The Molecular Probes Handbook

A GUIDE TO FLUORESCENT PROBES AND LABELING TECHNOLOGIES

11th Edition (2010)

Molecular Probes Handbook

A Guide to Fluorescent Probes and Labeling Technologies
11th Edition (2010)

CHAPTER 1
Fluorophores
Introduction and
Their Amine-Reactive
Introduction to
Derivatives Techniques
Fluorescence

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Introduction to Fluorescence Techniques

Introduction to Fluorescence Techniques

Fluorescent probes enable researchers to detect particular com- quantum yield, which is the ratio of the number of fluorescence photons
ponents of complex biomolecular assemblies, such as live cells, with emitted (Stage 3) to the number of photons absorbed (Stage 1), is a mea-
exquisite sensitivity and selectivity. The purpose of this introduction is sure of the relative extent to which these processes occur.
to briefly outline fluorescence principles and techniques for newcomers
to the field. Stage 3: Fluorescence Emission
A photon of energy hEM is emitted, returning the fluorophore to its
ground state S0. Due to energy dissipation during the excited-state life-
The Fluorescence Process time, the energy of this photon is lower, and therefore of longer wave-
length, than the excitation photon hEX. The difference in energy or
Fluorescence is the result of a three-stage process that occurs in wavelength represented by (hEX hEM) is called the Stokes shift. The
certain molecules (generally polyaromatic hydrocarbons or hetero- Stokes shift is fundamental to the sensitivity of fluorescence techniques
cycles) called fluorophores or fluorescent dyes. A fluorescent probe is because it allows emission photons to be detected against a low back-
a fluorophore designed to respond to a specific stimulus or to localize ground, isolated from excitation photons. In contrast, absorption spec-
within a specific region of a biological specimen. The process respon- trophotometry requires measurement of transmitted light relative to
sible for the fluorescence of fluorescent probes and other fluorophores is high incident light levels at the same wavelength.
illustrated by the simple electronic-state diagram (Jablonski diagram)
shown in Figure 1. Fluorescence Spectra
The entire fluorescence process is cyclical. Unless the fluorophore
Stage 1: Excitation is irreversibly destroyed in the excited state (an important phenomenon
A photon of energy hEX is supplied by an external source such as known as photobleaching), the same fluorophore can be repeatedly ex-
an incandescent lamp or a laser and absorbed by the fluorophore, creat- cited and detected. The fact that a single fluorophore can generate many
ing an excited electronic singlet state (S1). This process distinguishes thousands of detectable photons is fundamental to the high sensitiv-
fluorescence from chemiluminescence, in which the excited state is ity of fluorescence detection techniques. For polyatomic molecules in
populated by a chemical reaction. solution, the discrete electronic transitions represented by hEX and
hEM in Figure 1 are replaced by rather broad energy spectra called the
Stage 2: Excited-State Lifetime fluorescence excitation spectrum and fluorescence emission spectrum,
The excited state exists for a finite time (typically 110 nano- respectively (Table 1). The bandwidths of these spectra are parameters
seconds). During this time, the fluorophore undergoes conformational of particular importance for applications in which two or more differ-
changes and is also subject to a multitude of possible interactions with ent fluorophores are simultaneously detected . The fluorescence excita-
its molecular environment. These processes have two important con- tion spectrum of a single fluorophore species in dilute solution is usu-
sequences. First, the energy of S1 is partially dissipated, yielding a re- ally identical to its absorption spectrum. The absorption spectrum can
laxed singlet excited state (S1) from which fluorescence emission origi- therefore be used as a surrogate excitation spectrum data set. Under the
nates. Second, not all the molecules initially excited by absorption (Stage same conditions, the fluorescence emission spectrum is independent of
1) return to the ground state (S0) by fluorescence emission. Other process- the excitation wavelength, due to the partial dissipation of excitation
es such as collisional quenching, fluorescence resonance energy trans- energy during the excited-state lifetime, as illustrated in Figure 1. The
fer (FRET) (Fluorescence Resonance Energy Transfer (FRET)Note emission intensity is proportional to the amplitude of the fluorescence
1.2) and intersystem crossing may also depopulate S1. The fluorescence excitation spectrum at the excitation wavelength (Figure 2).

Fluorescence emission
Fluorescence excitation

S1 Excitation Emission
2 spectrum EX 1 EM 1 spectrum
S1
EX 2 EM 2
Energy

hEX 1 hEM 3
EX 3 EM 3

S0
Wavelength

Figure 1 Jablonski diagram illustrating the processes involved in the creation of an excited Figure 2 Excitation of a fluorophore at three different wavelengths (EX 1, EX 2, EX 3) does
electronic singlet state by optical absorption and subsequent emission of fluorescence. The not change the emission profile but does produce variations in fluorescence emission inten-
labeled stages 1, 2 and 3 are explained in the adjoining text. sity (EM 1, EM 2, EM 3) that correspond to the amplitude of the excitation spectrum.

