Absorption of Light
In addition to refraction, scattering, interference and diffraction phenomena in partially or completely transparent matter, which can create a color impression, such as rainbow, sky blue, shimmering soap bubbles, magnificent peacock feathers or thin oil films and layers, the colors in our environment primarily arise through the absorption or reflection of light.
The "colorfulness" of matter results from the selective absorption of light from the visible part of the spectrum of electromagnetic radiation. This range lies between ultraviolet and infrared light. At the molecular level, this means that molecules enter an electronically excited state: By the absorption of a light quantum (photon) an electron is lifted from the highest occupied molecular orbital (HOMO; energy E1) into the lowest unoccupied molecular orbital (LUMO; energy E2).

EPhoton = h υ = (h c) / λ = ΔE = E2 - E1

EPhoton = energy of the photon
h = Planck constant
υ = frequency of radiation
c = speed of light
λ = wavelength of the radiation
ΔE = energy difference between the two levels

The energy of the absorbed photon corresponds exactly to the energy difference between the two energy levels, i.e. the molecule can only exist in discrete energy states, the energy is "quantized" and can only be absorbed or released in defined portions.

UV/Vis spectroscopy
The absorption of light - e.g. in a dye solution - can be measured. The basis for quantitative UV/Vis spectroscopy is the Lambert-Beer law:

E = lg(I0 / I) = ε ⋅ c ⋅ d

E = absorbance, optical density (OD)
ε = molar extinction coefficient
c = concentration
d = layer thickness
I0 = light intensity in front of the sample
I = light intensity after sample

By transforming the equation one obtains the concentration c of a compound

c = E / (ε ⋅ d)

The Lambert-Beer law applies to monochromatic radiation. By applying the absorbance over the wavelength, the complete UV/Vis spectrum of a compound is obtained.
The Lambert-Beer law loses its validity with higher concentrated solutions as well as with the occurrence of concentration-dependent reactions, such as the aggregation of dyes.

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When passing through the solution, the light beam is attenuated, part of the light is absorbed by molecules in the solution, i.e. scattering and reflection are neglected:

A + T = 1 = (I0 – I)/I0 + I/I0

A = Absorption = absorbed part of the initial light = (I0 – I) / I0
T = Transmission = transmitted part of the initial light = I / I0

Therefore, it is

E = -lg (T)

The proportionality constant in the Lambert-Beer law is a wavelength-dependent material constant and describes the so-called transition probability with which excitation is permitted. Spectroscopic selection rules apply, i.e. a distinction is made between permitted and prohibited transitions. The most important selection rule is the maintenance of multiplicity (see below). The allowed transitions have a higher transition probability and thus a higher extinction coefficient. In the case of organic dyes, the extinction coefficient at the long-wave absorption maximum is > 105 L mol-1 cm-1; these are permitted transitions within the conjugated p-electron system.

Jablonski diagram
Absorption is a very fast process (10-15 to 10-14 s) that takes place without spin reversal. The energy absorbed by the molecule through the absorption of the photon is released in different forms after a limited lifetime of the excitation state. If photochemical reactions are excluded, it is possible that luminescence may occur in the form of fluorescence or phosphorescence besides to the release in the form of thermal energy. These photophysical elementary processes, in which the chemical identity of the molecule is retained, can be represented graphically in the form of a Jablonski diagram or Jablonski term scheme.

In addition to the electronic levels S0 - S2 and T1, the corresponding oscillation sub-levels are shown. A distinction is made between radiation processes (wavy lines) and non-radiative processes (dashes).
A singlet state S has only paired electrons, the total spin S = 0; a triplet state T has two unpaired electrons, the total spin S = 1; this results in the multiplicity M = 2 S + 1 (number of energy levels in the presence of a magnetic field). For singlet states, M = 1 and for triplet states, M = 3. The selection rule of maintaining the multiplicity thus only allows transitions within the singlet or triplet system.

