synthesis of lipoproteins, cholesterol, bile salts

4) Synthesis of steroid hormones

5) Uptake and release of calcium in muscle cells

Golgi functions

1) Post-translational modification of proteins (from RER via vesicles), specifically O-linked glycosylation

2) Synthesis of lipoproteins

Mitochondria functions

1) Energy production (oxidative phosphorylation)

2) Beta oxidation of long chain fatty acids

3) Steroid hormone synthesis

4) Regulate apoptosis

5) Storage and release of calcium

Lysosomes functions

1) Heterophagy (phagocytosis and degradation of bacteria by neutrophils)

2) Bone remodeling via osteoclasts

3) Autophagy (destructions of worn-out organelles)

Peroxisomes functions

1) Beta oxidation of long chain fatty acids

2) Degrade hydrogen peroxide, a product of oxidative reactions

Lysosome storage dieseases

Caused by mutation in the gene that encodes for proteins in lysosomes (hydrolases, membrane proteins, co-factors). Results in accumulation of the substrates for lysosomal digestion

Zellweger’s/Cerebrohepatorenal Syndrome

Fatal disease due to absence of peroxisomal enzymes. Reduced degradation of cytotoxic hydrogen peroxide and abnormal accumulation of very long chain fatty acids. Skeletal muscle weakness, hypotonia, inability to nurse properly, neurological disorders, enlarged kidney, etc.

Plasma membrane functions

1) Physical barrier: establishes a flexible boundary

2) Selective permeability: regulates entry and exit of ions, molecules, nutrients

3) Electrochemical gradient: establishes and maintains an electrical charge

4) Communication: contains receptors

Carbohydrates functions (in plasma membrane)

1) Attach to proteins, forming glycoproteins

2) Forms cell coat (glycocalyx)

3) Establishes extra cellular microenvironment

4) Aids in metabolism, cell recognition, cell association, receptor sites for hormones, etc.