Download Fluorescence spectroscopy

Fluorescence spectroscopy
Fluorescence spectroscopy
Spectral characteristics
A Jablonski diagram represents absorption and
emission in a fluorescent molecule
A typical molecule absorbs from the ground state (S0) into
the vibrational manifold above the first singlet state (S1).
It then emits from S1 into the vibrational levels above S0.
Since some of the energy of the absorbed photon is lost during
relaxation, the emitted photon has a longer wavelength: it is
redshifted).
The pictured molecule absorbs in the green and emits in the
red.
The gap between absorption and emission is the Stokes shift.
A large Stokes shift means emission is well separated
(spectrally) from absorption.
This process takes ~1 ns.
Not all absorbed photons result in an emitted photon.
Quenching (transfer to another species, often oxygen) or
nonradiative relaxation gets rid of excitation energy without
emitting a photon.
The ratio of emitted photons to absorbed photons is the
quantum efficiency or quantum yield Φ0.
A good dye will have Φ0 very close to 1.
The efficiency typically depends on pH, temperature, and
environment, but can be within a few % of 1.
Fluorescence spectroscopy
Excitation and emission spectra
Usually the vibrational manifolds are so dense that individual absorption lines cannot be
resolved.
To get an emission spectrum, pick a location in the absorption manifold (e.g. 600 nm here) and measure the power
emitted at each wavelength.
To get an absorption spectrum, scan through the absorption band, measuring the ratio of transmitted light to
incident light at each wavelength.
This gives the extinction coefficient
To get an excitation spectrum, pick a location in the
emission manifold (e.g. 700 nm here) and scan the
excitation wavelength through the excitation manifold,
measuring the emitted power at each wavelength.
A)
FITC excitation and emission spectra
A well-designed fluorophore will have little nonspecific
absorption, so its excitation and absorption spectra will be
quite similar.
Almost everyone reports absorption spectra even if the excitation
spectrum is more relevant.
Since the vibrational levels of S0 and S1 are often similar,
the absorption/excitation and emission spectra are often
mirror images of each other.
Java Jablonski at Olympus.
B)
This example has an excitation (rather than absorption) spectrum
because I grabbed this from the handbook of a filter manufacturer
(Chroma) who has thought hard about the fluorescence process.
4HE
CREA
DIC
IMA
)MA
BRIG
RELA
A¬LE
COLO
PUT
Fluorescence spectroscopy
Excitation and emission are largely decoupled by the relaxations between them.
The spectral characteristics of a fluorophore are
Absorption scan:
A(λ) = "(λ)
Excitation spectrum: F (λ; λem ) = Φ0 "(λ)f (λem )
Emission spectrum: F (λ; λex ) = Φ0 "(λex )f (λ)
Only the magnitude of the emission spectrum (not its shape) is affected by the choice of excitation wavelength.
Fluorescence spectroscopy
Population measurement
If two states have different spectra (usually emission spectra), measuring a full spectrum
reveals the fraction of population in each state.
An artificial example: Fluo-3 and Fura Red have different responses to Ca levels.
If the initial and final spectra cross at the isosbestic point, the intensity at that point should be unchanged
throughout the experiment.
If not, there must be an intermediate state.
Fluorescence spectroscopy
Spectrofluorimeters
Light from an excitation source (usually a Xenon or Mercury lamp) is filtered through a
monochromator and excites the sample.
Emission is harvested at 90º, filtered through a second monochromator, and recorded by a
photomultiplier tube (PMT).
The measured emission is usually normalized to the incident intensity using the reference PMT.
Purple inserts are for polarized fluorescence (coming soon)
Fluorescence spectroscopy
Fluorescence microscopy
On a microscope, you usually don’t enough light to split up the whole spectrum.
Pass excitation and emission through filters and record the total intensity.
In principle you could get the same information using a color camera
In practice, most people take sequential (or simultaneous) black and white shots with different filter sets and
combine them in a false-color image.