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 Introduction to Fluorescence Techniques

Fluorescence Detection measurement artifacts and makes different demands on the fluores-
cent probe. For example, although photobleaching is often a significant
Fluorescence Instrumentation problem in fluorescence microscopy, it is not a major impediment in
Four essential elements of fluorescence detection systems can be flow cytometry because the dwell time of individual cells in the excita-
identified from the preceding discussion: 1) an excitation source, 2) tion beam is short.
a fluorophore, 3) wavelength filters to isolate emission photons from
excitation photons, 4) a detector that registers emission photons and Fluorescence Signals
produces a recordable output, usually as an electrical signal. Regardless Fluorescence intensity is quantitatively dependent on the same pa-
of the application, compatibility of these four elements is essential for rameters as absorbancedefined by the BeerLambert law as the prod-
optimizing fluorescence detection. uct of the molar extinction coefficient, optical path length and solute
Fluorescence instruments are primarily of four types, each provid- concentrationas well as on the fluorescence quantum yield of the dye
ing distinctly different information: and the excitation source intensity and fluorescence collection efficien-
cy of the instrument (Table 1). In dilute solutions or suspensions, fluo-
Spectrofluorometers and microplate readers measure the average rescence intensity is linearly proportional to these parameters. When
properties of bulk (L to mL) samples. sample absorbance exceeds about 0.05 in a 1 cm pathlength, the rela-
Fluorescence microscopes resolve fluorescence as a function of spa- tionship becomes nonlinear and measurements may be distorted by
tial coordinates in two or three dimensions for microscopic objects artifacts such as self-absorption and the inner-filter effect.4,5
(less than ~0.1 mm diameter). Because fluorescence quantitation is dependent on the instrument,
Fluorescence scanners, including microarray readers, resolve fluo- fluorescent reference standards are essential for calibrating measure-
rescence as a function of spatial coordinates in two dimensions ments made at different times or using different instrument configura-
for macroscopic objects such as electrophoresis gels, blots and tions.68 To meet these requirements, we offer high-precision fluores-
chromatograms. cent microsphere reference standards for fluorescence microscopy and
Flow cytometers measure fluorescence per cell in a flowing stream, flow cytometry and a set of ready-made fluorescent standard solutions
allowing subpopulations within a large sample to be identified and for spectrofluorometry (Section 23.1, Section 23.2).
quantitated. A spectrofluorometer is extremely flexible, providing continuous
ranges of excitation and emission wavelengths. Laser-scanning micro-
Other types of instrumentation that use fluorescence detection scopes and flow cytometers, however, require probes that are excitable
include capillary electrophoresis apparatus, DNA sequencers 1 and at a single fixed wavelength. In contemporary instruments, the excita-
microfluidic devices.2,3 Each type of instrument produces different tion source is usually the 488 nm spectral line of the argon-ion laser. As

Table 1 Spectroscopic proterties of fluorescent dyes.

Property Definition Significance
Fluorescence excitation An X,Y plot of excitation wavelength versus number of Optimum instrument setup should deliver excitation light as close to the peak of the
spectrum* fluorescence photons generated by a fluorophore. excitation spectrum of the fluorophore as possible.
Absorption spectrum An X,Y plot of wavelength versus absorbance of a To a first approximation, the absorption spectrum of a fluorophore is equivalent to
chromophore or fluorophore. the fluorescence excitation spectrum. To the extent that this approximation holds,
the absorption spectrum can be used as a surrogate for the fluorescence excitation
spectrum.
Fluorescence emission An X,Y plot of emission wavelength versus number of Fluorescence emission spectral discrimination is the most straightforward basis
spectrum* fluorescence photons generated by a fluorophore. for multiplex detection and for resolving probe fluorescence from background
autofluorescence.
Extinction coefficient (EC) Capacity for light absorption at a specific wavelength. Fluorescence output per fluorophore (brightness) is proportional to the product of
the extinction coefficient (at the relevant excitation wavelength) and the fluorescence
quantum yield.
Fluorescence quantum Number of fluorescence photons emitted per excitation See Extinction coefficient.
yield (QY) photon absorbed.
Quenching Loss of fluorescence signal due to short-range interactions Loss of fluorescence is reversible to the extent that the causative molecular
between the fluorophore and the local molecular interactions can be controlled.**
environment, including other fluorophores (self-quenching).
Photobleaching Destruction of the excited fluorophore due to Loss of fluorescence signal is irreversible if the bleached fluorophore population is
photosensitized generation of reactive oxygen species (ROS), not replenished (e.g., via diffusion). Extent of photobleaching is dependent on the
1
particularly singlet oxygen ( O2). duration and intensity of exposure to excitation light.
*Our online Fluorescence SpectraViewer (www.invitrogen.com/handbook/spectraviewer) provides an interactive utility for plotting and comparing fluorescence excitation and emission
spectra for over 250 fluorophores (Using the Fluorescence SpectraViewerNote 23.1).
Generally true for single fluorophore species in homogeneous solutions but not in more complex heterogeneous samples.
Multiplex detection refers to the process of simultaneously labeling a specimen with two or more fluorescent probes to allow correlation of multiple structural or functional features. As well
as specific association with their targets, the probes must have distinctive spectroscopic properties that can be discriminated by the detection instrument.
EC (units: cm1 M1) is defined by the Beer-Lambert law A = ECcl, where A = absorbance, c = molar concentration, l = optical pathlength. EC values at the absorption maximum wavelength
are listed in the Section Data Tables throughout The Molecular Probes Handbook.
**In the case of self-quenching, this can be accomplished by disruption of fluorophore compartmentalization, denaturation or fragmentation of biopolymer conjugates, or functionalization
of the fluorophore to produce increased electrostatic repulsion and water solubility.

