Week 2 - ANHB3323 (3) - Microscopy and the Cell Cycle

Cell size

Cell size = 5-100um

Nucleus = 5um

Red Blood Cell = 8um

- cellular ruler

Light vs Electron Microscope visibility (vs human eye)

Light = down to 0.2um

Electron = down to 0.2nm

Human Eye = down to 0.2mm

Electron microscope

done in a vacuum

therefore cant do live cells

Fixation

immediate stop of living processes in cells/tissues

to preserve structure

Fixation will block...

degradation of enzymes such as: DNAses, RNAses, and proteases

and

Prevent 'rotting' in bacteria

Methods/Reagents used for Fixation of Cells (5)

1. Physical (heat/freezing etc.)

2. Solvents

3. Acids

4. Chemical

5. Metal Salts

What do you do to a Fixed Tissue Sample after Fixation? (roughly)

Process it to make it harder

Cut it to make it transparent (thinner)

Sectioning as Fixed Tissue (2 methods)

1. Cryostat:

- cuts frozen sections:

- 5 to 20um

2. Parafin/wax embedding:

- 3 to 10um sections

- permanent archival block

Immuno-staining

antibody to recognise specific antigen

antibody is visualised with fluorescent secondary antibody/conjugate

Fluorescence (3 stage process)

1. Excitation Photon of energy (hvEX)

2. Excitation-state Lifetime

3. Emission Photon of energy (hvEM)

Electron Microscopy Types (2)

Transmission EM (TEM):

- information of internal structure

- electron beam enters specimen

Surface Scanning EM (SEM):

- information of surface structure

- electron beam bounces off surface

Transmission Electron Microscope Sample Preperation

1. Fixation:

- Glutaraldehyde

- Paraformaldehyde

2. Post-Fixation:

- Osmium (secondary fixative)

Surface Scannaing Microscope Sample Preperation

1. Fixation:

- Glutaraldehyde

- Paraformaldehyde

2. Post-fixation:

- Osmium

- Tannic acid

[only difference with TEM = tannic acid]

Cell cycel phases

Interphase:

1. G1 = growth

2. S = DNA replication

3. G2 = final prep

Mitosis:

1. Prophase

2. Metaphase

3. Anaphase

4. Telophase

Duration of each phase in Interphase

G1 = indefinite (normal cell activity)

S-phase = 6 to 8 hours (DNA replication)

G2 = 3 to 4 hours

Cell shapes

Squamous, spheroid, polygonal, discoid, cuboidal, fusiform, columnar, fibrous and stellate

Nucleus

Control centre of cell:

• Nuclear envelope with pores

• chromatin (DNA wrapped around histones)

• nucleolus (make ribosomes)

Endoplasmic reticulum (ER)

Rough: studded with ribosomes, site of protein synthesis

Smooth: lipid synthesis and drug metabolism

Golgi Apparatus

Modify, sort, and package proteins and lipids for secretion or delivery to other organelles.

Non-ATP diffusion

Passive diffusion: small molecules across the cell membrane

Facilitated: use a protein channel, slightly larger molecules

Types of Carrier proteins

Channel: shape is constant

Carrier: changes shape to conform to molecule (active or passive)

Receptor: required for cell signalling → receptor is specific to it’s complimentary ligand therefore not signalled unless bound by appropriate molecule.

Active diffusion

Requires ATP, moves against concentration gradient, can transport larger molecules.

Exocytosis vs endocystosis

Exo: out of the cell

Endo: Into the cell

Endocytosis Methods

Phagocytosis: ingestion of large particles or cells;

Pinocytosis: uptake of small particles or fluids.

endocrine signalling

steroid hormones: require transport protein in bloodstream, can pass through cell membrane to intracellular receptors typically on nucleus.

peptide hormone: travel freely through blood, attached to receptor on cell membrane and trigger a cascade.

Messenger types

first/primary messenger: only one, typically outside the cell

Secondary messenger: once activated there can be multiple

Signalling

Endocrine: hormones

Exocrine: secretions into ducts and outside the body.

Electrical: used by nervous system, driven by movement of positively charged ions