BS101: Lecture 2 - Organisation of the cell and methods to study cells

What is the fundamental unit of life?

All organisms are made of cells

What is the cell?

The simplest collection of matter that can live

What is the importance of cell structure?

Correlates with function

What are the 2 types of Light Microscopes?

Upright and Inverted

What is the Upright Light Microscope?

Objective lens points towards sample from above

What is the Inverted Light Microscope?

Objective lens points from below

What are the types of Light Microscopy?

Brightfield, Optical contrast, Fluorescence

What are the types of Electron Microscopy?

Transmission, Scanning

Describe Unstained Brightfield Microscopy

• Complexicity?

• Colour?

• Contrast?

• Easy

• No colour - Difficult to see structures

• Light passes through - no contrast

Describe Stained Brightfield Microscopy

• Increased detection of subcellular structures

• Can kill cells - harsh chemicals

• Colour

Describe Phase-Constrast Microscopy

• Density

• Staining?

• Outline

• Amplifies variations in density

• Cells alive - no staining

• White outline

What is the benefit of Phase Contrast Microscopy?

Allows observation of transparent living cells

How does Phase Contrast Microscopy work?

• Where does light pass through?

• What does this cause?

• Result?

• Light passes through diaphragm which focuses a ring of light onto sample

• Causes phase shift to alter light wave path

• Results in improvement of contrast

How does Differential Interference Contrast Microscopy work?

• Interference

• Appearance

• Result

Interference between polarised light, objects appear in relief, casting shadows (3D)

How is the Optical Contrast in Differential Interference Contrast Microscopy?

Good, looks 3D

Optical Contrast Phase Constrast

White halo around cell

How does Immunocytochemistry work?

• Antigen

• Binding of Antibody

• What does dye indicate?

• Antigen on cell

• Enzyme/dye bound to antibody - to visualise binding of antibody

• Dye indicates where antigen is

How do Electron Microscopes work?

Electromagnetic lenses focus electron beam

How does the resolution of an Electron Microscope compare to a Light Microscope?

Higher resolution

How are samples viewed in Electron Microscopes?

• Stained w/

• Vacuum

• Stained w/ heavy metals for contrast

• Vacuum to prevent electron scatter

Scanning Electron Microscopes

Focus electron beam onto surface of specimen, 3D image

Transmission Electron Microscopes

Focus beam through specimen, mainly study internal structure of cells

Cell Fractionation

• What does it do?

• What does this mean for scientists?

• Role of biochem/cytology

• Takes cells apart and separates major organelles

• Enables scientists to determine functions of organelles

• Biochem + cytology help correlate cell function w/ structure

Process of Cell Fractionation

• What is the pellet rich in?

• Spinning

• Order of Organelles

• Pellet rich in nuclei and cellular debris

• Spin at increasing speeds for longer times

• Mitrochondria, Microsomes, Ribosomes

Which organisms consist of prokaryotes?

Only bacteria and archae

How is DNA stored in Eukaryotes?

In nucleus bound by membranous nuclear envelope

How are organelles stored in Eukaryotes?

Membrane bound

Where is the cytoplasm in Eukaryotes?

Region between plasma membrane and nucleus

How are Prokaryotes different than Eukaryotes?

• Nucleus

• DNA

• Organelles

• Cytoplasm

• No nucleus

• Unbound DNA (nucleoid)

• No membrane-bound organelles

• Cytoplasm bound by plasma membrane

Q: Which factor increases the numerical aperture (NA) of an objective lens?

Immersion oil between the slide and lens

Equation for Numerical aperture (NA)

• Equation

• n =

• θ =

• nsin(θ)

• n = refractive index of the medium btwn specimen and lens

• θ = half-angle of light entering lens

How does oil cause NA to go up?

Increases refractive index n

What is Numerical Aperture?

How much light a lens can gather

Relationship btwn Wavelength and Resolution

Resolution increases when wL decreases

Effect of closing the Condenser

Reduces illumination and NA, lowering resolution

Why does blue/violet light improve resolution?

Shorter wL

In a compound light microscope, which component primarily focuses light onto the specimen?

Condenser

What does the Objective Lens do?

Collects light after it’s passed through specimen and forms magnified image

Which component of the cytoskeleton is primarily responsible for cell shape and cell movement?

Actin Filaments