What can fluorescence imaging show us?

 

Fluorescence imaging shows what is going on in heart cells (left) and the nervous system (right)

 
 

How does fluorescence imaging work?

Some molecules can absorb energy in the form of light. This causes them to enter an 'excited state'. Molecules cannot permanently exist in the excited state, so they eventually return to the original (or 'ground') energy state and emit energy in the form of light.

Some of the energy is lost when molecules are in the excited state. This means that the emitted light will have less energy, and therefore be of a longer wavelength (different colour) from the excitation light.

 

How can we see proteins in cells?

Fluorescence is a natural property of some proteins, which scientists have borrowed to see the inner workings of cells.

A famous example of this is green fluorescent protein (GFP), isolate from the jellyfish Aequoria victoria, which gives this jellyfish a green glow. GFP can be attached to other proteins and acts like a 'fluorescent flag' to show where specific proteins are in cells.

GFP was the first, but proteins that fluoresce with many other colours are now used, enabling scientists to see different proteins simultaneously in the same cell.

The discovery and optimisation of GFP led to the award of the Nobel Prize in Chemistry to Osamu Shimomura, Martin Chalfie and Roger Ysien in 2008.

This image shows a cell expressing both a red and a green fluorescent protein. The green flurescent protein was targeted the the endoplasmic reticulum. The red fluorescent protein was targeted to mitochrondira.

 

Colours can label different parts of cells

In this heart cell (myocyte), calclium channels called ryanodine receptors are shown in green. Their regular distribution is vital to generate the calcium signals that promote contraction. The image also shows the cell's nuclei (blue) and a marker for hypertrophy - cell growth (orange).

 

Seeing dynamic changes in cellular messengers like calcium

In the sequence above, a calcium spark in the first image (top left) triggers a larger calcium wave, which spreads through the cell. Such calcium waves could disrupt the rhythm of the heart.

 

Which proteins interact with each other?

 

FRET (Förster Resonance Energy Transfer) is an important technique that can be used to estimate the proximity between two fluorescent molecules.

If the molecules are sufficiently close, the energe of one excited molecule can be passed to the other.

By monitoring the wavelength (colour) of the emitted light, it is possible to say whether the molecules are near or far apart.

 

What do cells look like in their native environment?

This is a 3-dimensional reconstruction of a thick section of the brain where a specific cell type, Purkinje neurones, have been made to produce green fluorescent protein.