Photo-physics of molecules

transfer efficiency

Relationship between ________ and donor- acceptor distance allows FRET to determine distance between molecules in order of wavelength in nm.

What is bioluminescence?

production of emission light by a living organism

where is GFP active?

GFP is constitutively active (emits fluorescence) in other organisms, not only in jelly fish.

substrate can be?

dye molecule, chromophore(light emission)

Catalyst can be?

Protein molecule

Intrinsic fluorophores?

Aromatic amino acids; most proteins possess intrinsic fluorescent due to fluorescence amino acids(Tryptophan, tyrosine and phenylalanine)

Application of intrinsic fluorophores

Restricted flexibility, e.g; fluorescence spectroscopy

Extrinsic fluorophores?

It is sensitive to environmental parameters(biosensors) and regulates fluorescence emission properties.

Application of extrinsic fluorophores?

Spectral flexibility, e.g; Immunofluorescence, flow cytometry

Recipe for bioluminescence?

Chemiluminescent precursor (substrate) and oxidizing agent(catalyst)

Process of bioluminescence

The substrate and the oxidizing agent interact and the substrate gets oxidized then excited. Energy is then released and a ground state complex is formed

Example of bioluminescence

Firefly and Blue jellyfish

What are the components of the blue jellyfish?

Substrate is and oxidizing agent is aquorin

GFP in blue jellyfish

can be used to visualize when it undergoes fluorescence emission. In this case the fluorophore is the protein and the chromophore is the polypeptide chain.

Use of immunofluorescence

In labs it is used to diagnose organs

Types of immunofluorescence

Direct- target + primary antibody & fluorophore

Indirect- target + primary antibody + secondary antibody & fluorophore

What are fluorescent labels

Molecules that aid in detection of biomolecules

FRET?

Forster Resonance Energy Transfer

Study of the distance between the chromophores. Energy transfer upon overlap(dipole-dipole interaction) of donor & acceptor

Photobleaching?

Irreversible photochemical destruction by emission.

Advantages and disadvantages of Photobleaching?

Disadvantage- decrease in intensity

Advantage- FRAP

Use of FRAP?

Used for study of cell membrane and diffusion