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v
Contents
Preface x
Acronyms xii
Symbols and Conventions
xiii
1
1
Introduction
2
2.1
2.2
2.3
2.3.1
2.3.2
2.3.3
2.3.4
2.3.5
2.4
2.5
2.5.1
2.5.2
2.5.3
2.5.4
2.5.5
2.5.6
2.6
2.6.1
2.6.2
2.6.3
2.7
2.7.1
2.7.2
2.7.3
2.7.4
2.7.5
2.7.6
Basic Definitions and Fundamentals 5
Introduction 5
Light Sources 5
Filtering and Dispersing Light 6
Absorber Filters 6
Interference Filters 8
Polarizers 8
Prisms 9
Grating 9
Light Detectors 10
Light Beams 12
Radiant Power and Radiance in Space: Divergent and Collimated Beams 12
Radiant Power and Radiance in Time: Continuous, Modulated, and Pulsed 13
Spectral Radiant Power (Emission Spectra) of Lamps, LEDs, and Lasers 14
Light Wavelength, Transmittance, and Absorbance 14
Spontaneous Decay and Stimulated Emission in Lasers and STED Nanoscopy 16
Energy, Momentum, Polarization, Spin, and Angular Momentum 17
Light Collection Set-Ups 17
Microscope Objectives 17
Fluorescence Detection Set-Ups 18
Fluorescence Imaging Set-Ups 18
Fundamentals of Fluorescence 21
Fluorescence: Fields of Application 22
Molar Absorption Coefficient 23
Excitation Spectra 24
Emission Spectra 25
Stokes Shift 25
Fluorescence Quantum Yield 27
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Contents
2.7.7
2.7.8
2.7.9
2.7.10
2.7.10.1
2.7.10.2
2.7.10.3
2.7.10.4
2.7.10.5
2.7.10.6
2.7.10.7
2.8
Brightness 27
Effective Brightness 27
Fluorescence Mean-Lifetime 28
Factors Affecting Fluorescence 29
Effect of Microenvironment 29
Influence of Liquid Viscosity on Fluorescence Quantum Yield and Fluorescence Mean-Lifetime 30
Influence of Electric Permittivity and Hydrogen Bonding 30
Effects of Temperature 31
Quenching 31
Self-Quenching 32
Singlet Oxygen Production by Sensitizer Dyes 32
Photostability 32
3
3.1
3.2
3.2.1
3.2.2
3.3
3.3.1
3.3.1.1
3.3.1.2
3.3.1.3
3.3.2
3.3.3
3.3.4
3.3.5
3.4
3.4.1
3.4.2
3.4.3
3.4.4
3.4.5
3.4.6
3.5
Target-Fluorophore Binding 37
Introduction 37
Choosing the Right Solvent 37
Water and PBS 37
Water Miscible Organic Solvents 39
Fluorogenic Reactions 45
Primary Amines 45
Fluorogenic Reactions of Primary Amines With Homocyclic o-Phthaldihaldehydes 45
Fluorogenic Reactions of Primary Amines With Heterocyclic o-Dicarboxaldehydes 49
Fluorogenic Reactions of Primary Amines With Other Reagents 49
Secondary Amines 52
Thiols 53
Cyanide 53
α-Dicarbonylic Compounds 53
Labeling Reactions 54
Covalent Labeling of Amines 56
Covalent Labeling of Thiols 57
Covalent Labeling of Carboxylic Acids 57
Covalent Labeling of Alcohols 58
Covalent Labeling of Reducing Saccharides 58
Others 60
Immunofluorescence 61
4
4.1
4.2
4.2.1
4.2.2
4.2.3
4.2.4
4.2.5
4.3
4.3.1
4.3.2
Classes and Molecular Structures 65
Introduction 65
Rhodamines 73
Rhodamines With Absorption Maximum Below 500 nm 73
Rhodamines With Absorption Maximum Between 500 and 550 nm 73
Rhodamines With Absorption Maximum Between 550 and 600 nm 73
Rhodamines With Absorption Maximum Above 600 nm 75
Rhodamines With a High Net Charge 78
HAS-Rhodamines 79
Carbo-Rhodamines 79
Silico-Rhodamines 79
