UV-VIS Luminescence Spectroscopy
Why do fluorometers measure fluorescence at a right angle
to eliminate transmittance from the reading, lowers signal to noise ratio
FRAP
-(Fluorescence Recovery After Photobleaching), used to quantify the lateral diffusion of a molecularly thin film containing fluorescently labeled probes, or to examine single cells. Great for studying cell membrane diffusion. Save a background image of the sample, then photobleach an area, watch from the dark spot to gradually regain brightness as the fluorescent probes diffuse into the area.
FACS
-(Fluorescence activated cell sorting) microarrays. Utilizes flow cytometry to sort a heterogeneous mixture of cells into two or more containers based on light scattering and fluorescent emissions.
FISH
-(Fluorescence in situ hybridization) Fluorescence Immunocytochemistry, used to detect and localize the presence or absence of specific DNA sequences on chromosomes. Fluorescent probes bind to only those parts of the chromosome that they are complimentary to.
What is the fluorescence intensity Proportional to
-F is proportional to the amount of light absorbed and the fluorescence quantum yield F~ΦA -It is also proportional to concentration F ~ Φ εF*lc
FRET
-Fluorescence Resonance Energy Transfer, describes energy transfer between two light-sensitive molecules. a donor chromophore in its excited state can transfer energy to an acceptor chromophore through non radiative dipole-dipole coupling. efficiency is proportional to (1/distance^6) therefore it is very sensitive to small changes in distance.
what is a fluorophore
-a molecule with a rigid conjugated structure, usually a polyaromatic hydrocarbon or heterocycle
processes involving photons radiation-less transitions
-absorption -fluorescence -phosphorescence -vibrational relaxation -intersystem crossing -internal conversion
Chemiluminescence
-an excited electron state is created via a chemical reaction -photon emission in order to reach the ground state -bioluminescence
what is the quantum yield of a fluorophore
-gives the efficiency of the fluorescence process -phi=(# photons emitted)/(#photons absorbed) -always between 0.05 - 1
What are the 5 properties of fluorophores
-high ring density of pi electrons -aromatic hydrocarbons -fused-ring structures -hydrocarbon conjugation -planarity and rigidity (increase ability of molecule to release energy as light)
what are the main advantages of fluorescence over spectrophotometry
-highly specific and sensitive
What is the stokes shift
-the difference in wavelengths between the emission and excitation wavelengths -will always be positive for fluorescence
extinction coefficient, dependent on?
-the efficiency of absorbing light energy -wavelength dependent
Excitation
-the process of light waves forcing valence electrons of a flourophore into a higher energy state
Emission
-the process of the fluorophore releasing energy as light through photon emissions that follow a brief period of vibrational relaxation
what are the disadvantages of fluorescence
-very sensitive to environmental changes -intensity and wavelength vary with pH, temperature (more loss of energy by collision), solvents, oxidizing agents
What happens when fluorescence occurs
-when light of shorter wavelength is absorbed by the fluorophore, valence electrons are excited. -light is emitted when the molecule releases the remainder of energy that was absorbed initially at S2 through VIBRATIONAL RELAXATION to S1 and then photon emission to ground state
Why is the emission wavelength longer than the excitation wavelength
-when the shorter (more energy) excitation wavelength is absorbed, some energy is lost through molecular vibrations -therefore the emission wavelength is longer (less energy)
What are the three techniques of luminescence spectroscopy
1. Fluorescence Spectroscopy 2. Phosphorescence spectroscopy 3. Chemiluminescence spectroscopy
What two major divisions of luminescence separate the 3 techniques of luminescence spectroscopy
1. Photoluminescence -fluorescence spectroscopy -phosphorescence spectroscopy 2. chemiluminescence
What are the parts of a Fluorometer
1. an excitation light source 2. a means to select the excitation wavelength (filter or monochromator) 3. a sample holder 4. a means to select the fluorescence wavelength to be monitored (filter or monochromator) 5. a detector: signal is proportional to the photons striking it (photomultiplier tube)
types of fluorescence microscopes
1. epifluorescence microscope (widefield) -reflected light illumination -a conventional compound microscope that has been equipped with a high-intensity light source -utilize incident illumination to illuminate the sample from above. In this way the objective lens is used as both the illumination condenser and the fluorescent light collector. 2. Confocal microscope -focus on a single plane through the use of an aperture to eliminate out of focus light -improves resolution -improves signal to noise -can image thick specimens -allows 3D imaging of samples
list the 4 advantages of fluorescence spec over UV-VIS spectrophotometry
1. highly specific -no two compound have the same fluorescence signature (combination go excitation and emission spectra) 2. Sensitive -10^3 to 10^4 times more sensitive than UV/VIS -emitted radiation is measured directly -measures the amount of light intensity present over a zero background (absorbance: @ low concentrations, the small difference between 100% T and the transmitted beam is difficult to measure accurately and precisely) 3. Greater range of linearity (less sample dilution) 4. Low signal to noise, "she may mean better SNR" (since emitted light is read at right angles to the exciting light
Cross talk/bleeding (over)
3 categories 1. Cross excitation- Crosstalk occurs when two (or more) fluorescent markers are excited and imaged when trying to capture the signal from only one. (less common) 2. Cross emission- interaction between two fluorophores' emission (more common) 3. Interaction between emission from one fluorophore and excitation of another fluorophore (more common)
what is the formula for fluorescence
F = Io*10^-εlc
Fluorescence vs. Phosphorescence
Fluoresecence: average lifetime of an excited singlet state = 10^-6 to 10^-9 sec -no triplet state Phosphorescence: lifetime of the excited triplet state can be up to 10 sec or more -does not immediately re-emit the radiation it absorbs
Bleed-through (cross talk) elimination
Single-label controls -> quantify bleeding through 'wrong filter'->remove computationally watch for unequal staining -> adjust protocol in general: select fluorophores with distinct emission spectra, and using filters, collect sequentially into dedicated channels
What is fluorescence
The property of a molecule absorbing light of a short wavelength and emitting light of a longer wavelength
Singlet state
all electrons in the molecule are spin-paired
what is quenching
anything that makes a fluorophore lose fluorescence intensity
what is photobleaching
irreversible decomposition of the fluorescent molecules because of light intensity in the presence of molecular oxygen (caused by cleaving of covalent bonds)
triplet state
one set of electron spins is unpaired
stokes shift
the difference in energy between excitation and emission
Quantum yield
the efficiency of energy release of a fluorophore (0-1)
