Electronic Microscopy Seminar

Biology Seminars

• There are four seminars in Biology:
  • Electronic Microscopy
  • Cancer Biology
  • Autosomal and sex-linked inheritance
  • Human genome and mutagenesis

Seminar 1: Electronic Microscopy

Basic Concepts

• Magnification refers to the extent to which an image is enlarged.
• Resolution is the minimum distance at which two points can be distinguished as separate.
• Numerical aperture refers to the amount of light an objective can absorb.
• Depth of field refers to the ability to keep distinct planes of a preparation or sample in focus simultaneously.

Electronic Microscopes

• Electronic microscopy uses a beam of electrons instead of visible light to form high-resolution and high-magnification images
• Electron microscopy differs in:

Transmission Electron Microscopy (MET/TEM).

• MET uses a beam of electrons that passes through the sample.
• It provides two-dimensional images with a high resolution of 0.2 nm.
• Used to study the internal structure of cells and tissues, allowing observation of cellular organelles like mitochondria, lysosomes, and the endoplasmic reticulum.
• It has magnifications from 1,000x to 1,000,000x.
• It has the same lens arrangement as an optical microscope.

MET Configuration

• Electron source (cannon)
• Electromagnetic lens system.
• Observation chamber.

Image Formation in MET

• Based on the interaction of primary electrons with the sample.
• Yields a flat (2D) image.

Scanning Electron Microscopy (MEB/SEM)

• MEB uses an electron beam that scans the surface of the sample.
• MEB generates three-dimensional images with great depth of field
• Resolution goes down to 3 nm.
• Magnification ranges from 10x to 100,000x.

MEB Configuration

• Features a tungsten filament electron gun.
• It has condenser lenses (column) and a secondary electron detector.
• It has a vacuum chamber and cooling system
• Image Formation in MEB
  • Relies on the interaction of the beam with secondary electrons that come from the superficial layers of the sample and reveal the topography, or backscattered electrons that come from the nucleus of the sample and bear data about the composition.
  • Image contrast depends on the number of reflected electrons
  • Yields 3D images

Environmental Scanning Electron Microscope (ESEM)

• ESEM allows observation of samples in their natural state.
• ESEM doesn't require complex preparation.
• ESEM can be used to study both biological and non-biological materials.
• It has a resolution of 3 nm.
• ESEM has applications in:
  • Biological and forensic sciences.
  • Nanotechnology and semiconductor materials.
  • Geology and analysis of contaminating particles.

Sample Processing for MET and MEB

• Vitrification involves cooling a sample to prevent water crystal formation.
• It stops all activity.
• Vitrification is necessary in electron microscopy because all cells observed have to be dead, but their vital functions must be visible, as that is the focus of study.

MET Sample Processing

• Fixation: use of abundant fixatives like:
  • In-vitro
  • Perfusion: introducing the fixative directly into the bloodstream.
  • Chemical Method: employment of fixative solutions.
    • Glutaraldehyde to fix protein
    • Osmium tetroxide to fix lipids
    • Fixation quality is affected by the size of the sample, fixation time, fixative concentration, temperature, osmolarity, and pH changes (use of Buffer solutions)
  • Physical Method: use of low temperatures to effect vitrification
• Dehydration
• Inclusion: use of epoxy resins
• Cutting:
  • Semi-thin cut
    • Uses a crystal blade
    • Sections are 0.7 µm thick
    • Sample observed under an MO
  • Ultra-thin cut
    • Uses a diamond blade
    • Sections are 60-90 nm thick
    • Sample observed under a MET
• Grid Assembly
• Grid Contrasting
  • Uranyl Acetate
  • Lead Citrate
  • After some time, samples are washed and air-dried, then ready to be observed

MEB Sample Processing

• Samples must be conductive to avoid the electron beam negatively charging it and distorting image formation.
• Fixation: presumed to be the same as MET.
• Drying
  • The critical point method involves using liquids to cause the sample to pass to gas without modifying their density at a certain pressure and temperature.
  • Liquid CO2 is introduced and removed, displacing all the water. CO2 then evaporates.
• Sample Assembly
  • Physical union, not just a mechanical one, between the sample and the support.
  • Heat transmission through:
    • Carbon adhesives
    • Double-sided tape
    • The material surrounding the sample must also be conductive.
• Sample Coating
  • Ion sputtering involves coating the sample (20nm) with a metal element like Au, Pd, Pt, or Ag.
  • Evaporation: Heating the carbon thread beyond its melting point, causing it to fall onto the sample and form a continuous film as it condenses.
• Observation

Use Case: Study of the SARS-CoV-2 viral cycle

• Images from cells infected with SARS-CoV-2 are presented.
• MET is used to observe viral particles inside infected cells.
• MEB is used to analyze the interaction between viruses and the cell membrane.