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Contents
4.3.3
4.4
4.5
4.6
4.7
4.8
4.8.1
4.8.2
4.8.3
4.8.4
4.9
4.10
4.11
4.12
4.13
4.13.1
4.13.2
4.13.3
4.14
4.14.1
4.14.2
4.14.3
4.14.4
4.15
4.15.1
4.15.2
4.15.3
4.16
4.17
4.18
4.18.1
4.18.2
4.18.3
4.19
4.20
4.21
4.22
4.23
4.23.1
4.23.2
4.24
4.25
4.26
4.26.1
4.26.2
4.26.3
4.27
Other HAS-Rhodamines 79
Pyronines 79
HAS-Pyronines 82
Sulforhodamines 84
HAS-Sulforhodamines 84
Fluoresceins 84
Non-Halogenated Fluoresceins 88
Halogenated Fluoresceines 89
Mercaptofluoresceins 91
Fluorescein-Analogs 91
HAS-Fluoresceins 91
Sulfofluoresceins 91
Fluorones 92
HAS-Fluorones 93
Cyanines 93
Trimethine Cyanines 95
Pentamethine Cyanines 95
Heptamethine Cyanines 97
Borondipyrromethenes 97
Small Water-Soluble Borondipyrromethenes 100
Medium-Sized, Water-Soluble Borondipyrromethenes 104
Large Water-Soluble Borondipyrromethenes 106
Other Classes Derived From Borondipyrromethene 108
Rhodols 109
The First Rhodols Synthesized 109
Rhodols Synthesized More Recently 109
Rhodol Analogs 112
HAS-Rhodols 113
Rosamines 114
HAS-Rosamines 114
Silico-Rosamines 114
Phospha-Rosamines 115
Other HAS-Rosamines 115
Rosols 118
HAS-Rosols 118
Pyrodols and Pyrodones 119
Trianguleniums 120
Acridines 120
Simple Acridines 121
Acridones 122
Merocyanines 122
Phenoxazines 122
Coumarins 125
7-Hydroxy Coumarins 125
Small 7-Amino Coumarins 125
More Elaborated 7-Amino Coumarins 126
Sulforhodols 128
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Contents
4.28
4.29
4.30
4.31
4.32
4.33
4.34
4.35
4.36
4.37
4.38
4.39
4.39.1
4.39.2
4.39.3
4.40
4.40.1
4.40.2
4.40.3
4.41
4.42
Pyrenes 128
Quinolines 129
Benzothiazoles 132
Chromones 133
Naphthalimides 133
Indoles 134
Naphthalenes 135
Squaraines 137
Pteridines 137
Isoquinolines 139
Benzene Derivatives 140
Other Single Structures 140
Small Structures 140
Medium-Sized Structures 142
Large Structures (Na > 80) 142
Hybrid Structures 145
Hybrid Structures: Fusion of Two Existing Dyes 146
Hybrid Structures: Single Bond Connected Dyes 146
Hybrid Structures: Polymethine Bridged Dyes 147
Non-Disclosed Structures 148
Fluorescent Structures Other Than Small-Molecule Organic Dyes 149
5
5.1
5.2
5.2.1
5.2.2
5.2.3
5.2.4
5.2.5
5.2.6
5.2.7
5.2.8
5.3
Scattergrams of the Photophysical Properties 163
Introduction 163
Photophysical Properties Along the Spectrum 164
Molecular Sizes vs. λa,max 164
Molar Absorption Coefficients vs. λa,max 169
Fluorescence Quantum Yield vs. λa,max 169
Brightness vs. λa,max 172
Stokes Shift vs. λa,max 173
Stokes Shift vs. Brightness 174
Fluorescence Mean-Lifetime vs. λa,max 177
Fluorescence Mean-Lifetime vs. Brightness 178
Fluorophore Charges 180
6
6.1
6.2
6.3
Band Shapes and Excitation and Emission Ranges 185
Introduction 185
Typical Absorption and Emission Spectra of Some Classes 187
Coarse Prediction of Excitation and Emission Ranges 191
7
7.1
7.2
7.3
Measuring Photostability and Mitigating Photobleaching
Introduction 195
Measuring Photostability 196
Mitigating Photobleaching 202
195
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Contents