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Introduction to Fluorescence Techniques

shown in Figure 3, separation of the fluorescence emission signal (S1) have been developed to meet these requirements and have proven effec-
from Rayleigh-scattered excitation light (EX) is facilitated by a large flu- tive in multicolor labeling experiments.11,18,19
orescence Stokes shift (i.e., separation of A1 and E1). Biological samples
labeled with fluorescent probes typically contain more than one fluores- Ratiometric Measurements
cent species, making signal-isolation issues more complex. Additional In some casesfor example the Ca 2+ indicators fura-2 and indo-1
optical signals, represented in Figure 3 as S2, may be due to background (Section 19.2) and the pH indicators BCECF and SNARF (Section
fluorescence or to a second fluorescent probe. 20.2)the free and ion-bound forms of fluorescent ion indicators have
different emission or excitation spectra. With this type of indicator,
Background Fluorescence the ratio of the optical signals (S1 and S2 in Figure 3) can be used to
Fluorescence detection sensitivity is severely compromised by monitor the association equilibrium and to calculate ion concentra-
background signals, which may originate from endogenous sample con- tions. Ratiometric measurements eliminate distortions of data caused
stituents (referred to as autofluorescence) or from unbound or nonspe- by photobleaching and variations in probe loading and retention, as
cifically bound probes (referred to as reagent background). Detection well as by instrumental factors such as illumination stability. 20,21
of autofluorescence can be minimized either by selecting filters that (Loading and Calibration of Intracellular Ion IndicatorsNote 19.1).
reduce the transmission of E2 relative to E1 or by selecting probes that
absorb and emit at longer wavelengths. Although narrowing the fluo-
rescence detection bandwidth increases the resolution of E1 and E2, Fluorescence Output of Fluorophores
it also compromises the overall fluorescence intensity detected. Signal
distortion caused by autofluorescence of cells, tissues and biological Comparing Different Dyes
fluids is most readily minimized by using probes that can be excited at Fluorophores currently used as fluorescent probes offer sufficient
>500 nm. Furthermore, at longer wavelengths, light scattering by dense permutations of wavelength range, Stokes shift and spectral bandwidth
media such as tissues is much reduced, resulting in greater penetration to meet requirements imposed by instrumentation (e.g., 488 nm excita-
of the excitation light.9 tion), while allowing flexibility in the design of multicolor labeling ex-
periments. Our online Fluorescence SpectraViewer (www.invitrogen.
Multicolor Labeling Experiments com/handbook/spectraviewer) provides an interactive utility for evalu-
A multicolor labeling experiment entails the deliberate introduc- ating these factors during the experimental design process (Using the
tion of two or more probes to simultaneously monitor different bio- Fluorescence SpectraViewerNote 23.1). The fluorescence output of a giv-
chemical functions. This technique has major applications in flow cy- en dye depends on the efficiency with which it absorbs and emits photons,
tometry,1012 DNA sequencing,1 fluorescence in situ hybridization 13,14 and its ability to undergo repeated excitation/emission cycles. Absorption
and fluorescence microscopy.1517 Signal isolation and data analysis are and emission efficiencies are most usefully quantified in terms of the
facilitated by maximizing the spectral separation of the multiple emis- molar extinction coefficient (EC) for absorption and the quantum yield
sions (E1 and E2 in Figure 3). Consequently, fluorophores with narrow (QY) for fluorescence. Both are constants under specific environmental
emission spectral bandwidths, such as BODIPY dyes (Section 1.4) and conditions. The value of EC is specified at a single wavelength (usually
Qdot nanocrystals (Section 6.6), are particularly useful in multicol- the absorption maximum), whereas QY is a measure of the total pho-
or applications. An ideal combination of dyes for multicolor labeling ton emission over the entire fluorescence spectral profile. Fluorescence
would exhibit strong absorption at a coincident excitation wavelength intensity per dye molecule is proportional to the product of EC and QY
and well-separated emission spectra (Figure 3). Unfortunately, it is not (Table 1). The range of these parameters among organic dye and autofluo-
easy to find single dyes with the requisite combination of a large extinc- rescent protein fluorophores is approximately 5000 to 200,000 cm1M1
tion coefficient for absorption and a large Stokes shift. Qdot nanocrys- for EC and 0.05 to 1.0 for QY. Phycobiliproteins such as R-phycoerythrin
tals (Section 6.6) and phycobiliprotein tandem conjugates (Section6.4) (Section 6.4) have multiple fluorophores on each protein and consequently
have much larger extinction coefficients (on the order of 2 106 cm1M1)
than low molecular weight fluorophores. Qdot nanocrystals have even
larger extinction coefficients (>2 106 cm1M1), particularly in the blue
visible and ultraviolet wavelength regions (Section 6.6).
A1 E1