Fluorescence spectroscopy
Excitation and emission filters are usually combined in a filter cube:
ROSCOPE¬ IS¬ THE¬ SET¬
O¬ CALLED¬ THE¬ FILTER¬
HROIC¬BEAMSPLITTER¬
¬
RPTION¬ &OR¬ EXAMPLE¬ THINFILM¬ INTERFERENCE¬ FILTERS¬
¬ ONLY¬ THOSE¬ AND¬
WAVE ACOUSTOOPTICAL¬ FILTERS¬ BLOCK¬ BY¬ DIF
FLECTION¬
E¬ A¬ SPECIFIC¬ DYE¬
E¬TERM¬hOPTICAL¬DENSITYv¬IS¬MORE¬PRECISE¬"OTH¬OF¬
ON¬FILTER¬
RE¬DESCRIBED¬IN¬DETAIL¬IN¬THE¬SECTION¬BEGINNING¬ON¬
!0)¬AND
TENUATION¬
¬ETC
LEVEL¬ IS¬ CROSSTALK¬ &IGURE¬ ¬ WHICH¬
M¬ATTENUATION¬LEVEL¬OVER¬A¬SPECIFIC¬RANGE¬OF¬TWO¬
BANDPASS¬ FILTER¬ DE
'*(63&
IN¬SERIES¬4HIS¬VALUE¬IS¬IMPORTANT¬WHEN¬MATCHING¬
The
excitation light is several orders of
3CHEMATIC¬OF¬A¬FLUORESCENCE¬FILTER¬CUBE
magnitude more intense than emission, so the
EMISSION¬FILTERS¬FOR¬A¬FLUORESCENCE¬FILTER¬SET
TTER¬ATTENUATES¬ALL¬
FICIENTLY¬TRANSMITS¬
filters must have selectivities of many O.M.
This is often expressed as optical density, a log10
CRIBES¬THE¬SHARPNESS¬OF¬THE¬TRANSITION¬FROM¬TRANS
scale of absorption:
¬ ALWAYS¬ OF¬ LONGER¬
HESE¬ CAN¬ BE¬ EITHER¬
IGURE¬¬ILLUSTRATES¬TWO¬SETS¬OF¬FILTERS¬WITH¬THE¬SAME¬
OD = log10(I/I0)
COLORS¬ARE¬BLUE¬OR¬
POINT¬
BUT¬ DIFFERENT¬ SLOPE¬ )N¬ THE¬ &IGURE¬ NOTE¬ THAT¬
BLOCK¬AND¬ORANGE¬
¬FILTERS¬LOOK¬VERY¬SIMILAR¬ON¬A¬¬TRANSMISSION¬
ICATED¬ON¬THE¬OPTICAL¬DENSITY¬SCALE¬ARE¬SIGNIFICANTLY¬
OR¬OR¬DICHROMATIC
E¬ SPECIFIED¬ BY¬ STATING¬ THE¬ WAVELENGTH¬ AT¬ WHICH¬ A¬
'*(63&
&ILTER¬SETS¬WITH¬VARYING¬SLOPE¬SHOWN¬IN¬!¬PERCENT¬TRANSMIS
SION¬AND¬"¬IN¬OPTICAL¬DENSITY
Fluorescence spectroscopy
The excitation and emission filters (and dichroic) must be
matched to the fluor.
A)
Allowing extra excitation risks damaging fluor and letting in stray light.
&
If illuminating with a pre-defined wavelength (like from a laser line) the
excitation filter may be unnecessary.
4HE¬ PR
CREATE¬
DICHROI
IMAGE¬
B)
)MAGE¬
BRIGHTN
RELATIV
A¬LESSE
COLOR¬B
PUT¬IE
AS¬ LOW
IMPROV
C)
ADEQUA
CONTRAS
FILTER¬D
APPLICA
MATION
)MAGE¬
ITY¬ REQ
INSERTE
D)
SPECIFIC
WELL¬ GR
AN¬UND
TECHNIQ
OF¬APPL
Fluorescence spectroscopy
Fluorescent proteins (FPs)
The development of a range of stable, fast-folding variant of jellyfish green fluorescent
protein (mostly by Rogen Tsien) has revolutionized in vivo fluorescent measurements.