Appendix A
A1. Short Name, Name, Class, Molecular Formula, and References 207
Appendix B
B1. Ranked by Excitation Maximum
Appendix C
C1. Ranked by Emission Maximum 297
Appendix D
D1. Ranked by Stokes Shifts
Appendix E
E1. Ranked by Brightness
Appendix F
F1. Ranked by Fluorescence Mean-Lifetime 419
Appendix G
G1. Ranked by Molecular Net Charge 433
253
335
371
Index 479
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xii
Acronyms
A
Acetate buffer
ACN
Acetonitrile; ethanenitrile
ADC
Antracene-2,3-dicarboxaldehyde
APD
Avalanche photodiode
B
Borate buffer
BODIPY 4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene
C
Carbonate buffer
CCD
Charged coupled device
CBQCA 3-(4-Carboxybenxoyl)quinoline-2-carboxaldehyde
DMF
Dimethylformamide; N,N-dimethylmethanamide
DMSO
Dimethylsulfoxide; dimethyl(oxido)sulfur
FRET
Förster resonant energy transfer
HAS
Heteroatom-substituted
HEPES
4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid
IA
Iodoacetamido
Isothyocianate
ITC
ME
Maleimido
NDA
Naphthalene-2,3-dicarboxaldehyde
N-Hydroxysuccinimidyl ester
NHS
Near-infrared
NIR
NMP
N-Methyl-2-pyrrolidinone
o-Phthaldialdehyde
OPA
P
Phosphate buffer
PBSPhosphate-buffered saline (usually: 8.0 g/L NaCl, 0.2 g/L KCl, 1.42 g/L Na2HPO4,
and 0.24 g/L KH2PO4)
PD
Photodiode
PC
Propylene carbonate; 4-methyl-1,3-dioxolan-2-one
PMT
Photomultiplier tube
RT
Room temperature
SE
N-Hydroxysuccinimidyl ester
THF
Tetrahydrofuran; 1,4-epoxybutane
Tris
Tris(hydroxymethyl)aminomethane; 2-amino-2-(hydroxymethyl)propane-1,3-diol
UV
Ultraviolet
UVA
UV radiation in the 315 and 400 nm range. Informally also known as “black light”
Vis
Visible
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xiii
Symbols and Conventions
Symbol Name and definition [Units]
A(λ)
Absorbance at wavelength λ. A(λ) = Log10(Po(λ)/P(λ)) = Log10(1/T(λ)). [dimensionless]
b
Cuvette inner length. [cm]
B(λ)Molecular fluorescent brightness at wavelength λ. B(λ) = ε(λ) Φf. [м–1cm–1].
BmaxMolecular fluorescent brightness at wavelength λa,max. Bmax = ε(λx,max) Φf = εmax Φf. [м–1cm–1].
cAmount concentration. Expressed in mol dm–3 or mol L–1, while the non-SI unit м (small cap M)
is used as an abbreviation for mol dm–3. [mol dm–3]
h
Planck constant. h = 6.626069 × 10–34 J s. [Js]
LRadiance. Defined as the radiant power (P) leaving or passing through a small element of surface
(dS) divided by the solid angle of this surface (dΩ) and the orthogonally projected area of this element
of surface in a plane normal to the beam direction (dS cosθ). L = d2p/(dΩ dS cosθ) [Js–1m–2sr–1 or
Wm–2sr–1]. For a parallel beam of radiation the radiance is simple given by L = dp/(dS cosθ). [Js–1m–2
or Wm–2]
L(λ)Spectral radiance. Derivative of radiance, L, with respect to wavelength, λ. [Js–1m–2nm–1 or
Wm–2nm–1]
LpPhoton radiance. Defined as the number of photons leaving or passing through a small element
of surface (dS) per second divided by the solid angle of this surface (dΩ) and the orthogonally
projected area of this element of surface in a plane normal to the beam direction (dS cosθ).