Seminar 3: Autosomal Inheritance and Sex-Linked Inheritance

Autosomal Dominant Inheritance

• The trait is expressed in heterozygotes.
• There's a 50% chance of inheriting the trait.
• It affects men and women equally.
• It's vertical, found in all generations.
• No carriers; everyone expresses it

Neurofibromatosis

• Causes nervous system problems, specifically tumors develop along the nervous system.
• There are two types:
  • Type 1: A defect in the gene to produce of neurofibromin (anti-tumor factor), so tumors occur; leads to bone lesions, skin lesions, and optic pathway gliomas.
  • Type 2: A defect in the gene to produce schwannomin (anti-tumor factor), so tumors occur, leading to VIII cranial pair tumor, and meningiomas.

Huntington's Chorea

• A neurodegenerative disease contrasting with Parkinson's.
• It leads to cognitive, psychiatric, and motor alterations, and progressive dementia.
• Use speech and occupational therapy, exercise, psychotherapy, antidepressants or antipsychotics for its treatment
• The CAG triplet in a normal person features 10 to 35 repeats, so affected individuals may have between 36 and 120 repeats, leading to excessive brain protein products.
• It's linked to dopamine.
• Genetically lethal in homozygosis, but the disease manifests in heterozygosis

Marfan Syndrome

• It affects the connective tissue, with large hands and feet.
• It involves unusual limb length, skeletal phenotype, eye and cardiovascular lesions, delayed walking, learning disabilities
• For its treatment: medical check-ups for prevention, medicines that lower the arterial pressure, glasses, surgery, and psychological treatments...
• A mutation in a gene related to fibrillin, a critical protein for the connective system

Achondroplasia

• It causes dwarfism with the following symptoms:
  • Abnormal growth of long bones
  • Lumbar lordosis
  • Hypotonia
  • Macrocephaly
  • Typical facial features
  • Recurring otitis
  • Strabismus
  • Trident sign (separation between the third and fourth fingers)
• Treatment involves:
  • Correcting the direction of bone growth.
  • Espatilization and correction of spinal shape.
  • Increasing the size of vertebrae openings to relieve pressure.
• Mutation in a gene that impedes osteocyte growth, leaving bones undeveloped.
• Sublethal

Von Willebrand Disease

• This condition causes a blood clotting disorder like mild hemophilia, but affects more women than men.
• This blood disorder leads to hemorrhages, nosebleeds, and gum bleeds.
• Its treatment involves oral contraceptives, concentrated coagulation factors and antifibrinolytic drugs before surgery.
• There are several types:
  • Type 1: deficiency of a coagulation factor.
  • Type 2: deficiency in the synthesis of this factor.
  • Type 3: absence of this factor, which is impossible in heterozygotes, and rare in homozygotes.

Retinoblastoma

• This condition leads to a malignant tumor in the retina
• This tumor can spread to the neck and bone marrow.
• Its treatment involves chemotherapy, radiotherapy and surgery.
• It is caused by a mutation in a tumor-suppressor gene, with 60% mutation.

Autosomal Recessive Inheritance

• Expresses only when the genotype features two recessive alleles.
• Characterized by carriers rather than heterozygotes.
• Shows a horizontal pattern, not found in earlier generations.
• Enhanced risk of the hereditary condition by consanguinity.
• It affects men and women equally.
• Only descendants of affected parents are affected.

Albinism

• This condition involves reduced or absent melanin in skin, hair, and eyes
• Albinism involves oculocutaneous symptoms
• There are two types:
  • Type 1: reduced melanin production, caused by mutations in a melanocyte gene.
  • Type 2: absent melanin production, with mutations affecting transport needed for melanogenesis.
• Blacks are more likely to have albinism.

Phenylketonuria

-This condition relates to disabled phenylalanine metabolism, and resulting buildup of phenylalanine.

• It shows symptoms like elevation of phenylalanine in plasma, delayed psychomotor development, neurological manifestations, a musty odor in the urine, skin, hair, and light eyes, cutaneous eruptions
• Treatment options are lowering phenylalanine levels with diet, and supplementing with tyrosine.
• It can be reversed if phenylalanine is not ingested

Sickle cell anemia or drepanocytic anemia

• This conditions affects hemoglobin production, resulting in abnormal erythrocytes that cannot function well.
• This anemia has multiple symptoms, for example:
  • Painful swelling of hands and feet
  • Pallor
  • Fatigue
  • Headaches
  • Rapid pulse
  • Chest pain
  • Breathing difficulty
  • Pulmonary hypertension
  • Splenomegaly
  • Jaundice
  • Delayed puberty
  • Abdominal pain
  • Infections
  • Strokes
  • Cerebrovascular episodes
• Treatment:
  • Preventing infections
  • Pain treatment
  • Red blood cell transfusions
  • Marrow transplantation
• It can be caused by an A to T substitution in the β globin chain gene
• This causes an amino acid change that also alters the protein.
• Sickle cells are eliminated after 10-20 days.

