1
Quantitative Measurement of Fluorescence Brightness of Single
2
Molecules
3
Yuxi Tian, Johannes Halle, Michal Wojdyr, Dibakar Sahoo and Ivan G. Scheblykin*
4
Chemical Physics, Lund University, Box 124, SE-22100, Lund, Sweden *
5 6
Email:
[email protected]
Abstract
7
Single-molecule fluorescence spectroscopy and imaging measure many characteristics of the
8
fluorescence from individual molecules like relative intensity, polarization, lifetime and spectrum.
9
However, such an important and fundamental parameter as absolute fluorescence intensity (or in
10
other words fluorescence brightness), which is proportional to the absorption cross section and
11
fluorescence quantum yield, has not yet been sufficiently exploited in the field. One reason for that
12
is the difficulty of absolute fluorescence brightness measurements. In the present work a detailed
13
description of fluorescence brightness measurements of single molecules is given. We discuss
14
several important factors like the power density and polarization of excitation light, the substrates
15
and the local environment. It is shown that the fluorescence brightness of a single molecule indeed
16
can be measured with sufficient accuracy and used as a powerful parameter for characterization of
17
materials at single molecule/particle level. The brightness of a single object can give similar
18
information as the fluorescence quantum yield that is crucial for understanding the photophysical
19
properties for individual multi-chromophoric systems in inhomogeneous environments.
1
1
1
Introduction
2
Fluorescence, emission due to transition from the first singlet excited state to the ground state,
3
was firstly observed in 1565[1] and named by George Gabriel Stokes in 1852[2]. Fluorescence
4
measurements have become very popular in science, technology and medicine in the past decades
5
due to their extraordinary sensitivity. Since the development of single molecule spectroscopy
6
(SMS)[3,4], fluorescence plays the most important role in this field. Investigating the fluorescence
7
properties of individual molecules gives lots of new opportunities in physics[3,5], chemistry[6,7],
8
biology[8–11] and material sciences[12–14] which are also recently reviewed[15].
9 10
11
To characterize the fluorescent ability of a sample, fluorescence quantum yield (QY), is defined as:
number of emitted photons number of absorbed photons
12
In liquid solutions, QY is a widely used standard parameter that can be measured by either
13
absolute[16] or relative techniques[17]. However, QY measurement at the single-molecule level is
14
not an easy task, even though it is very important for unraveling excitation quenching mechanisms
15
in non-homogeneous environment especially for multi-chromophoric nano systems.
16
The main problem is the measurement of single-molecule absorption. Due to the small
17
absorption cross section, it is very difficult to directly measure the absorption of a single molecule.
18
Recently, three groups have approached absorption detection of single molecules in different ways
19
which makes it possible to measure the fluorescence QY of single objects directly[18–20]. There
20
have been also attempts to measure absolute QY at the single molecule level by other techniques
21
without direct absorption measurement e.g. using sensitivity of the radiative transition to the local
2
1
electromagnetic field mode density[21–23]. However, all these methods are still very difficult to
2
realize in non-specialized laboratories.
3
Instead of fluorescence QY, one can measure the fluorescence excitation cross section that is a
4
product of the absorption cross section and the fluorescence QY. The advantage is that it avoids the
5
absorption cross section measurement. The fluorescence excitation cross section has been used to
6
describe the fluorescence ability of bright objects such as organic/inorganic nanoparticles[24]. The
7
same parameter although expressed in different units is called single-molecule brightness (B) and
8
has been exploited for unraveling complicated quenching processes in conjugated polymers[25–27]
9
and for quantitative characterization the number of dye molecules [28] and the energy migration in
10
linear dye aggregates[29].
11
Single-molecule brightness measurements rely on measuring the absolute fluorescence
12
intensity of individual molecules. The fluorescence intensity strongly depends on the orientation
13
of the molecule relative to the electric field of the excitation light and the optical axis of the
14
objective lens. Other effects also influence the fluorescence intensity, for example the microscope
15
collection efficiency, local environment, excitation power density and so on. Even though absolute
16
intensity measurements are difficult and subject to errors, using carefully controlled sample
17
preparation and calibrated instruments one can get reproducible results. In this contribution we
18
will discuss practical questions of the brightness measurement of single molecules.
19
2
20
2.1 Sample preparation
Experimental
21
For correct measurements of brightness, it is especially important to decrease contributions
22
from luminescent impurities as much as possible. The substrates (glass, 22 × 22 × 0.15 mm, 3
1
Menzel-Glasser, Germany, or quartz, 22 × 22 × 0.17 mm, Fujian, China) were firstly treated by 1%
2
Hellmanex III (Hellma) solution in ultrasonic bath. Then very thorough rising in Mill-Q water was
3
performed to remove the residual water-soluble substances. UV irradiation (260 nm) was then
4
applied to oxidize the remaining emissive impurities on the cover slips. All the other materials
5
(vials, pipette tips, etc) used during the preparation were cleaned by chloroform (Sigma-Aldrich,
6
≥99%). For the single molecule measurement, toluene (Sigma-Aldrich, 99.8%) solutions with ~10-10
7 8
M
of
N,N’-Bis(2,6-dimethylphen-yl)-perylene-3,4,9,10-tetra-
9
Sigma-Aldrich) and 9 mg/ml of Poly(methyl methacrylate (PMMA, Sigma-Aldrich, cleaned by
10
precipitation in hexane) were prepared. This solution was then spin-casted onto a cleaned substrate
11
leading to 40-50 nm thin film (measured by profilometry).
carboxylicdiimide
(DXP,
12
The concentration of the sample should be very well controlled. Too high concentration
13
certainly causes overlap between molecules (within the diffraction-limited spot) making the B to
14
be an overestimation. However, too low concentration increases the contribution from the
15
luminescent impurities. The correlation of the number of spots per image and the concentration
16
should be investigated in order to find suitable concentration as described e.g. in ref.[30]
17
For ensemble measurements (lifetime and excitation power density dependence), the
18
concentration of the dye in the solution for spin casting was increased to ~10-8 M.