Fluorescence (10-9 to 10-7 s) represents the light emission observable during deactivation of the excited molecule by transition from the lowest vibrational level of the first excited singlet state S1 to vibrational levels of the electronic ground state S0 (i.e. between states of equal multiplicity) ("rule of Kasha"). Thus, the observed fluorescence is independent of the excitation wavelength.
The occurrence of fluorescence, which G.G. Stokes called after the luminescent mineral fluorite (CaF2), can be empirically related to certain structural features of the organic molecules: non-aromatic hydrocarbons show no fluorescence; even if, as in the case of lycopene, they exhibit eleven conjugated double bonds, while aromatic compounds are almost exclusively fluorescent.
A rigid and flat molecular structure, which is often present in aromatic compounds, can therefore in many - but not all - cases be regarded as a prerequisite for good fluorescence capability.
It has also been shown that the introduction of certain groups into aromatic hydrocarbons weakens fluorescence. One of these is the nitro group, which prevents e.g. nitrobenzene from fluorescing.

Intersystem Crossing (ISC)
Bromine or iodine atoms also reduce fluorescence. This is referred to as the internal heavy atom effect. These substituents facilitate the transition into the triplet system, which is actually forbidden by spin, through a larger spin orbit coupling. This mutual transition between isoenergetic vibration levels of the singlet system and the triplet system is called "intersystem crossing" (ISC). Paramagnetic substances with unpaired electrons, such as molecular oxygen O2, also mediate this transition as catalysts.

A light emission occurring during the deactivation of the vibrational ground state of the triplet state T1 in vibrational levels of the electronic ground state S0 is called phosphorescence. In contrast to fluorescence, which can only be observed as long as the excitation occurs, phosphorescence may persist for a longer time even after the excitation has ended ("afterglow"). This in turn is related to the rule of maintaining the multiplicity, which causes a longer lifetime of the triplet state (10-4 to 102 s) and thus a delayed deactivation into the singlet ground state.

Since fluorescence or phosphorescence always takes place from the vibrational ground state v0 of the electronically excited state S1 or T1, the emission band is always shifted to higher wavelengths compared to the excitation band, i.e. bathochrom. Compared to fluorescence, phosphorescence is shifted even more strongly to long wavelengths.

Internal Conversion (IC)
Furthermore, a deactivation called "internal conversion" (IC) can take place e.g. by a transition from the vibrational ground state of the first electronically excited state S1 to isoenergetic vibronic levels of the electronic ground state S0. From such higher excited vibrational levels, a vibrational relaxation (10-12 s) takes place with the release of heat. This type of radiation-free deactivation is always faster than luminescence processes. This process seems to be particularly effective with very flexible molecules. For this reason, aliphatic compounds almost exclusively undergo internal transformation to the electronic ground state and do not fluoresce. For most other molecules, too, radiation-free deactivation processes dominate luminescence processes.

Fluorescent Dyes
Only in very few organic dyes is radiation-free deactivation slow enough, so that transitions from the excited state to the ground state gain importance, where the excess energy is emitted by emission of a photon as fluorescence.

A fluorescent dye is characterized by its spectroscopic properties such as excitation and emission spectrum, fluorescence quantum yield (ηfl) and fluorescence decay time (τfl). The fluorescence of the dye is independent of the wavelength of the excitation.

What has to be considered when selecting a dye as a fluorescence label is described in the corresponding section.

Spectral properties
The spectral properties of a fluorescent dye are strongly dependent on the molecular structure. In order for the absorption of a molecule to be in the visible wavelength range (400 - 700 nm), the energy difference between the ground and excited state must be sufficiently low. The most striking structural feature of an organic dye is a conjugated p-electron system, the so-called "chromophore" (greek: color carrier).

Absorption of rhodamine dyes
In most ATTO dyes, the chromophore has a rigid molecular backbone. Many representatives belong to the family of rhodamine dyes. As a common structural element, these dyes have a carboxyphenyl substituent at the central carbon atom of a xanthene moiety.
The carboxyl group (red) is in the 2- or ortho-position and significantly influences the physical-chemical properties of all rhodamines.
Due to the free ortho-carboxyl group, e.g. ATTO 565 and ATTO 590 as well as their derivatives exhibit special characteristics that need to be taken into account when dealing with these two fluorophores. Both dyes carry an additional carboxy group in the 4- or 5-position within the phenyl ring as a site of coupling, making ATTO 565 and ATTO 590 suitable as fluorescent labels:


Dependency of Absorption Wavelength on pH
The ortho-carboxy group is in close proximity to the chromophore and as a result the protonation-deprotonation equilibrium of this acid functionality strongly influences the optical properties of a rhodamine dye.

