Topic 01 - Molecular Fluorescence - Slide Format

8/7/2019 Topic 01 - Molecular Fluorescence - Slide Format

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Figure from W.L. Jorgensen and L. Salem, The Organic ChemistsBook of Orbitals, New York, Academic Press, 1973.


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Figure from W.L. Jorgensen and L. Salem, The Organic ChemistsBook of Orbitals, New York, Academic Press, 1973.


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Absorption of electromagnetic radiation is afast process typically on the order of 10-15s i.e. the time required for a photon to cross a

distance of 10 (typical size of a moleculartransition dipole).

Frank Condon Principle Followed:

Electronic motions/transitions occur muchfaster than nuclear motions(i.e. 10-16 10-14 s versus 10-13 10-12 s)and therefore occur most favourably whenthe nuclear structure of the initial and final

states are most similar. i.e. spin quantum numbers dont change

during electronic transitions.


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The transition dipole moment isa vector representing the thespatial change in charge densitybetween the ground and excitedstate of a molecule.

Molecules preferentially absorblight which is polarized (electricfield) parallel to the direction oftheir transition dipole.

There is zero probably ofabsorbing light polarized

perpendicular to the transitiondipole moment.

Figure from Olympus Microscopy Resource Center: www.olympusmicro.com


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Kashas Rule: Only the lowest excited singlet state (S1) orTriplet State (T1) need be considered for mostphotochemical reactions owing to rapid radiationlessconversion of higher-order electronic states (S2,T2, etc.) tothe S1 and T1 state.


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R The fluorescence lifetime () is the time after which the fluorescence of asample population has decayed to 1/e or 37% of its initial intensity:

where Iis the intensity and tis the time after excitation.

Some systems have multi-component lifetimes:

where ai is the fractional contribution to the total intensity.

The fluorescence lifetime provides information about local environment of thefluorophore.

I(t) = aiIoexp t

i

( )i=1

n


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90 Geometry with respect toexcitation and emission paththrough the sample

Rhodamine B Cell is a QuantumCounter, used to correct for sourcepower variability as a function ofwavelength.

Quantum counter consist of a highlyconcentrated solution of fluorophore

that will absorb any wavelength oflight incident on it with near 100%efficiency over the wavelengthrange of interest and emit light with100% efficiency (i.e. = 1.0) at acharacteristic emission wavelengththat will be of an intensity directlyproportional to the source intensityat the excitation wavelength.

Corrected spectra achieved byusing the ratio of the Sample PMTto that of the Ref. PMT

Typically, 3g/L Rhodamine B inethylene glycol provides for effectivecorrection over the excitation rangefrom 220 to 620nm, with emission at630nm

Figure from Instrumental Analysis G.D. Christian, J.E. OReilly,Eds., Second Ed., Allyn and Bacon, Inc., Boston, 1986, p.


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Quartz tubes filled with high-pressure gas(e.g. mercury or xenon). Operate at severalhundred degrees Celsius.

Powered by a DC current (the arc) betweentwo electrodes. Typically, 50-200 W.

High voltage strike needed to ignite. Ionization of gaseous vapour to create

plasma.

Free electrons and positive ions stream tocathode and anode. Collisions excite gaseousatoms which emit light to relax.

Mercury arc lamps contain small amount ofliquid mercury and an inert gas such as argonor xenon. The arc produces enough heat toproduce mercury vapour. Xenon arc lampscontain only xenon gas.

Sources for fluorescence spectroscopy: Arc

Lamps

Figure from Olympus Microscopy Resource Center: www.olympusmicro.com; photo: R. Algar


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Lamps

Mercury does not provideeven intensity across thespectrum. Xenon is better inthis respect, but has onlyweak intensity in the UVregion.

Mercury and xenon lampsare good high intensitypolychromatic sources forfluorescence spectroscopy.

When high intensitymonochromatic sources arerequired, lasers are used.



