Flourescence

Excitation, Emission Energy Diagram

(Picture)

How does a flourescence microscope work?

Light is transmitted through an excitation filter which allows only 1 wavelength through, that wavelength then bounces off a dichroic beam splitter which send the light to the sample where it is reflected as 2 different wavelengths (dependent on the sample) back at the splitter and to the emission filter which allows only one wavelength through to the detector

How does a flourescence spectometer work?

Light source shines light through a monochrometer which is set to let a specific wavelength through which goes through the sample to an excitation mirro, shining the light back through the sample to a monochrometer which shines the light at the detector which transmits the light to a readout device

What is photobleaching?

an irreversable decomposition of the fluorescent molecules because of light intensity in the prescence of molecular oxygen, cause by cleaving of covalent bonds

What is quenching?

any process that decreases the fluorescence intensity of a substance, caused by the high sensitivity

Excitation

when a substance absorbs energy

Emission

when a substance releases excess energy via photons to return to ground state

Stokes Shift

The difference in the relative intensity peaks of the emission curve and the excitation curve

Disadvantages of flourescence?

-Extremely sensitive to the slightest changes in environment -Intensity and wavelength vary with: pH, temp., solvents and oxidizing agents

Quantum Yield

-Gives the efficiency of the fluorescence process -Phi = (# of photons emitted)/(Number of photons absorbed)

Advantages of Flourescence?

-Highly specific : no two compounds have the same fluorescence signature -Sensitive: 10^3 to 10^4 times more sensitive than UV/VIS -Greater range of linearity: less sample dilution -Low signal to noise

Flourophore Chemistry

-Molecule with a rigid conjugated structure, usually a polyaromatic hydrocarbon -Characteristics: -High ring density of Pi electrons -Aromatic Hydrocarbons -Fused-ring structures -Hydrocarbon conjugation -Planarity and rigidity

Fluorescence Intensity

-Porportional to the amount of light absorbed and the fluorescence quantum yield -F = Io*10^(-elc)

What is cross-talk?

3 categories: -Cross excitation - interaction between different excitation light and fluorophores -Cross Emission - interaction between two fluorophores emission -Interaction between emission from one fluorophore and excitation of another

What is fluorescence?

A form of luminescence. Occurs when a substance that has absorbed light or other electromagnetic radiation emits that light by releasing the energy.