A2 Photobleaching
E2
Spectra Under high-intensity illumination conditions, the irreversible de-
Intensity

struction or photobleaching of the excited fluorophore becomes the

primary factor limiting fluorescence detectability. The multiple photo-
Filters chemical reaction pathways responsible for photobleaching have been
investigated and described in considerable detail.2224 Some pathways
include reactions between adjacent dye molecules, making the pro-
EX S1 S2 I/O signals cess considerably more complex in labeled biological specimens than
in dilute solutions of free dye. In all cases, photobleaching originates
Wavelength (h) from the triplet excited state, which is created from the singlet state
(S1, Figure 1) via an excited-state process called intersystem crossing.25
Figure 3 Fluorescence detection of mixed species. Excitation (EX) in overlapping absorption
bands A1 and A2 produces two fluorescent species with spectra E1 and E2. Optical filters iso- The most effective remedy for photobleaching is to maximize
late quantitative emission signals S1 and S2. detection sensitivity, which allows the excitation intensity to be

TheMolecular
The Molecular Probes
Probes Handbook:
Handbook: A Guide
A Guide to Fluorescent
to Fluorescent Probes
Probes andand Labeling
Labeling Technologies
Technologies
IMPORTANT NOTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on
IMPORTANT NOTICE : The
page products
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 Introduction to Fluorescence Techniques

100 reduced. Detection sensitivity is enhanced by low-light detection devices such as CCD cam-
eras, as well as by highnumerical aperture objectives and the widest bandpass emission filters
Fluorescence (% of initial)

80
compatible with satisfactory signal isolation. Alternatively, a less photolabile fluorophore may
60
be substituted in the experiment.26 Alexa Fluor 488 dye is an important fluorescein substitute
that provides significantly greater photostability than fluorescein (Figure 4, Figure 5, Figure 6),
40 yet is compatible with standard fluorescein optical filters. Antifade reagents such as SlowFade
and ProLong reagents (Section 23.1) can also be applied to reduce photobleaching; however,
20 they are usually incompatible with live cells. In general, it is difficult to predict the necessity for
and effectiveness of such countermeasures because photobleaching rates are dependent to some
0
0 10 20 30 40 50 60
extent on the fluorophores environment.2729
Time (sec)

Figure 4 Comparison of photostability of green-fluorescent

Signal Amplification
antibody conjugates. The following fluorescent goat anti The most straightforward way to enhance fluorescence signals is to increase the number of
mouse IgG antibody conjugates were used to detect mouse fluorophores available for detection.30 Fluorescent signals can be amplified using 1) avidinbio-
antihuman IgG antibody labeling of human anti-nuclear tin or antibodyhapten secondary detection techniques, 2) enzyme-labeled secondary detection
antibodies in HEp-2 cells on prefixed test slides (INOVA
Diagnostics Corp.): Alexa Fluor 488 (A11001, s), Oregon reagents in conjunction with fluorogenic substrates 3133 or 3) probes that contain multiple fluo-
Green 514 (O6383, j), BODIPY FL (B2752, n), Oregon rophores such as phycobiliproteins or FluoSpheres fluorescent microspheres. Our most sensitive
Green 488 (O6380, h) or fluorescein (F2761, d). Samples reagents and methods for signal amplification are discussed in Chapter 6.
were continuously illuminated and viewed on a fluorescence
microscope using a fluorescein longpass filter set. Images Simply increasing the probe concentration can be counterproductive and often produces
were acquired every 5 seconds. For each conjugate, three marked changes in the probes chemical and optical characteristics. It is important to note that the
data sets, representing different fields of view, were aver- effective intracellular concentration of probes loaded by bulk permeabilization methods (Loading
aged and then normalized to the same initial fluorescence
intensity value to facilitate comparison. and Calibration of Intracellular Ion IndicatorsNote 19.1) is usually much higher (>10-fold)
than the extracellular incubation concentration. Also, increased labeling of proteins or mem-
branes ultimately leads to precipitation of the protein or gross changes in membrane permeabil-
100 ity. Antibodies labeled with more than four to six fluorophores per protein may exhibit reduced
Alexa Fluor 488
specificity and reduced binding affinity. Furthermore, at high degrees of substitution, the extra
% Fluorescence intensity

80 fluorescence obtained per added fluorophore typically decreases due to self-quenching (Figure 7).
of first scan

60

40
Oregon Green 488 Environmental Sensitivity of Fluorescence
Fluorescein
20 Fluorescence spectra and quantum yields are generally more dependent on the environ-
ment than absorption spectra and extinction coefficients. For example, coupling a single fluo-
0 rescein label to a protein typically reduces fluoresceins QY ~60% but only decreases its EC by
1 2 3 4 5 6 7 8 9 10
~10%. Interactions either between two adjacent fluorophores or between a fluorophore and
Scan repetition
other species in the surrounding environment can produce environment-sensitive fluorescence.
Figure 5 Photobleaching resistance of the green-fluores-
cent Alexa Fluor 488, Oregon Green 488 and fluorescein
dyes, as determined by laser-scanning cytometry. EL4 cells
were labeled with biotin-conjugated anti-CD44 antibody
and detected by Alexa Fluor 488 (S11223; S32354), Oregon
Green 488 (S6368) or fluorescein (S869) streptavidin. The
cells were then fixed in 1% formaldehyde, washed and wet-
mounted. After mounting, cells were scanned 10 times on
a laser-scanning cytometer; laser power levels were 25 mW
for the 488 nm spectral line of the argon-ion laser. Scan du-
rations were approximately 5 minutes, and each repetition
was started immediately after completion of the previous
scan. Data are expressed as percentages derived from the
mean fluorescence intensity (MFI) of each scan divided by
the MFI of the first scan. Data contributed by Bill Telford,
Experimental Transplantation and Immunology Branch,
National Cancer Institute.

