Fluorescence spectroscopy Flashcards
What is fluorescence spectroscopy?
Fluorescent molecules have both absorption and emission spectra.
Fluorescence is 1000x more sensitive than UV spec.
Why is fluorescence spectroscopy more effective than UV?
It takes only nano seconds to excite the fluorescence.
It is very sensitive so only needs tiny amounts.
Emission intensity is related to excitation intensity.
What are flurophores?
Materials that have fluorescent properties.
Also called dyes or stains.
Some molecules are naturally fluorescent while others are designed to fluoresce.
How much energy do fluorophores absorb?
Fluorophores always absorb more energy than they emit.
A fluorophore always absorb light at a shorter wavelength and emits light at a longer wavelength.
Wavelengths are specific to each fluorophore.
What is stokes shift?
The difference between the absorption wavelength and emission wavelength.
This can tell you how good a material is as a fluorophore.
Why is the detector at 90.?
The detector is at 90. to decrease interference.
This is because it increases the signal to noise ratio.
Why is fluorescence spectroscopy used?
It can monitor in real time what is happening to the biological sample.
Samples can be in solution or under a microscope.
Low concentrations can be used (as it is 1000x more sensitive than UV spec).
How can fluorescence spec see colour?
The excitation and absorption are at different wavelengths.
On the Electromagnetic spectrum the colour for emission is different so the colour seen will change.
What is tryptophan fluorescence used for?
Can monitor what stage of cancer an individual is at without doing a biposy.
e.g. for colorectal cancer.
What does tryptophan fluorescence show about cancer?
If the tumour is small, there will only be a small amount of tryptophan, so a small intensity of emission.
As the size grows, more tryptophan binds and so there is a higher intensity of emission.
What are intrinsic fluorophores?
Naturally fluoresce
What are extrinsic fluorophores?
A dye or target must be added in order to see fluorescence.
What are examples of intrinsic protein fluorophores?
Ampicillin, morphine, aspirin.
These proteins are useful for finding the location of the protein as well as its conformation.
What are non protein examples of intrinsic fluorophores?
Isothiocyanates - anticarcionogenic agent from cruciferous vegetables.
Metabolites in urine samples for drug screening
What are examples of extrinsic fluorophores?
Green Fluorescent Protein (GFP) originally from jellyfish and is linked to specific proteins.
Mutations have been developed so that multiple colours can be seen measuring the intensity and location of specific proteins.