Lp = number of photons per second/(dΩ dS cosθ) [s–1m–2sr–1]. For a parallel beam of radiation the
photon radiance is simple given by Lp = number of photons per second/(dS cosθ). [s–1m–2]
Lp(λ)Spectral photon radiance. Derivative of photon radiance, Lp, with respect to wavelength, λ.
[s–1m–2nm–1]
P
Radiant power. Power emitted, transferred, or received as electromagnetic radiation. [Js–1 or W]
P(λ)Spectral radiant power, at λ. Derivative of radiant power, P, with respect to wavelength, λ. In spectroscopy: P(λ) = {total radiant power between [λ –(Δλ/2)] and [λ +(Δλ/2)] that exits a sample or
cuvette filled with a solvent containing the solute}/Δλ. [W nm–1]
Po(λ)Spectral radiant power at λ that exits a blank sample or cuvette “filled with solvent only.”
Po(λ) = {total radiance, between [λ –(Δλ/2)] and [λ +(Δλ/2)], that exits a blank sample or cuvette
filled with solvent only}/Δλ. [W nm–1]
NaNumber of atoms of the fluorescent structure (counter-ions of salts are not counted) in its prevalent ionic form in an aqueous solution at a specified pH. [dimensionless]
NnNumber of negative elementary charges of a fluorescent structure in its prevalent ionic form in an
aqueous solution at a specified pH. [dimensionless]
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xiv
Symbols and Conventions
NpNumber of positive elementary charges of a fluorescent structure in its prevalent ionic form in an
aqueous solution at a specified pH. [dimensionless]
qNet number of elementary charges of a fluorescent structure in its prevalent ionic form in an
aqueous solution at a specific pH (q = Np – Nn). [dimensionless]
T
Temperature. [oC and sometimes K]
T(λ)
Transmittance at wavelength λ. T(λ) = P(λ)/Po(λ). [dimensionless]
ΔSStokes shift. ΔS = νx,max – νe,max. [cm–1]. The following are also used: ΔS = λe,max – λx,max and
ΔS = λe,max – λa,max. [nm]
ε(λ)
Molar decadic absorption coefficient at wavelength λ. ε(λ) = A(λ) b–1c–1. [м–1cm–1].
εmaxMolar decadic absorption coefficient at the absorption maximum of the longest-wavelength band.
εmax = A(λa,max) b–1c–1. [м–1cm–1].
λ
Vacuum wavelength of the electromagnetic radiation. [nm]
λa,1Wavelength of absorption maximum of the longest wavelength absorption band (λa,1 ≡ λa,max).
[nm]
λa,maxWavelength of absorption maximum of the longest wavelength absorption band (λa,max ≡ λa,1).
[nm]
λa,–αWavelength at height α of the left side of the longest wavelength absorption band (0 < α ≤ 1). [nm]
λa,+αWavelength at height α of the right side of the longest wavelength absorption band (0 < α ≤ 1).
[nm]
Wavelength of emission maximum (λe,1 ≡ λe,max). [nm]
λe,1
Wavelength of emission maximum. [nm]
λe,max
Wavelength at height α of the left side of the emission band (0 < α ≤ 1). [nm]
λe,–α
Wavelength at height α of the right side of the emission band (0 < α ≤ 1). [nm]
λe,+α
Wavelength of excitation maximum of the longest wavelength band. [nm]
λx,max
Wavenumber. It is related to wavelength λ [nm] by ν = 107/λ [cm–1]
ν
Wavenumber of absorption maximum of the longest wavelength absorption band. [cm–1]
νa,max
Wavenumber of emission maximum. [cm–1]
νe,max
Wavenumber of excitation maximum of the longest wavelength band. [cm–1]
νx,max
τ1/2Fluorescence half-lifetime. τ1/2 = τf ln(2). Time required for half the entities, of a sample of initially excited entities, to decay. [ns]
Fluorescence mean-lifetime. τf = τ1/2 /ln(2). [ns]
τf
Photobleaching mean-lifetime or mean-lifetime before photobleaching. [s]
τpb
τpb,rRelative photobleaching mean-lifetime. Relative to a dye exposed to an identical radiance and
other conditions. [dimensionless]
ΦfFluorescence quantum yield. Φf = number of fluorescent photons emitted/number of photons absorbed. [dimensionless]
ΦtTriplet quantum yield. Φt = number of triplets created/number of photons absorbed.