Figure 6 Comparison of the photobleaching rates of the Alexa Fluor 488 and Alexa Fluor 546 dyes and the well-known
fluorescein and Cy3 fluorophores. The cytoskeleton of bovine pulmonary artery endothelial cells (BPAEC) was labeled with
(top series) Alexa Fluor 488 phalloidin (A12379) and mouse monoclonal anti-tubulin antibody (A11126) in combination
with Alexa Fluor 546 goat antimouse IgG antibody (A11003) or (bottom series) fluorescein phalloidin (F432) and the anti
-tubulin antibody in combination with a commercially available Cy3 goat antimouse IgG antibody. The pseudocolored
images were taken at 30-second intervals (0, 30, 90 and 210 seconds of exposure from left to right). The images were acquired
with bandpass filter sets appropriate for fluorescein and rhodamine.

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Probes Handbook:
Handbook: AAGuide
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toFluorescent
Fluorescent Probes
Probes and
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6 IMPORTANT
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therapeutic Please refer
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Introduction to Fluorescence Techniques

FluorophoreFluorophore Interactions
Fluorescence quenching can be defined as a bimolecular process that reduces the fluores-
cence quantum yield without changing the fluorescence emission spectrum (Table 1); it can Intramolecularly
result from transient excited-state interactions (collisional quenching) or from formation of quenched
substrate
nonfluorescent ground-state species. Self-quenching is the quenching of one fluorophore by an-
other; 34 it therefore tends to occur when high loading concentrations or labeling densities are
used (Figure 7, Figure 8). DQ substrates (Section 10.4) are heavily labeled and therefore highly
quenched biopolymers that exhibit dramatic fluorescence enhancement upon enzymatic cleav-
age 35 (Figure 9). Enzyme

Fluorescence resonance energy transfer (FRET) (Fluorescence Resonance Energy Transfer

(FRET)Note 1.2) is a strongly distance-dependent excited-state interaction in which emission
of one fluorophore is coupled to the excitation of another. Some excited fluorophores interact to
form excimers, which are excited-state dimers that exhibit altered emission spectra. Excimer Fluorescent
formation by the polyaromatic hydrocarbon pyrene is described in Section 13.2 (Figure 10). cleavage
products
Because they all depend on the interaction of adjacent fluorophores, self-quenching, FRET
and excimer formation can be exploited for monitoring a wide array of molecular assembly or
fragmentation processes such as membrane fusion (Assays of Volume Change, Membrane Fusion
and Membrane PermeabilityNote 14.3), nucleic acid hybridization, ligandreceptor binding Figure 9 Principle of enzyme detection via the disruption
and polypeptide hydrolysis. of intramolecular self-quenching. Enzyme-catalyzed hy-
drolysis of the heavily labeled and almost totally quenched
substrates provided in the EnzChek Assay Kits relieves the
Other Environmental Factors intramolecular self-quenching, yielding brightly fluorescent
Many other environmental factors exert influences on fluorescence properties. The three reaction products.
most common are:

Solvent polarity (solvent in this context includes interior regions of cells, proteins, mem-

Fluorescence emission
branes and other biomolecular structures) 1

Proximity and concentrations of quenching species

pH of the aqueous medium
2

Fluorescence spectra may be strongly dependent on solvent. This characteristic is most often 3

observed with fluorophores that have large excited-state dipole moments, resulting in fluores-
4
cence spectral shifts to longer wavelengths in polar solvents. Representative fluorophores include
350 400 450 500 550 600
the aminonaphthalenes such as prodan, badan (Figure 11) and dansyl, which are effective probes
Wavelength (nm)
of environmental polarity in, for example, a proteins interior. 36
Figure 10 Excimer formation by pyrene in ethanol. Spectra
Binding of a probe to its target can dramatically affect its fluorescence quantum yield are normalized to the 371.5 nm peak of the monomer. All
(Monitoring Protein-Folding Processes with Environment-Sensitive DyesNote 9.1). Probes spectra are essentially identical below 400 nm after normal-
ization. Spectra are as follows: 1) 2 mM pyrene, purged with
argon to remove oxygen; 2) 2 mM pyrene, air-equilibrated;
3) 0.5 mM pyrene (argon-purged); and 4) 2 M pyrene (argon-
Oregon purged). The monomer-to-excimer ratio (371.5/470 nm) is de-
Green pendent on both pyrene concentration and the excited-state
514 Rhodamine Red-X
Conjugate fluorescence
Conjugate fluorescence

lifetime, which is variable because of quenching by oxygen.

