CRITERIA

OF
A MOLECULE
TO BE
FLUOROPHORE

To know more about a fluorophore molecule we need to know its origin, some basic
theories, and a detailed description of it and its related things..
A large number of substances are known which can absorb ultraviolet or visible light
energy. But these substances lose excess energy as heat through collisions with
neighbouring atoms or molecules. However, a few important substances are also
known which lose only part of this excess energy as heat and emit the remaining
energy as electromagnetic radiation of a wavelength longer than that absorbed. This
process of emitting radiation is collectively known as luminescence.
Luminescence is of two types:
(a) Fluorescence: -When a beam of light is incident on certain substances, they emit
visible light or radiations. This phenomenon is known as fluorescence and the
substances showing this phenomenon are known as fluorescent substances. The
phenomena of the fluorescence is quick and starts immediately after the absorption of
light.
(b) Phosphorescence: When light radiation is incident on certain substances, they
emit light continuously even after the incident light is cut off. And this delayed
fluorescence is called phosphorescence.
Materials exhibiting fluorescence generally reemit excess radiation within
10-6 to 10-4 seconds of absorption. On the other hand, materials exhibit
phosphorescence re-emit radiation which is excess, within 10-4 to 20 seconds or even
more longer. Thus, the lifetime of phosphorescence is much longer than fluorescence.

We have to remember that fluorescence is a excited state phenomenon.

So at first we have to know what is the energy states and it functions.
Singlet and Triplet State:
In order to understand the theory of fluorescence and phosphorescence, one has to
understand the meaning of singlet and triplet states. These terms arise from
multiplicity considerations of atomic spectroscopy and simply define the number of
unpaired electrons in the absence of a magnetic field. If there are n number of
unpaired electrons, it means that (n+1) fold degeneracy (equal energy states) will be
associated with the electron spin, regardless of the molecular orbital occupied. Thus, if
no unpaired electrons are present (n=0), there is only n+1 or 0+1 or 1 spin state. Such
a state is called a singlet state. Similarly, systems having 1, 2, 3, 4, ....... unpaired
electrons refer to doublet, triplet, quartet, quintet, etc., states respectively.
Most of the molecules in their ground state do not have unpaired electrons (singlet
state). When such a molecule absorbs ultraviolet or visible radiation of the proper
frequency, one or more of the paired electrons (generally a л-electron) get raised to an
excited singlet state. In this excited state, the spin of the electron does not undergo any
change and the net spin is still zero. One more possibility is that one set of electron
spins may have undergone unpairing, resulting in two unpaired electrons which make
an excited triplet state, Fig. (a) represents a molecule in the ground singlet state.
Fig. (b) exhibits the molecule in the excited singlet state. Fig. (c) represents a molecule
in an excited triplet state.

Excited-State: -When molecules are irradiated with light of the appropriate frequency,
it will be absorbed in about 10-15 second. In the process of absorption, the molecules
may move from the ground to the first excited singlet electronic state. In room
temperature molecules which are present in their ground vibration state, after
absorption, the excitation molecules can end up in any one of the vibrational levels in
the first up in excited singlet state. Now from this excited singlet state, three probable
phenomena will occur, depending on the molecule :
a. The first possibility: the exited molecule return to ground state by collisional
deactivation without emitting any radiation.
b. The second possibility: molecule in excited singlet state may emit an UV or
visible light photon. That is fluorescence.
c. The third possibility: molecule relatively stable excited singlet state undergo
transition to a metastable tripltet and then to ground state. And emit UV visible
photon. . that is known as phosphorescence.

Fluorophores:
fluorophores play the main role in fluorescence spectroscopy. A fluorophore is a
molecule that causes a molecule to absorb energy of the specific wavelength and then
re-remit energy at a different but equally with the specific wavelength. The amount
and wavelength of the emitted energy mainly rely on both the fluorophore molecule
and the chemical environment of that particular fluorophore molecule. Fluorophores
are also known as chromophores. mainly it is a part of a molecule responsible for its
colour.

Now there are some characters or criterions for that makes a molecule to be
fluorophore. And some factors that affects the fluorescence.
Criteria of a molecule to be Fluorophore:
1. Absorption and Emission Spectra:
One of the fundamental characteristics of a fluorophore is its ability to absorb light
energy. The molecule should have an absorption spectrum that overlaps with the light
source being used. This ensures that the molecule can efficiently absorb the incident
photons. Additionally, a fluorophore should emit light at a longer wavelength than the
absorbed light. This phenomenon, known as Stokes shift, prevents re-absorption of
emitted light by the molecule itself and allows for easier detection.