[dimensionless]
ΦpPhosphorescence quantum yield. Φp = number of phosphorescent photons emitted/number of
photons absorbed. [dimensionless]
ΦpbPhotobleaching quantum yield. Expresses the number of molecules photobleached per absorbed
photon. [dimensionless]
м
Unit of amount concentration. [mol dm–3 or mol L–1]
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1
1
Introduction
Fluorescent dye labels and stains are used in a great variety of fields such as RNA or DNA detection and analyses
(real-time PCR, fluorescence in situ hybridization, DNA microarrays, DNA sequencers, and DNA analyzers),
proteomics, antibody labeling and immunoassays, flow cytometry, cell sorters, electrophoresis (in platforms capillary,
microchip, and slabs), high-performance liquid chromatography, fluorescence microscopy, and fluorescence nanoscopy, to mention a few. Fluorescence is used in so many fields due to its extraordinary sensitivity, high selectivity, simplicity, and wide linear working ranges, sometimes reaching up to six orders of magnitude in concentration. In most
cases, fluorescent dyes are used to label or stain a myriad of targets for their separation, detection, quantification,
imaging, or tracking in space and/or in time. Measurement of intensity changes is still the predominant mode of usage,
although measurements related to changes in polarization, emission lifetimes, and spectra are also used.
High contrasts in imaging, high signal-to-noise ratios, and optimal detection limits in analytical methods are
achieved when, together with the use of proper instruments and excitation sources, the best fluorophores are
included. However, it is a boring and time demanding task watching dozens of uncomplete spectra viewers, going
through catalogs, books, and hundreds of articles to find the best dye for a given application. Each dye has many
characteristics that must be confronted with the characteristics of the other hundreds. This book, among the databases provided, gives panoramic views (using scattergrams) of all dyes together.
This present database contains the molecular structures, photophysical properties, and characteristics of over 700
fluorescent dye labels and stains with medium to high brightness in aqueous solutions (plain water, phosphate-buffered
saline, or any aqueous buffer containing less than 1% of organic modifiers and without complexing agents) and with
absorption maximum in the 300–900 nm range of the spectrum. This database ended up including fluorescent entities
from the following main classes: acridines, acridiziniums, acridones, benzothiazoles, benzenes, boron-dipyrromethenes,
carbazoles, chromones, coumarins, cyanines, diketopyrrolopyrroles, fluorenes, fluoresceins (and fluorones), hybrid
structures, indoles, isoquinolines, mercaptofluoresceins, merocyanines, naphthalenes, naphthalimides, phenazines,
phenoxazines, phospholes, pteridines, pyrenes, pyridines, pyrodols, pyrodones, pyronines, quinolines, rhodamines (and
rosamines), rhodols (and rosols), squaraines, sulfofluoresceins, sulforhodamines, sulforhodols, and trianguleniums, to
mention a few. Hetero-atom substituted (HAS) xanthenes are also included, such as HAS-fluoresceins (and HASfluorones), HAS-pyronines, HAS-rhodamines (and HAS-rosamines), and HAS-rhodols (and HAS-rosols).
The photophysical parameters are tabulated and ranked by their importance for the users, such as: the optimal
excitation range (including the wavelength of excitation maximum); optimal detection wavelength range
(emission range, including the wavelength of emission maximum); Stokes shifts; molar absorption coefficients;
fluorescence quantum yields; molecular fluorescent brightness; fluorescence mean-lifetimes; sizes; and net
charges of the dyes in aqueous solutions at neutral pH. The photophysical and molecular properties were also
examined using scatter plots. Wavelengths of excitation maxima were compared with Stokes shifts, molar
absorption coefficients, fluorescence quantum yields, brightness, fluorescence mean-lifetimes, molecular sizes
(number of atoms), and charges (at the pH where the photophysical parameters were measured). The photophysical
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2
1 Introduction
parameters were also compared with each other; for instance, brightness vs. Stokes shift, brightness vs. molecular
size, and so on.