Cystic Fibrosis

• Effects functioning of exocrine glands due to an abnormal chloride and sodium transport.
• There are thick and viscous secretions and clinical manifestations, especially at the respiratory and digestive levels
• The respiratory ailment symptoms are: chronic obstruction, lowered pulmonar capacity infections.
• The digestive ailment symptoms are: pancreactic insufficiency, fat malabsorption and altered growth More symptoms listed are: rhinitis, sinusitis, nasal polypes, high electrolytes, bowel obstruction and acropaquia Treatment involves medications, physiotherapy, pulmonary rehab, surgery, diet, and more Mutations occur in a gene that encodes a protein related to ion transmembrane This autosomic-recessive genetic disease expresses variably, but is more frequent among Caucasians.

X-linked Dominant Inheritance

• If the father is affected, there's a 100% chance of his daughters being affected, and 0% sons
• Heterozygous females have a 50% chance of having affected children
• Homozygous females have a 100% chance
• More frequent in women

Hypophosphatemic Rickets

• Causes bone deformities due to improper bone mineralization.
• In children there are :
  • Headaches
  • Chiari malformation
  • Craniosynostosis
  • Bowed legs
  • Valgus knees
  • Bone pain
  • Joint pain
  • Muscular weakness
  • Growth delay
  • Short stature
  • Irregular spacing between teeth
• In adults:
  • Dental abscess
  • Early osteoartritis
  • Inflammation of bones and joints
  • Muscular pain
  • Periodontitis
  • Osteomalacia
  • Enthesopathy
  • Ossesous and articular pain auditory loss
  • Fractures and pseudofractures.
• Treatment includes:
  • Physiotherapy, surgery, and antibodies.
• The condition is caused by mutations in a gene related to phosphate concentration regulation mutation de novo.

Fragile X Syndrome

• The leading cause of hereditary mental impairment caused by the following symptoms:
  • Elongated face
  • Broad forehead
  • Large and projecting ears, mental and psychomotor delay, hyperactivity
  • Autistic traits
  • Poor eye contact
  • Redundant speech
  • Cardiac problems
  • Increased joint mobility
  • Low muscle tone
  • Macroorchidism
  • Early menopause
• Treatment: Specilized education treatment with medication, therapist
• Mutations in a gene that leads to repeated triplets silences gene expression (methylation).

X-linked Recessive Inheritance.

• Heterozygous women lead to 50% affected and 50% carrier sons.
• Affected men: 0% affected sons.
• A mutation can only be passed from father to son.

Duchenne Muscular Dystrophy

• The leading cause of death is a respiratory faliure, and the second heart-related.
• This dystrophy has specific symptoms: winged scapula, hyperlordosis, conduction disorders (hypertrophic or dilated myocardiopathy), delayed walking (3 years), loss of walking (13 years), scoliosis, impaired ventilator function, intellectual difference, pseudohypertrophy of the calves.
• Treatment involves: physiotherapy, corticosteroids, orthopedics, surgery...
• Mutations in the gene that encodes dystrophin, a protein that stabilizes muscle fiber membranes

Daltonism

• This condition causes a perception disorder characterized by the inability to distinguish differences between some colors.
• It is caused by mutations in genes encoding proteins found in cones.

Y-linked Inheritance

• Mutations are only transmitted from fathers to sons.

Ichthyosis

• This condition involves the skin becoming dry and scaly

Seminar 2: Cancer

Definition of Cancer

• Cancer happens when some of the body’s cells grow uncontrollably and spread to other parts of the body.
• Caused by genetic aberrations.
• Ordinary tissue consists of balanced cell proliferation and mortality
• Multiple mechanisms regulate the different stages of the cell cycle.
• The most common cancers occur in the breast, lung, colon, rectum, and prostate.
• Cancer is always produced by cell division errors.

Risk Factors for Cancer

• Environmental factors affecting DNA:
  • Chemical compounds.
  • Radiations
  • Viruses or bacteria.
  • Diet
• Mutations in genes that regulate the cell cycle:
  • Process of DNA repair.
  • Division itself.
  • Apoptosis.
• Cancer is produced by interactions with genetic inheritance and external factors.
• There are carcinogens:
  • Physical carcinogens: UV and ionizing radiation exposure.
  • Chemical Carcinogens: arsenic, asbestos or products found in tobacco smoke.
  • Biological carcinogens: viruses.
• Because of this, cancer probability rises with age, caused by accumulating risk factors, summed with inefficiency of cellular repair.