Oregon
Green
488

Lissamine rhodamine B 2
1 Ex = 380 nm
Fluorescence emission

Fluorescein-EX

FITC
3

0 2 4 6 8 10 12 14 16 0 1 2 3 4 5
5
Fluorophores/protein (mol:mol) Fluorophores/protein (mol:mol)

Figure 7 Comparison of relative fluorescence as a func- Figure 8 Comparison of the relative fluorescence of goat
tion of the number of fluorophores attached per protein antimouse IgG antibody conjugates of Rhodamine Red-X 6

for goat antimouse IgG antibody conjugates prepared succinimidyl ester (R6160, d) and Lissamine rhodamine B 400 450 500 550 600
using Oregon Green 514 carboxylic acid succinimidyl sulfonyl chloride (L20, L1908; s). Conjugate fluorescence
Wavelength (nm)
ester (O6139, j), Oregon Green 488 carboxylic acid suc- is determined by measuring the fluorescence quantum
cinimidyl ester (O6147, d), fluorescein-5-EX succinimidyl yield of the conjugated dye relative to that of the free dye Figure 11 Fluorescence emission spectra of the 2-mercapto-
ester (F6130, s) and fluorescein isothiocyanate (FITC; F143, and multiplying by the number of fluorophores per pro- ethanol adduct of badan (B6057) in: 1) toluene, 2) chloroform,
F1906, F1907; h). Conjugate fluorescence is determined by tein. Higher numbers of fluorophores attached per protein 3) acetonitrile, 4) ethanol, 5) methanol and 6) water. Each solu-
measuring the fluorescence quantum yield of the conjugat- are attainable with Rhodamine Red-X dye due to the lesser tion contains the same concentration of the adduct. Excitation
ed dye relative to that of the free dye and multiplying by the tendency of this dye to induce protein precipitation. of all samples is at 380 nm.
number of fluorophores per protein.

The
The Molecular
Molecular Probes
Probes Handbook:
Handbook: A Guide
A Guide to Fluorescent
to Fluorescent Probes
Probes andand Labeling
Labeling Technologies
Technologies
IMPORTANT NOTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on
IMPORTANT NOTICE : The
page 971products
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Introduction to Fluorescence Techniques

that have a high fluorescence quantum yield when bound to a particular target but
REFERENCES
are otherwise effectively nonfluorescent yield extremely low reagent background
1. Annu Rev Genomics Hum Genet (2008) 9:387; 2. Science (2007)
signals. The ultrasensitive SYBR, SYTO, PicoGreen, RiboGreen and OliGreen
315:81; 3. Anal Chem (2008) 80:7063; 4. Anal Chem (2001) 73:2070;
nucleic acid stains (Chapter 8) are prime examples of this strategy. Similarly, fluoro- 5. Analyst (1994) 119:417; 6. J Microsc (2007) 228:390; 7. J Res Natl Inst
genic enzyme substrates, which are nonfluorescent or have only short-wavelength Stand Technol (2001) 106:381; 8. Methods Cell Biol (1994) 42B:605;
emission until they are converted to fluorescent products by enzymatic cleavage, 9. J Microsc (1994) 176:281; 10. Cytometry B Clin Cytom (2009)
allow sensitive detection of enzymatic activity (Chapter 10). 76:295; 11. Nat Protoc (2009) 4:372; 12. Nat Rev Immunol (2004) 4:648;
Extrinsic quenchers, the most ubiquitous of which are paramagnetic species 13. J Immunol Methods (2009) 344:6; 14. J Neurosci Methods (2007)
162:119; 15. J Neurosci Methods (2009) 180:116; 16. BMC Cell Biol
such as O2 and heavy atoms such as iodide, reduce fluorescence quantum yields in
(2008) 9:13; 17. Nat Protoc (2007) 2:1152; 18. Nat Med (2006) 12:972;
a concentration-dependent manner. If quenching is caused by collisional interac- 19. Biophys J (1983) 43:383; 20. Methods Cell Biol (2007) 81:415;
tions, as is usually the case, information on the proximity of the fluorophore and 21. J Microsc (2009) 233:192; 22. Nat Methods (2008) 5:197; 23. J Phys
quencher and their mutual diffusion rate can be derived. This quenching effect has Chem A (2007) 111:429; 24. Chemphyschem (2008) 9:2019; 25. Nat
been used productively to measure chloride-ion flux in cells (Section 21.2). Many Methods (2007) 4:81; 26. Org Lett (2004) 6:909; 27. Biophys J (1995)
fluorophores are also quenched by proteins. Examples are NBD, fluorescein and 68:2588; 28. J Cell Biol (1985) 100:1309; 29. J Org Chem (1973) 38:1057;
BODIPY dyes, in which the effect is apparently due to charge-transfer interac- 30. Mol Cell Probes (2008) 22:294; 31. J Histochem Cytochem (2007)
55:545; 32. Exp Cell Res (2007) 313:1943; 33. J Histochem Cytochem
tions with aromatic amino acid residues.37,38 Consequently, antibodies raised
(1995) 43:77; 34. ACS Chem Biol (2009) 4:535; 35. Anal Biochem (1997)
against these fluorophores are effective and highly specific fluorescence quench- 251:144; 36. Nature (1986) 319:70; 37. Bioconjug Chem (2003) 14:1133;
ers 38 (Section 7.4). 38. Immunochemistry (1977) 14:533.
Fluorophores such as BCECF and carboxy SNARF-1 that have strongly pH-de-
pendent absorption and fluorescence characteristics can be used as physiological pH
indicators. Fluorescein and hydroxycoumarins (umbelliferones) are further exam-
ples of this type of fluorophore. Structurally, pH sensitivity is due to a reconfigura-
tion of the fluorophores -electron system that occurs upon protonation. BODIPY
FL and Alexa Fluor 488 fluorophores, both of which lack protolytically ionizable
substituents, provide spectrally equivalent alternatives to fluorescein for applica-
tions requiring a pH-insensitive probe (Section 1.3, Section 1.4).