The resulting panoramic views provided by the scattergrams reveal many compelling properties for the users,
adding efficiency to the work of fluorescent label and stain selection. The resulting panoramic view is also interesting
to research groups engaged with the development and synthesis of new dyes as there are many scarcely populated
and even empty areas in the scattergrams. There are no fundamental constraints within current theories that prohibit
the existence of fluorophores in some of these empty areas. Therefore, these empty or scarcely populated ranges
of a desired photophysical or molecular property could be filled with newly synthetized water-soluble organic
fluorescent molecules and this, in turn, could lead to new applications in both basic and applied sciences.
Chapter 2 presents the most common light sources, such as lamps, LEDs, and lasers. The most commonly used
pieces of hardware for light filtering and dispersion are also presented and explained (filters, polarizers, prisms,
and diffraction gratings). Light detectors are fundamental in the field and are therefore also reviewed. Additionally,
the properties of light and light beams are of fundamental importance as they allow users to maximize the
performance of currently available instruments. Fluorescence imaging and detection set-ups are also briefly
­presented to show the available options. Finally, the fundamental concepts and definitions of the field, such as
excitation and emission spectra, molar absorption coefficient, Stokes shift, fluorescence quantum yield, brightness, and the many factors affecting fluorescence, are presented. Descriptions are based on the most recent theories describing electromagnetic radiation and are presented in a simple, very didactic manner with a large
number of illustrations.
In Chapter 3, the techniques of target-fluorophore binding are succinctly presented. It begins with a review of
the properties of the following solvents: water, phosphate-buffered saline (PBS), and 29 water mixable organic
solvents. Their melting points, boiling points, vapor pressure, and viscosity are also provided. In addition, the
evaporation rates of the following solvents are tabulated: methanol, acetone, tetrahydrofuran, acetonitrile, ethanol, 2-propanol, water, PBS, propylene carbonate, N,N-dimethylformamide, 1-methyl-2-pyrrolidinone, formamide, and dimethylsulfoxide. Fluorogenic reactions (reactions in which only the product of the reaction is
fluorescent) are reviewed, focusing on reactions with primary amines, secondary amines, thiols, and α-dicarbonylic
compounds. At the end of this chapter, the available labeling reactions are reviewed. A three-columned table contains common functional groups that can be found on target molecules, their respective reactive moiety options
for fluorescent labels, and the final functional groups produced by the resulting covalent linkage. Examples of
target functional groups are alcohols, aldehydes, amines, carboxylic acids, glycols, ketones, phenols, and thiols.
Chapter 4 presents over 700 fluorescent dyes with medium to high brightness in aqueous solutions, separated
into over 40 classes (1 per section). Each section details the molecular structures and photophysical parameters of
each fluorescent dye. An additional section contains the names and suppliers of the dyes and stains whose structures have not been disclosed (only 44 of the 777). Molecular structures are presented in the ionic form at which
photophysical parameters were measured. For almost all 700 fluorescent dyes, this is within the neutral pH range.
Below each molecular structure the following very useful photophysical parameters of the corresponding dye are
given: wavelength of excitation maximum (of the longest wavelength band) or wavelength of absorption maximum
(if the excitation maximum was not available in the references), Stokes shift, and maximum molecular brightness
(expressed in the convenient and practical unit of mм–1cm–1). Molecular brightness is defined as the product of
molar absorption coefficient and fluorescence quantum yield. It expresses both how good a fluorescent dye is at
absorbing radiation (absorption cross-section) and the probability of emission of a fluorescent photon upon each
photoexcitation. The three photophysical parameters presented are very important from a practical point of view.
Therefore, they are shown alongside the aqueous solution in which measurements were taken. The joint presentation of the molecular structure, photophysical parameters, and solution used provides the maximum amount of
information in a concise manner. This will be useful for both fluorescent dye users and dye developers (Organic
Chemists). Finally, all references from which data was taken are given in the text. Note that there is one entry for
each dye, with some rare exceptions when photophysical parameters were measured at two distinct pH values.