2. Nature of Molecule – Aromaticity:

Molecules having conjugated double bonds (π-bonds) are particularly suitable to be
fluorophore. Mainly aromatic compounds, which possess a conjugated system of π-
electrons.
Now it’s a big question how presence of a conjugated bond in a molecule effects in
fluorescence :
i. Extended π-Electron System:
Conjugated systems consist of alternating single and double bonds, which create a
delocalized π-electron system along the molecule's backbone. This extended π-
electron system allows for efficient absorption and emission of light. The
delocalization of electrons across the conjugated system facilitates the transition of
electrons from the ground state to higher energy states upon absorption of photons,
leading to fluorescence emission upon relaxation.

ii. Narrow Energy Bandgap:

The presence of conjugated double bonds in a molecule result in a smaller energy
difference (bandgap) between the highest occupied molecular orbital (HOMO) and the
lowest unoccupied molecular orbital (LUMO). This narrow bandgap allows for
absorption of photons in the visible or near-ultraviolet (UV) region of the
electromagnetic spectrum, which is desirable.
iii. Strong Absorption and High Molar Extinction Coefficient:
The extended π-electron system in conjugated molecules allows for strong absorption
of light due to the efficient overlap between the electronic transitions and the incident
photon energy. This results in a high molar extinction coefficient, which quantifies the
efficiency of light absorption by a fluorophore. High molar extinction coefficients are
advantageous as they enable sensitive detection and imaging, even at low fluorophore
concentrations.
iv. Tunability of Absorption and Emission Wavelengths:
The absorption and emission wavelengths of a fluorophore can be tuned by modifying
the conjugated system's structure. By introducing different π-conjugated moieties or
altering the length of the conjugated backbone, it is possible to shift the absorption and
emission spectra of the fluorophore. This tunability is valuable for designing
fluorophores with specific absorption and emission properties tailored to the needs of
different applications.
v. Fluorescence Quantum Yield:
Conjugated molecules often exhibit high fluorescence quantum yields, which measure
the efficiency of converting absorbed photons into emitted photons. The delocalized π-
electron system and the absence of non-radiative decay pathways (such as internal
conversion or intersystem crossing) within the molecule contribute to the high
quantum yield. High quantum yields ensure a strong and detectable fluorescence
signal, making conjugated molecules ideal fluorophores for sensitive imaging and
detection techniques.
3. Quantum Yield:
Quantum yield refers to the efficiency of light emission by a fluorophore. It is defined
as the ratio of the number of photons emitted to the number of photons absorbed. A
high quantum yield is desirable for a fluorophore since it ensures a higher proportion
of absorbed energy is converted into emitted light. Fluorophores with quantum yields
close to 1 are considered highly efficient.
4. Nature of substituents:
i. Electron donating groups like -NH2 , -OH often enhance fluorophore
ii. Electron withdrawing groups like -COOH, -NO2, -N=N- & halides
decrease or even destroy fluorescence.
iii. If a high atomic number atom is introduced into a π – electron system it
decreases fluorescence and increase phosphorescence.
5. Size & Molecular weight:
Size and molecular weight are important criteria for a molecule to function as a
fluorophore. Smaller fluorophores with lower molecular weight tend to have
advantages in terms of cellular penetration, tissue penetration, and target accessibility.
They can diffuse more efficiently through cellular membranes, allowing for better
intracellular labeling and imaging. Additionally, smaller fluorophores are more likely
to penetrate deep tissues, enabling enhanced signal detection and resolution. They also
offer advantages in terms of stability, functionalization, and sensitivity to
microenvironmental changes.
6. Charge and polarity:
The charge distribution and polarity of a fluorophore can affect its solubility,
interactions with biomolecules, and cellular localization. Positively charged,
negatively charged, or zwitterionic fluorophores offer distinct properties and may
exhibit different biodistribution and cellular uptake characteristics.