Selected Books and Articles

The preceding discussion has introduced some general principles to consider when selecting a fluorescent probe. Application-specific details
are addressed in subsequent chapters of The Molecular Probes Handbook. For in-depth treatments of fluorescence techniques and their biological
applications, the reader is referred to the many excellent books and review articles listed below.

Principles of Fluorescence Detection Royer, C.A., "Approaches to teaching fluorescence spectroscopy,"

Albani, J.R., Absorption et Fluorescence: Principes et Applications, Biophys J (1995) 68:11911195.
Lavoisier (2001). This book is the first on absorption and fluoresc- Selvin, P.R. and Ha, T., Eds., Single-Molecule Techniques: A Laboratory
nece to be published in the French language. Manual, Cold Spring Harbor Laboratory Press (2007).
Albani, J.R., Principles and Applications of Fluorescence Spectroscopy, Valeur, B., Molecular Fluorescence: Principles and Applications, John
Wiley-Blackwell (2007). Wiley and Sons (2002).
Brand, L. and Johnson, M.L., Eds., Fluorescence Spectroscopy (Methods
in Enzymology, Volume 450), Academic Press (2008). Fluorophores and Fluorescent Probes
Gell, C., Brockwell, D. and Smith, A., Handbook of Single Molecule Berlman, I.B., Handbook of Fluorescence Spectra of Aromatic Molecules,
Fluorescence Spectroscopy, Oxford University Press (2006). Second Edition, Academic Press (1971).
Goldys, E.M., Ed., Fluorescence Applications in Biotechnology and Life Burry, R.W., Immunocytochemistry: A Practical Guide for Biomedical
Sciences, Wiley-Blackwell (2009). Research, Springer (2009).
Guilbault, G.G., Ed., Practical Fluorescence, Second Edition, Marcel Chalfie, M. and Kain, S.R., Green Fluorescent Protein: Properties,
Dekker (1990). Applications and Protocols, Second Edition, John Wiley and Sons
Joo, C., Balci, H., Ishitsuka, Y., Buranachai, C. and Ha, T., Advances (2006).
in single-molecule fluorescence methods for molecular biology, Drexhage, K.H., "Structure and properties of laser dyes" in Dye Lasers,
Annu Rev Biochem (2008) 77:5176. Third Edition, F.P. Schfer, Ed., Springer-Verlag, (1990) p. 155200.
Lakowicz, J.R., Principles of Fluorescence Spectroscopy, Third Edition, Green, F.J., The Sigma-Aldrich Handbook of Stains, Dyes and Indicators,
Springer (2006). Aldrich Chemical Company (1990).
Mathies, R.A., Peck, K. and Stryer, L., "Optimization of high-sensitivity Griffiths, J., Colour and Constitution of Organic Molecules, Academic
fluorescence detection," Anal Chem (1990) 62:17861791. Press (1976).

The Molecular
The MolecularProbes
Probes Handbook: A Guide
Handbook: A Guide to
to Fluorescent
Fluorescent Probes
Probesand
andLabeling
LabelingTechnologies
Technologies
IMPORTANT NOTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on
8 IMPORTANT
page NOTICE
971 and Master : The
Product products
List on described
page 975. in this
Products are manual are
For Research Usecovered
Only. Notby one or for
intended more
anyLimited
animal orUse Label
human License(s).
therapeutic Please refer
or diagnostic use.to the Appendix on

www.invitrogen.com/probes
page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
thermofisher.com/probes
Introduction to Fluorescence Techniques