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Lamps

Light Amplification by Stimulated Emission ofRadiation High-intensity Coherent (same frequency and same phase) Monochromatic (bandwidth < 0.01 nm) Continuous wave (CW) or pulsed (picosecond and femtosecond pulses are

possible)

coherent

incoherent(different freq.)

incoherent(different phase)


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Lamps

Pump source: electrical, radiant (laser, flashlamp), chemical energy

Gain or Lasing medium must be pumped for lasing action determines type of laser determines wavelength of laser laser output can be frequency-doubled by certain non-linear crystals

(e.g. BBO = -barium borate, KDP = potassium dihydrogen phosphate)

The laser cavity:

Partially Reflective MirrorFully Reflective Mirror

Gain/Lasing Medium

Pump Source

Laser Beam


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Lamps

Stimulated emission

Excited species are hit by fluorescencephotons traversing the cavity

This results in the emission of a secondphoton which is precisely in phase and inthe direction of the incident photon

Coherent radiation builds up as stimulatedphotons traverse the cavity and yieldfurther stimulated emission

Absorption

Ground state species in the lasing mediumcan absorb photons to produce themetastable excited state

The number of photons from stimulatedemission must exceed the number of

photons re-absorbed by the lasing mediumfor a net gain (i.e. amplification)

The necessary population inversion isachieved/maintained by pumping

Four-level systems are more efficient thanthree-level systems


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Lamps

Gas lasers A gaseous atom, ion, molecule, or excimer is the lasing medium e.g. HeNe (632.8 nm), Argon ion (454.6, 488.0, 514.5 nm),

Nitrogen (337.1 nm), XeF (351 nm)

Solid-state lasers The lasing medium is often an ion in a host crystal (e.g. Nd3+ in

yittrium aluminum garnet or Ti3+ in sapphire) e.g. Nd:YAG (1064 nm), Ti:sapphire (650-1050 nm),

Semiconductor lasers Based on p-n junctions e.g. GaN, GaAs, AlGaAs

Dye lasers Organic fluorophores in solution are the active lasing medium Continuously tunable over a certain wavelength range (typically

40-80 nm) Pumped by another laser

Also: chemical lasers, metal-vapour lasers, free-electron lasers


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Lamps

Photoelectric effect to generate a current

Amplification through an electron cascade effect at a series of dynodes Current proportional to light intensity Spectral response not uniform; depends on cathode material



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LampsThe PIN photodiode is a three layer structure consisting

of: a layer of p-type semiconductor; a layer of intrinsic(undoped) semiconductor; and a layer of n-typesemiconductor.

In the p-type layer, positive charges (holes) are mobileand allow electric current to flow.

In the n-type layer, negative charges (electrons) aremobile and allow electric current to flow.

In the intrinsic layer, there are no charge carries andthus the layer is an insulator, preventing current fromflowing through the diode.

Absorption of light in the intrinsic layer creates chargecarriers by the excitation of electrons, making the deviceconductive and generating an electric current.

The magnitude of the current is proportional to theamount of incident light.

Semiconductors will be covered in much greater

detail in a later lecture.


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Lamps Similar design to PIN photodiode, except

the intrinsic region is thinner.

While operating voltages for reversedbiased PIN photodiodes are on the orderof 0-10 V, avalanche photodiodes areoperated at 102-103 V.

Electrons generated upon absorption of aphoton are accelerated by the highpotential difference and producesecondary charge carriers in collisions.

The amplification is analogous to dynodesin a PMT.

The gain is typically 102-103, providingbetter sensitivity than PIN diodes.However, dark currents and noise arehigher. The response is also non-linear.



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Quenching species can includehalide salts (e.g. Cl-, Br-, I-), H+,oxygen, some metal ions (e.g.Mn2+, Cu2+), acrylamide, amines,nitrate, histidine, cysteine,halogenated hydrocarbons, and

more.

Heavy atoms often quench byinducing intersystem crossing(especially Br- and I-). Metals ionscan quench by electron transfer.

Not all species are quenched bythe same species, or by the samemechanism(s).

Quinine sulfate:

(a) 0.1 M H2SO4(b) 0.1 M H2SO4

+ 0.5 M NaCl

(c) 0.1 M H2SO4+ 0.5 M KI

Fluorescein:

d) 0.1 M NaOHe) 0.1 M NaOH

+ 0.5 M NaCl

f) 0.1 M NaOH+ 0.5 M KI

(a) (b) Cl- (c) I- (d) (e) Cl- (f) I-


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The orientation factor depends on theangles and distance between thetransition dipoles for the donor andacceptor.


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Applications of Fluorescence

Quenching and Energy Transfer

Methods:

Wavelength-Shifting Molecular Beacons

From: S. Tyagi, S.A.E. Marras and F. RussellKramer Wavelength-shifting molecular beaconsNature Biotechnology,18 (2000) 1191-1196.

Topic 01 - Molecular Fluorescence - Slide Format

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