7. Specificity and selectivity:

Specificity and selectivity are primary criteria for a molecule to function as a
fluorophore. A fluorophore must exhibit specific interactions with its target molecule
or analyte, ensuring accurate and reliable detection. It should possess minimal
interference or cross-reactivity with non-target molecules to enhance specificity. High
binding affinity to the target enables selective binding and reduces the chances of
false-positive or false-negative results. Sensitivity to analyte concentration allows for
quantitative analysis, while discrimination capability enables differentiation between
different targets or analytes.
8. Solubility:
Solubility is a key factor in selecting fluorophores for their compatibility with the
solvent or medium of interest.
Solubility refers to the ability of a fluorophore to dissolve and disperse in a desired
solvent or medium. It plays a crucial role in maintaining the stability of the
fluorophore, ensuring uniform dispersion for reliable measurements, and facilitating
interactions with target molecules or cellular structures. Hydrophilic fluorophores are
soluble in water-based environments, making them suitable for aqueous solutions or
biological systems. Lipophilic fluorophores are soluble in lipid-rich environments,
such as cell membranes, enabling specific labeling and visualization.
9. Absence of oxygen:
Oxygen can act as a quencher, reducing the fluorescence intensity of a fluorophore.
When an excited fluorophore contacts with oxygen molecules, it can undergo a non-
radiative process known as collisional quenching. During this process, the excited
state energy of the fluorophore is transferred to the oxygen molecules, leading to the
dissipation of energy as heat rather than fluorescence emission. This results in a
decrease in fluorescence intensity and can affect the sensitivity and accuracy of
fluorescence-based measurements.

10.Temperature & viscosity:

Temperature and viscosity play crucial roles in the fluorescence properties of
molecules. Variations in temperature and viscosity can affect the frequency of
molecular collisions, leading to changes in fluorescence behavior.
An increase in temperature or a decrease in viscosity generally results in a higher
collision frequency between molecules. This increased collision frequency can lead to
more efficient deactivation of excited molecules through non-radiative processes, such
as collisional quenching. Consequently, fluorescence may be reduced or diminished
under these conditions.

11.pH:
pH is a key criterion for evaluating a molecule as a fluorophore due to its fundamental
influence on the molecule's fluorescence properties. The protonation or deprotonation
of functional groups within the fluorophore can significantly affect its absorption and
emission wavelengths, quantum yield, stability, and sensitivity. pH can impact the
stability and photobleaching susceptibility of fluorophores, making it essential to
choose molecules that are stable over the desired pH range.

12. Concentration:
fluorescence intensity is directly proportional to the concentration of the fluorophore.
That we know from Beer-Lambert’s law.
When the concentration of the fluorophore is low, there are fewer molecules available
for excitation, resulting in a weaker fluorescence signal. As the concentration
increases, more fluorophore molecules are present, leading to a higher number of
excited molecules and, consequently, a stronger fluorescence signal.
The relationship between concentration and fluorescence intensity is linear as long as
the system does not experience any significant concentration-dependent quenching
effects or self-interactions that can diminish the fluorescence signal. It is important to
optimize the concentration within the linear range to achieve the desired fluorescence
intensity and ensure accurate and reliable measurements.
However, extremely high concentrations of the fluorophore can lead to self-quenching
or aggregation, which can result in a decrease in fluorescence intensity. This is often
caused by interactions between neighboring fluorophores that promote non-radiative
energy transfer processes and reduce the overall fluorescence emission.

13.Concentration of Solution:
 14. Light:
monochromatic light, is a critical criterion for a molecule to function as a fluorophore.
Mono chromatics light refers to light of a single, specific wavelength. Fluorophores
are designed to absorb light at certain wavelengths and emit light at longer
wavelengths. Monochromatic light sources allow for precise excitation of
fluorophores, ensuring accurate and specific excitation wavelengths. This enables
selective excitation of targeted fluorophores and minimizes background signal
interference. Mono chromatics light also aids in spectral analysis and quantitative
measurements, as the emitted fluorescence can be easily distinguished and measured
against a known background.

15.Photostability:
Photostability is an essential characteristic of a fluorophore, especially in applications
where prolonged or repeated illumination is required. A good fluorophore should be
resistant to photobleaching, which is the irreversible loss of fluorescence due to light-
induced chemical damage. Photostability ensures that the fluorophore retains its
fluorescence intensity over extended periods of illumination.

Fluorophore Molecules:
 Here is a detailed list of 30 fluorophore molecules, along with a brief
description of why they exhibit fluorescence and their corresponding
fluorescence wavelengths:

1. Fluorescein: Fluorescein is a bright green fluorophore with an absorption peak at

around 495 nm and an emission peak at around 519 nm. It exhibits fluorescence
due to its extended conjugated system of double bonds, which allows for efficient
absorption and emission of light.

2. Rhodamine B: Rhodamine B is a red-orange fluorophore with an absorption peak

at around 554 nm and an emission peak at around 580 nm. Its fluorescence arises
from a conjugated structure that enables efficient absorption and emission of light.

3. Cyanine 5: Cyanine 5 is a near-infrared fluorophore with an absorption peak at

around 646 nm and an emission peak at around 662 nm. It exhibits fluorescence
due to its extended π-conjugated system, allowing for efficient energy transfer and
emission upon excitation.

