Concept review: Fluorescence
• Chromophore. The structural feature of a molecule responsible for the absorption of UV or visible light.
• Fluorophore. A chromophore that remits an absorbed photon at a longer wavelength.
• Extinction coefﬁcient (ε). The absorbance (=-log(I /I ) of light of a particular wavelength by 1 cm of a 1
M solution of a chromophore (units of M cm ).
• Quantum Yield. The fraction of absorbed photons that are reemitted by a ﬂuorophore. 29
photon in photon out
Michael W. Davidson (micro.magnet.fsu.edu)
The process responsible for the ﬂuorescence of ﬂuorescent probes and other ﬂuorophores is illustrated by a
• Absorption. A photon is absorbed by the ﬂuorophore, creating an excited electronic sing1et state (S ).
This process distinguishes ﬂuorescence from chemi- or bioluminescence, in which the excited state is
populated by a chemical reaction. The initial excitation may result in the molecule in a higher energy
• Excited-State. The excited state exists for a ﬁnite time (typically 1–10 nanoseconds). During this time,
the ﬂuorophore undergoes conformational changes and the energy of S is partially dissipated by the
vibrational relaxation. Fluorescence emission originates from the lowest energy vibratio1al state of S .
Processes such as collisional quenching, ﬂuorescence resonance energy transfer (FRET) and
intersystem crossing may depopulat1 S .
• Fluorescence Emission. A photon of energy is emitted, returning the ﬂuorophore to its gr0und state S .
Due to energy dissipation during the excited-state lifetime, the energy of this photon is lower, and
therefore of longer wavelength, than the excitation photon. The difference in energy or wavelength
represented by the absorbed and emitted photon is called the Stokes shift. 30
• The entire ﬂuorescence process is cyclical. Unless the ﬂuorophore is irreversibly destroyed in the excited
state (an important phenomenon known as photobleaching), the same ﬂuorophore can be repeatedly
excited and detected. The fact that a single ﬂuorophore can generate many thousands of detectable
photons is fundamental to the high sensitivity of ﬂuorescence detection techniques.
• http://probes.invitrogen.com/handbook/sections/0001.html Fluorescence spectra
Image source: http://www.probes.com/handbook/sections/0001.html
• For polyatomic molecules in solution, the discrete electronic transitions of the previous slide are replaced
by rather broad energy spectra called the ﬂuorescence excitation spectrum and ﬂuorescence emission
spectrum, respectively. The bandwidths of these spectra are parameters of particular importance for
applications in which two or more different ﬂuorophores are simultaneously detected.
• With few exceptions, the ﬂuorescence excitation spectrum of a single ﬂuorophore species in dilute
solution is identical to its absorption spectrum.
• Generally speaking, the ﬂuorescence emission spectrum is independent of the excitation wavelength ,
due to the partial dissipation of excitation energy during the excited-state lifetime. This is known as
Kasha’s rule after Michael Kasha. The emission intensity is proportional to the amplitude of the
ﬂuorescence excitation spectrum at the excitation wavelength. Simply put, the greater the number of
molecules that absorb a photon, the greater the number of molecules that will emit a photon as
Lifetimes of excited state processes
• A key feature of ﬂuorescence is that the molecule spends a measurable amount of time in the singlet
excited state. This time is typically in the range of 1-10 ns.
• A number of different things can happen to molecule while it is in the excited state. Fluorescence is, of
course, one thing that can happen to the molecule. Other ways of depopulating the excited state include
non-radiative relaxation (essentially an internal conversion from 1 to S0) or quenching or intersystem
crossing to a triplet state.
• If a triplet state is formed it can emit a photon through the process of phosphorescence or it can non-
radiatively relax. 33
Instrumentation for detecting ﬂuorescence
A spectroﬂuorometer platereader
• Spectroﬂuorometers are the most common instrument for measuring of ﬂuorescence. Essentially, they are
instruments that are similar to UV-vis spectrophotometers in design except that the emission detector is
positioned at a 90 degree angle from the direction of the excitation source.
• It is also quite common to detect ﬂuorescence using a platereader type device. For this type of device, the
cuvette is replaced with a microplate with perhaps 96 or 384 (or more) wells on it. Both excitation and
collection of emission occur from the same direction. Most instruments could measure from either the top
or the bottom (assuming that the bottom of the plate is clear plastic or glass).
• The third common class of instruments for detection of ﬂuorescence are confocal or wideﬁeld ﬂuorescence