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1 Introduction
Chapter 5 presents scattergrams of the database. These panoramic perspectives give a good overall picture of the
dye options available and also reveal many interesting properties that can otherwise be hard to see. For example,
the scattergram of molecular absorption coefficient (εmax) vs. wavelength of excitation maximum (λx,max) shows
how εmax behaves with λx,max. Furthermore, different marks and colors are used for each class to make the
behavior of each class more visible. Scattergram analysis is also used to examine the behavior of fluorescence
quantum yield (Φf) vs. λx,max. The scattergrams simultaneously give a detailed view of the fluorescence quantum
yield of each dye and class along the whole spectrum (300–900 nm).
The product of εmax and Φf, which is defined as brightness (Bmax), provides the answer to some extremely important practical questions: which are the brightest fluorescent dyes available? Which classes contain the brightest
structures? Which excitation wavelength ranges have the brightest entities? Which are the brightest fluorophores
for a given laser wavelength or light source? Another important scatter plot is Stokes shift vs. λx,max. In the present
book, Stokes shift is presented as the difference between emission maximum and excitation maximum
(ΔS = λe,max – λx,max). Large Stokes shifts are very important for some applications. This scattergram shows the
classes with the largest Stokes shifts and the excitation wavelength ranges containing the entities with the largest
Stokes shifts. It also exhibits the typical Stokes shifts of each class. Another interesting scattergram is brightness
vs. Stokes shift. Unfortunately, this shows that it is still not possible to have the best of both worlds, as the brightest dyes have medium to small Stokes shifts and the dyes with high Stokes shifts have medium to low brightness.
Other graphs worth mentioning here are the scattergram of brightness vs. molecular size, as well as a scattergram
presenting brightness vs. dye charges (positive and negative) and λx,max in the same graph! The above-mentioned
scattergrams are only a few of the many presented in this chapter.
Chapter 6 discusses brightness values observed in practice, or effective brightness. In reality, laser lines are usually fixed and rarely operate exactly at the λx,max of the fluorescent dyes used. Therefore, effective brightness
(termed B(λ)) in fact goes from zero (0) to Bmax. The maximum brightness reported is Bmax = 112,000 м–1cm–1 or,
more conveniently, 112.0 mм–1cm–1, which was found in the rhodamine class. This chapter also proposes a
method to predict excitation ranges (good for excitation) and emission ranges (ideal for detection) in order to
obtain predictable effective brightness values from the scarce information available in the literature. The
absorption bands and emission bands within a given class somehow have a regular shape if their spectral radiance
is plotted against frequency or wavenumber instead of wavelength. This is used to predict, with the aid of an
equation which utilizes class information, λx,max, and λe,max, both the excitation range and detection ranges of
fluorescent dyes in aqueous solutions. This is very useful from a practical point of view. Of course, conjugation
and protein-rich environments produce some bathochromic shifts that are hard to predict. Nonetheless, band
parameters collected from hundreds of excitation and emission bands facilitated the prediction of not only the full
width at half maximum, but also the wavelengths at one-quarter of the height of the left side of the excitation
bands (λx,–0.25), wavelengths at one-half the height of the left side of the excitation bands (λx,–0.5), and wavelengths
at one-half the height of the right side of the excitation bands (λx,+0.5), and so on. The same is predictable from the
emission band, i.e., λe,–0.25, λe,–0.5, λe,+0.5, λe,+0.25, and λe,+0.1.
Chapter 7 presents and discusses the photostability, or photorobustness, of the fluorescent dyes. Usually, fluorescence radiance is measured and compared with other fluorescent dyes under identical illumination conditions.
The concepts of photodegradation cross-section, relative photodegradation rates, relative photodegradation
mean-lifetimes, or relative photodegradation half-lifetimes are discussed and compared. There is still a lack of
precise and reproducible measurements of these photophysical parameters as the results are impacted by the
experimental conditions used. Examples of these environmental conditions are: if the aqueous solutions are
degassed or not; the illumination conditions (radiance and wavelength used); and additional solutes (ingredients)
present in the aqueous solutions. In addition, some dyes are also affected by the pH of the aqueous solution.
Each Appendix presents the fluorescent dyes ranked by a different photophysical parameter. Examples of these
parameters include excitation ranges, emission ranges, Stokes shift, brightness, fluorescence mean-lifetime, and
dye net charges.
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