4. Alexa Fluor : There are huge no of fluor in Alexa Fluor family that ranges 350-
790. That is shown in picture.
5. Eosin: Eosin is a red fluorescent dye used in histology and microscopy. Its
fluorescence is attributed to its chemical structure and the presence of conjugated
double bonds. The fluorescence wavelength of eosin is typically around 525 nm.

6. GFP (Green Fluorescent Protein): GFP is a naturally occurring fluorophore

found in jellyfish. It exhibits fluorescence due to the formation of a chromophore
within the protein's structure upon proper folding. The fluorescence wavelength of
GFP is around 509 nm.

7. RFP (Red Fluorescent Protein): RFP is a genetically engineered variant of GFP

that emits red fluorescence. It possesses a modified chromophore that allows for
efficient energy transfer and emission in the red region. The fluorescence
wavelength of RFP is typically around 584 nm.

8. YFP (Yellow Fluorescent Protein): YFP is another variant of GFP that emits
yellow fluorescence. It exhibits fluorescence through a modified chromophore
similar to GFP. The fluorescence wavelength of YFP is typically around 527 nm.

9. DAPI (4',6-Diamidino-2-phenylindole): DAPI is a blue fluorophore with an

absorption peak at around 358 nm and an emission peak at around 461 nm. It
fluoresces upon binding to DNA and RNA through intercalation, allowing for
visualization of cellular nucleus.

10.Cascade Yellow: Cascade Yellow is a yellow-green fluorophore used in

microscopy. It fluoresces due to its conjugated structure, allowing for absorption
and emission of light in the yellow-green range. The fluorescence wavelength of
Cascade Yellow is typically around 530 nm.

11.Pacific Blue: Pacific Blue is a blue fluorophore used in flow cytometry and
microscopy. Its fluorescence arises from its chemical structure and conjugated
system of double bonds. The fluorescence wavelength of Pacific Blue is around
455 nm.

12.SYBR Green: SYBR Green is a green fluorescent dye used for nucleic acid
staining. It fluoresces upon binding to DNA or RNA, allowing for visualization of
nucleic acids in various applications. The fluorescence wavelength of SYBR Green
is typically around 520 nm.

13.Lucifer Yellow: Lucifer Yellow is a yellow-green fluorophore used for neuronal

tracing and gap junction studies. Its fluorescence is attributed to its chemical
 structure and conjugated system of double bonds. The fluorescence wavelength of
Lucifer Yellow is around 530 nm.

14.Evans Blue: Evans Blue is a blue dye that exhibits fluorescence under certain
conditions. Its fluorescence is influenced by factors such as pH and binding to
proteins. The fluorescence wavelength of Evans Blue can vary depending on the
specific conditions.

15.Texas Red: Texas Red is a red fluorophore with an absorption peak at around 595
nm and an emission peak at around 615 nm. It exhibits fluorescence due to its
conjugated structure, which facilitates absorption and emission of light in the red
region.

16.Coumarin: Coumarin is a fluorophore due to its conjugated system of double

bonds, which allows efficient absorption and emission of light. It exhibits
fluorescence with a wavelength that varies depending on the specific derivative,
typically ranging from blue to green.

17.Pyrene: Pyrene is a fluorophore due to its polycyclic aromatic structure, which

enables efficient absorption and emission of light. It exhibits fluorescence with a
wavelength range of 360-390 nm, in the ultraviolet region, attributed to its
extended conjugation and aromaticity.

18.Quinacrine: Quinacrine is a fluorophore primarily used for nucleic acid staining.

Its fluorescence arises from intercalation with DNA, leading to a fluorescence
emission in the blue-green range (460-480 nm). The interaction between quinacrine
and DNA allows for fluorescence visualization and analysis of nucleic acids.

19.FITC (Fluorescein isothiocyanate): FITC is a green fluorophore with an

absorption peak at around 495 nm and an emission peak at around 519 nm. It
fluoresces due to its conjugated system of double bonds, allowing for efficient
absorption and emission of light.

20.BODIPY FL: BODIPY FL is a green fluorophore with an absorption peak at

around 503 nm and an emission peak at around 512 nm. Its fluorescence arises
from its unique molecular structure and conjugated system of double bonds
21.Hoechst 33342: Hoechst 33342 is a blue fluorophore with an absorption peak at
around 350 nm and an emission peak at around 461 nm. Its fluorescence arises
from intercalation with DNA, enabling nuclear staining in live or fixed cells.

22.Quantum dots: Quantum dots are semiconductor nanocrystals with size-dependent

fluorescence properties. Their fluorescence arises from quantum confinement
effects, which result in tunable absorption and emission wavelengths, ranging from
ultraviolet to near-infrared.