Hence, the absorption maximum of ATTO 565 and ATTO 590 is different for the protonated and deprotonated form. For instance the absorption maximum of deprotonated (addition of triethylamine) ATTO 565 in Ethanol is shifted by 16 nm to shorter wavelength (hypsochromic) relative to the protonated dye (addition of trifluoroacetic acid):

The Dye-Spirolacton Equilibrium
ATTO 565 and ATTO 590 in their deprotonated form can build a colorless spirolacton: After nucleophilic attack by the carboxylate anion at the centre carbon atom a five membered ring (lactone) is formed, interrupting the xanthene chromophore. As a result the compound turns colorless, i.e. no longer absorbs and fluoresces in the visible range of the spectrum:

The percentage of spirolacton in the equilibrium strongly depends on the solvent, pH, temperature and chemical structure of the dye. In polar aprotic solvents the equilibrium is almost completely in favor of the spirolacton. Consequently solutions of ATTO 565 and ATTO 590 in anhydrous acetone are virtually colorless. However, in polar protic solvents like water or ethanol, the equilibrium is almost completely in favor of the dye form.

The Fluorescence Spectrum
In most cases, the fluorescence spectrum of a dye is a mirror image of the long-wavelength absorption band, as can be seen very nicely in the case of ATTO 514:

The fluorescence maximum is typically shifted by 25 - 40 nm to longer waves compared to the absorption maximum. This so-called Stokes shift is also influenced by the reorientation of the solvent molecules surrounding the dye during the lifetime of the excited state. The emission therefore occurs from a lower-energy "solvent relaxed state". The more the electron distribution changes during excitation, the greater the energy difference and thus the Stokes shift. This is applied in a practical way with our ATTO LS dyes.

Fluorescence quantum yield (ηfl)
One of the most important properties of a fluorescent dye is the fluorescence quantum yield (ηfl). The quantum yield describes the ratio of emitted photons (nfl) to the number of absorbed photons (nabs).

ηfl = nfl / nabs

Thus, the fluorescence quantum yield can never exceed 100 %. As indicated above, radiationless deactivation processes often compete with fluorescence and thus reduce the quantum yield.
For the investigation of a dye by fluorescence, a high fluorescence quantum yield is of course advantageous and desirable.

The experimental determination of the fluorescence quantum yield is discussed in more detail elsewhere.

Fluorescence decay time (τfl)
The emission of a fluorescence photon after excitation is a statistical process. Thus, the duration for which a single molecule remains in the excited state is also a statistical quantity. However, if one considers an ensemble of many identical molecules, a well-defined decay statistic results. After excitation with a short laser pulse, the number of excited molecules decreases exponentially in the simplest case. The time after which the number of excited molecules (n1) has dropped to the factor 1/e (approx. 37%) is called the fluorescence decay time (τfl).

n1(t) = n1(0) exp(- t / τfl)

The fluorescence decay time is an important property of a dye and can be used to identify it. Values from τfl for our ATTO dyes are typically in the nanosecond range.
The determination of the fluorescence decay time is described elsewhere.

Molecular Interactions
The fluorescence decay time, as well as the fluorescence quantum yield of a dye, is not a constant value, but is influenced by the environment of the dye, i.e. solvent and temperature. Decay time and quantum yield of the dye are not independent from each other, they are linked by the following relationship:

τfl = τ0 × ηfl

τ0 is the so-called natural life time that would occur in the absence of radiationless deactivation (ηfl = 100%).
As a result, changes in the fluorescence decay time can provide information about changes in the local environment of the dye molecule.

General literature:
M.F. Vitha, Spectroscopy, Principles and Instrumentation, Wiley-VCH, 2018, ISBN 978-1-119-43664-5.
T. Förster, Fluoreszenz organischer Verbindungen, Vandenhoeck & Ruprecht, Göttingen, 1997 (Reprint), ISBN 3525423128
K. H. Drexhage, Structure and Properties of Laser Dyes, in: F. P. Schäfer, Dye Lasers, Springer Verlag, Berlin, Heidelberg, 1973.
M. Klessinger, J. Michl, Excited States and Photochemistry of Organic Molecules, Wiley-VCH, Weinheim, 1995, ISBN 0471185765.
W.A. Bingel, Theorie der Molekülspektren, Verlag Chemie, Weinheim, 1967.
Textbooks of Physical and Theoretical Chemistry.