Bioimaging Lecture 1

What are the three basic components of a simple bioimaging system ?

Energy source, sample, detector

What scales of biological material are included in this imaging overview ?

Lipids, proteins and nucleic acids

Viruses, bacteria and yeast

Cells

Organoids/spheroids

Tissue

Organs

Model organisms

What is the size range for bacterial cells ?

About 1 - 10 micrometres long

About 0.2 - 1 micro metres wide

Name six properties of light

Colour

Wavelength

Frequency

Intensity

Phase

Relationship between frequency and wavelength

Shorter wavelength means higher frequency; longer wavelength means lower frequency

What is the photoelectric effect ?

Energy from light ejects an electron from a metal surface; the energy required differs between metals

What equation for photon energy is present ?

E=hc/delta

What wave like phenomena of light is highlighted?

Diffraction, interference and polarisation

What is light described as being made of?

Oscillating electric and magnetic fields; light is an electromagnetic wave

Spectroscopy

Spectroscopy uses absorption of different wavelengths of light to understand chemical composition and concentration.

Hematoxylin

Hematoxylin is dark blue or violet

Binds to basophilic substances such as DNA and RNA

Eosin

Eosin is red or pink

Eosin binds acidophilus substances such as positively charged amino-acid side chains e.g. lysine and arginine

Brightfield contrast

Brightfield contrast is the visual difference created in a microscope when a sample absorbs or scatters transmitted white light, causing a darker specimen to stand out against a bright illuminated background

Thin, transparent, unstained samples often show little Brightfield contrast as they produce little change in light intensity

Stained and clear objects

Stained objects change amplitude

Clear objects change phase

Clear sample visualisation

To visualise phase differences in a clear sample you must convert a phase change into an amplitude/contrast change

Scratch wound assay

A scratch wound assay is used to test the migration of cells in culture

Scratch wound assay performed ?

A confluent cell layer is scratched or wiped clean; live-cell Brightfield (phase) imaging tracks cell

Why is a scratch wound assay useful ?

It helps assess cell motility and potential to invade surrounding tissue

What is fluorescence ?

A type of photoluminescence in which light excites a molecule and a photon is emitted from an excited state

What are the three types of luminescence

Luminescence is light emission from a molecule

Photoluminescence (light energy)

Chemiluminescence (chemical energy)

Electroluminescence (electrical energy)

What are the main steps in fluorescence ?

Light raises an electron to an excited state; the excited state loses energy to the environment; a photon is emitted

Different energy levels and their ordering

Electronic (visible and UV) > vibrational (IR) > rotational energy levels (microwave)

Jablonski diagram

So is the ground state and S1 and S2 are the excited states

What happens when a fluorophore absorbs a photon is suitable energy ?

Before excitation electrons are normally found at the lowest energy state

When a fluorophore absorbs a photon of suitable energy it is promoted from the ground state to an excited state such as S1 or S2