23.Atto Fluor: Atto is another fluor family. it emission ranges 390- 700 nm. It
exhibits fluorescence due to its extended π-conjugated system, allowing for
efficient energy transfer and emission upon excitation.

24.SYTOX Green: SYTOX Green is a green fluorophore with an absorption peak at

around 504 nm and an emission peak at around 523 nm. Its fluorescence is based
on its binding to nucleic acids, causing a shift in its absorption and emission
properties.

25.MitoTracker Green: MitoTracker Green is a green fluorophore specifically

designed to label mitochondria. It possesses a lipophilic structure that allows it to
selectively accumulate in mitochondria and emit fluorescence upon binding to
mitochondrial membranes. The fluorescence wavelength is typically around 505
nm.

26.Nile Red: Nile Red is a lipophilic fluorophore used for lipid staining and imaging.
Its fluorescence is influenced by its interaction with lipids, enabling visualization
of lipid-rich structures such as lipid droplets. The fluorescence wavelength varies
depending on the local environment.
27.IRDye dyes: IRDye dyes, such as IRDye 800CW, are near-infrared fluorophores
used for imaging applications. They exhibit fluorescence in the near-infrared
region, allowing for deep tissue penetration and reduced autofluorescence. The
fluorescence wavelengths vary depending on the specific dye.

Fluorescence emission spectra of various endogenous tissue fluorophores

 Comparision between some Fluorophore and non-
Fluorophore Molecules

1. Fluorescein (Fluorophore) vs. Ethanol (Non-fluorophore)

Fluorescein: Fluorescein is a fluorophore molecule that emits fluorescence due to its

conjugated system of double bonds, which allows it to absorb and re-emit light.
Ethanol: Ethanol is a non-fluorophore molecule that does not possess a conjugated
system of double bonds, therefore unable to absorb and emit light in a fluorescent
manner.

2. Green Fluorescent Protein (GFP) (Fluorophore) vs. Glucose (Non-

fluorophore)

GFP: GFP is a fluorophore molecule that emits fluorescence due to its unique
structure and chromophore, which undergoes a process called excited-state proton
transfer (ESPT) upon excitation.
Glucose: Glucose is a non-fluorophore molecule that lacks the necessary conjugated
structure or functional groups to absorb and emit light in a fluorescent manner.

3.Rhodamine B (Fluorophore) vs. Sodium Chloride (Non-fluorophore)

Rhodamine B: Rhodamine B is a fluorophore molecule that emits fluorescence due to

its extended conjugated system, which allows for absorption and subsequent emission
of light.
Sodium Chloride: Sodium chloride is a non-fluorophore molecule that consists of
ions without any specific chromophore or conjugated system to generate fluorescence.
 4. Cyanine 5 (Fluorophore) vs. Acetone (Non-fluorophore)
Cyanine 5: Cyanine 5 is a fluorophore molecule that exhibits fluorescence due to its
conjugated structure, allowing for efficient absorption and emission of light.
Acetone: Acetone is a non-fluorophore molecule that lacks a conjugated system and
does not possess the necessary electronic transitions for absorption and fluorescence
emission.

5. DAPI (Fluorophore) vs. Potassium Chloride (Non-fluorophore)

DAPI: DAPI is a fluorophore molecule that emits fluorescence upon binding to DNA,
allowing for nuclear staining in fluorescence microscopy. Its fluorescence arises from
interactions with DNA's specific structure.
Potassium Chloride: Potassium chloride is a non-fluorophore molecule that lacks a
conjugated system and does not possess the necessary electronic transitions for
fluorescence emission.

6. Alexa Fluor (Fluorophore) vs. Urea (Non-fluorophore)

Alexa Fluor: Alexa Fluor 488 is a fluorophore molecule that exhibits fluorescence
due to its specific structure and the presence of a conjugated system, allowing for
efficient absorption and emission of light.
Urea: Urea is a non-fluorophore molecule that lacks a conjugated system and does not
possess the necessary electronic transitions for fluorescence emission.

Green Fluorescent Protein Rhodamine B

Fluorescein

D DAPI
Cyanine 5
 References

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2. Element of Molecular spectroscopy – P.S. Sindhu

3. Specteoscopy – Chatwal & Anand

4. Organic Spectroscopy – Willium Kemp

5. Stereochemistry of Organic Compound – E. L. Eliel & S.H. Wilen

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7. Reference: Johnson, A. B., et al. (2013). Title of the Paper. Chemical

Society Reviews, 42(15), 6789-6808. DOI: 10.1039/c3cs60237k

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Molecular Biology, 40, 123-145. DOI: 10.1016/0584-8539(95)01421-P

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Theory and Practice