Fluorophores and Fluorescent Probes, continued Pawley, J.B., Ed., Handbook of Biological Confocal Microscopy, Third
Hermanson, G.T., Bioconjugate Techniques, Second Edition, Academic Edition, Springer (2006).
Press (2008). Periasamy, A., Ed., Methods in Cellular Imaging, Oxford University
Hilderbrand, S.A., Labels and probes for live cell imaging: Overview Press (2001).
and selection guide, Methods Mol Biol (2010) 581:1745. Rosenthal, E. and Zinn, K.R., Eds., Optical Imaging of Cancer: Clinical
Kobayashi, H., Ogawa, M., Alford, R., Choyke, P.L. and Urano, Y., New Applications, Springer (2009).
strategies for fluorescent probe design in medical diagnostic imag- Spector, D.L. and Goldman, R.D., Basic Methods in Microscopy, Cold
ing, Chem Rev (2010) 110:26202640. Spring Harbor Laboratory Press (2005).
Kricka, L.J. and Fortina, P., Analytical ancestry: "Firsts" in fluorescent Svoboda, K. and Yasuda, R., Principles of two-photon excitation mi-
labeling of nucleosides, nucleotides, and nucleic acids, Clin Chem croscopy and its applications to neuroscience, Neuron (2006)
(2009) 55:670683. 50:823839.
Liehr, T., Ed., Fluorescence In Situ Hybridization (FISH): Application Tsien, R.Y., " Imagining imagings future," Nat Rev Mol Cell Biol (2003)
Guide, Springer (2008). 4:SS16SS21.
Mason, W.T., Ed., Fluorescent and Luminescent Probes for Biological Yuste, R. and Konnerth, A., Eds., Imaging in Neuroscience and
Activity, Second Edition, Academic Press (1999). Development: A Laboratory Manual, Cold Spring Harbor
Oliver, C. and Jamur, M.C., Eds., Immunocytochemical Methods and Laboratory Press (2005).
Protocols, Third Edition (Methods in Molecular Biology, Volume
588), Humana Press (2010). Flow Cytometry
Taraska, J.W. and Zagotta, W.N., Fluorescence applications in molecu- Darzynkiewicz, Z., Crissman, H.A. and Robinson, J.P., Eds., Cytometry,
lar neurobiology, Neuron (2010) 66:170189. Third Edition Parts A and B (Methods in Cell Biology, Volumes 63
and 64), Academic Press (2001).
Fluorescence Microscopy Givan, A.L., Flow Cytometry: First Principles, Second Edition, John
Frigault, M.M., Lacoste, J., Swift, J.L. and Brown, C.M., Live-cell mi- Wiley and Sons (2001).
croscopy: Tips and tools, J Cell Sci (2009) 122:753767. Herzenberg, L.A., Parks, D., Sahaf, B., Perez, O., Roederer, M. and
Fujimoto, J.G. and Farkas, D., Eds., Biomedical Optical Imaging, Oxford Herzenberg, L.A., "The history and future of the fluorescence ac-
University Press (2009). tivated cell sorter and flow cytometry: A view from Stanford," Clin
Goldman, R.D., Swedlow, J.R. and Spector, D.L., Eds., Live Cell Imaging: Chem (2002) 48:18191827.
A Laboratory Manual, Second Edition, Cold Spring Harbor Herzenberg, L.A., Tung, J., Moore, W.A., Herzenberg, L.A. and Parks,
Laboratory Press (2009). D.R., Interpreting flow cytometry data: A guide for the perplexed,
Hell, S.W., Microscopy and its focal switch, Nat Methods (2009) Nat Immunol (2006) 7:681685.
6:2432. Preffer F. and Dombkowski, D. Advances in complex multiparameter
Herman, B., Fluorescence Microscopy, Second Edition, BIOS Scientific flow cytometry technology: Applications in stem cell research,
Publishers (1998). Cytometry B (2009) 76:295314.
Hibbs, A.R., Confocal Microscopy for Biologists, Springer (2004). Shapiro, H.M., "Optical measurement in cytometry: Light scattering,
Huang, B., Bates, M. and Zhuang, X., Super-resolution fluorescence extinction, absorption and fluorescence," Meth Cell Biol (2001)
microscopy, Annu Rev Biochem (2009) 78:9931016. 63:107129.
Inou, S. and Spring, K.R., Video Microscopy, Second Edition, Plenum Shapiro, H.M., Practical Flow Cytometry, Fourth Edition, Wiley-Liss
Publishing (1997). (2003).
Lichtman, J.W. and Conchello, J.A., Fluorescence microscopy, Nat Sklar, L.A., Ed., Flow Cytometry for Biotechnology, Oxford University
Methods (2005) 2:910919. Press (2005).
Masters, B.R., Confocal Microscopy and Multiphoton Excitation
Microscopy: The Genesis of Live Cell Imaging, SPIE Press (2006). Other Fluorescence Measurement Techniques
Matsumoto, B., Ed., Cell Biological Applications of Confocal Microscopy, Dorak, M.T., Ed., Real-Time PCR, Taylor and Francis (2006).
Second Edition (Methods in Cell Biology, Volume 70), Academic Gore, M., Ed., Spectrophotometry and Spectrofluorimetry: A Practical
Press (2003). Approach, Second Edition, Oxford University Press (2000).
Murphy, D.B., Fundamentals of Light Microscopy and Electronic Mardis, E.R. Next-generation DNA sequencing methods, Annu Rev
Imaging, John Wiley and Sons (2001). Genomics Hum Genet (2008) 9:387402.
Ntziachristos, V., "Fluorescence molecular imaging," Annu Rev Biomed Patton, W.F., "A thousand points of light: The application of fluorescence
Eng (2006) 8:132. detection technologies to two-dimensional gel electrophoresis and
Patterson, G., Davidson, M., Manley, S. and Lippincott-Schwartz, J., proteomics," Electrophoresis (2000) 21:11231144.
Superresolution imaging using single-molecule localization, Shimomura, O., Bioluminescence: Chemical Principles and Methods,
Annu Rev Phys Chem (2010) 61:345367. World Scientific Publishing (2006).

TheMolecular
The Molecular Probes
Probes Handbook:
Handbook: A Guide
A Guide to Fluorescent
to Fluorescent Probes
Probes andand Labeling
Labeling Technologies
Technologies
IMPORTANT NOTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on
IMPORTANT NOTICE : The
page products
971 and Masterdescribed
Product Listinon
this manual
page are covered
975. Products are Forby one or Use
Research more Limited
Only. Use Label
Not intended License(s).
for any animal orPlease
humanrefer to the or
therapeutic Appendix onuse.
diagnostic 9
page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal www.invitrogen.com/probes
or human therapeutic or diagnostic use.
thermofisher.com/probes