Genes, Mutations and Cancer

• Genes are DNA segments that control the body’s creation of proteins.
• Genetic change can be a mutation, or a genetic variant in DNA.
• A point mutation occurs when changing a single nucleotide.
• People can have bad genes and not realize, because the other gene compensates.
• Cancer rarely develops due to point mutations.

Division without control

• Cell mutation enables tumor growth without check.
• The three genes that intervene are:
  • Proto-oncogenes: Natural genes create proteins to regulate cycles of normal cell growth and division to ensure multiplication. The genes may mutate or have too many copies.
  • Oncogenes: Cancerous proto-oncogenes prompt uncontrolled cell division.
• Tumor-suppressor genes: Repair DNA, control division speed, and help in apoptosis if those proteins fail or mutate.
• Tumor-suppressor genes can fail at a loss of activity or mutation, causing uncontrolled division.
  • Examples are EI BRCAI/BRCA2 (Breast cancer genes), leading to hereditary mutation
• Inhibitors of tumor suppression act by intervening at monitoring stations and by coding repair genes.
• Reactivated cell division requires repairing new cells in the cellular density increase.
• Deregulation in cancer cells inhibits proliferation.

When do mutations of predisposition for cancer occur?

• Somatic mutation mostly occurs in tumor cells.
• Hereditary mutations occur mostly in all the cells of the body, but can be mosaic type.

What kind of genetic changes cause concretely the cancer?

• Point mutations (a mutation, such as a single nucleotide substitution, that affects only one point in a genetic sequence). The Kras protein is a very common mutation, telling sells to multiple.
• Chromosomic reassortment (Aneuploidias)
  • When an interchange of two genes causes leukaemia cell multiplication without ceasing control.
• DNA Change outside of the genes.
• Epigenetic changes that don't affect DNA codes.

Morphological changes in the tissues and cancerous cells

Before cells turn to the cancerous state, these cells go through abnormal changes.

• Hyperplasia cells increase in number.
• Dysplasia - microscopically change in a noncancerous state.
• Cancer cells undergo normal changes, with an increase in size with multiple nucleolos. These cancer cells go through changes that can lead to the developement of cancer.

Types And Terminologies

• A carcinoma is the most frequent type of cancer, referring to tumors in skin cells or on organs.
• Adenomas are benign, yet can become malignant over time
• Features of cancer cells
  • Self sufficiency in signals of growth
  • Lack of sensibility in signals of growth inhibitor
  • Evasion of apoptosis
  • Unlimited replicative capability
• Continued angiogenesis through the creation of blood vessels.
• Altered cell metabolisms
• Inflammatory microenvironment
• Immunitarian evasion or immunity protection from cancer cells.
• Phenotypic unblocking plasticity causes the ability to specialize in a type of cell it should not causing: d
  • Desdiferentiation of the cells.
  • Terminal Blockage
  • Transdifferentiation

Diseemination Of Cancer/ Metastasis

• Causes death for the majority
• Treatments help in long term survival. The goal is prevention of more spreading and relief pain
• Cancer metastasis shares same name as of origin
• Through microscopy similar characteristics are visible
• Tumors from metastases can cause severe damage

Genetics in Cells

• Number of mutations for development differs through the tumor from a median of 4 mutations.
• Anomalous cell division accumulates chromosmic anomalities

Childhood Cancers

• Most common: Encephal, Leukemia, or tumors in the lymphatic system
• Five percent are hereditary, with no clear reason

Gene Examples involved in Cancer

• Genes P53 stop genetic production with multiple changes, and responds to ADN alteration by stopping cells trying to fix, or enabling apoptosis.
• The genes “MYC” code for the oncotype proteins that produce initiation and maintenance of cancer through mechanisms that alter checkpoints of specific receptors

Hereditary Example for hereditary cancers

• Exposure, habits, smoking and obesity
• There can arise genes as a hereditary anomaly and it can generate heritable defects of the parents.
• Features of cancer include:
  • Presenting the same cancer in many members
  • Appearing prior to normal years
  • Appears in over one kind of cancer
  • Can be shown with body parts both sided
• They pass through new generations
• Must be able to recopliate that familiar info along with age group and race.

Preventive Detection

• CTC (circulating tumor cell= circulating cancerous cell): Isolation used for knowing genetic history and make treatment simpler