19
2.2 Experimental setup
20
A home-built wide-field epifluorescence microscope based on Olympus X71 with Ar-ion laser
21
as the excitation source was used as described previously[14]. The fluorescence of the sample was
22
collected by an oil immersion objective lens (Olympus UPlanFLN 60x, numerical aperture (NA) 4
1
1.25) and imaged by a CCD camera (ProEM:512B, Princeton Instruments). The laser is linearly
2
polarized and used directly for all the measurements except for the polarization effect experiment.
3
In this experiment elliptically polarized (1.6:1) excitation light was obtained by putting a λ/4 plate
4
in the beam path. For lifetime measurement, a pulsed diode laser (Picoquant 485 nm, 40 MHz) was
5
used as excitation source for time-correlated single-photon counting (TCSPC) measurements
6
carried out with the same microscope as for the single-molecule detection[14] and detected by a
7
fast avalanche photodiode (APD, Micro Photon Devices) together with PicoHarp 300 (PicoQuant
8
GmbH) photon counting electronics. To measure the instrument response function, we used
9
Raman scattering of the excitation laser from water. The instrument response function (IRF) had
10
the width of ~50 ps. For the excitation power density dependence experiment, to minimize the
11
possible irreversible photo bleaching, the fluorescence brightness of the samples was measured
12
upon increasing of the excitation power. The sample was not moved during the measurement. The
13
reverse measurement upon decreasing of the excitation power was also performed at the same spot
14
to check the contribution from the irreversible photo bleaching. For all the measurements, the
15
samples were kept in nitrogen atmosphere to prevent photo-oxidation.
16
2.3 Single-molecule Brightness
17
Single-molecule brightness (B) is a measure of the ability of a single molecule (or a single
18
object in general) to emit light via photo-excitation. Obviously it is proportional to the absorption
19
cross section and fluorescence quantum yield or in other words it is proportional to the
20
fluorescence excitation cross section of the molecule. Since this term is not yet widely used in the
21
field of SMS, we would very much like to avoid any confusion by spending some time to explain
22
exactly meaning of “B”. Following our previous publications[25,29], we define “B” as the ratio 5
1
between the number of experimentally detected photons per second (F) and the excitation power
2
density (Iex in W∙cm-2).
B
3
F Ck I ex I ex
(1)
4
Where C is the CCD counts and k is the conversion factor between the counts and detected photons,
5
CCD specific.
6
The advantage of this definition is that it is straightforward and contains directly measured
7
parameters only. The disadvantage, however, is that B is setup specific, which means that exactly
8
the same molecule under exactly the same geometrical and chemical conditions may possess
9
different B when measured at different microscopes. Indeed, besides the photophysical
10
parameters (absorption cross section σ (in cm2) at the excitation photo energy (hv) and
11
fluorescence quantum yield Φ) of the molecule, B depends on the light detection efficiency of
12
the setup ηdet :
13
B
I hv F F0 det ex det det I ex I ex I ex hv
(2)
14
Where F0 is the number of the emitted photons per second; ηdet = ηcollTηCCD, ηcoll - light collection
15
efficiency of the objective lens; T - transmission of the microscope and ηCCD - quantum efficiency
16
of the CCD camera.
17
However, brightness experiments carried out in different laboratories still can be compared if
18
B of a standard dye has been also measured and used as a reference. For our setup, the collection
19
efficiency of the objective lens is measured to be 0.57 (as described in SI section 1), the
20
transmission of the whole setup is 0.66 (as also described in SI section 2) and the quantum
6
1
efficiency of the CCD camera is 0.95. So, the total detection efficiency is ηdet = 0.36. With these
2
values, we can calculate the theoretical value for B. Assuming that the orientation of the DXP is
3
random in the PMMA matrix and the absorption cross section was not affected, then the average
4
absorption cross section of DXP in solution (8.19×10-17 cm2 at 458 nm[31]) can be used. The
5
average B value of DXP was calculated using equation (2) to be 68 cm2W-1s-1.
6
2.4 Brightness measurement and data analysis
7
For brightness measurements, all the DXP samples were measured with the wide-field
8
epifluorescence microscope and the fluorescence image were obtained by the CCD camera as
9
shown in Figure 1A. A blank sample was always measured under the same condition used for the
10
estimation of the impurities as shown in Figure 1B. The fluorescence images were analyzed by a
11
home-built software which automatically indentified the molecules, calculated the fluorescence
12
intensity and the excitation power density for each individual molecule. Not only the intensity
13
threshold but also the intensity profile were used as criteria for the identification of molecules.
14
When a molecule was indentified, the fluorescence intensity of a selected molecule was obtained
15
by integrating the whole point spread function (PSF) (signal inside the solid circle in Figure 1A)
16
and subtraction of the background obtained from the area between the solid and dashed circles
17
shown in Figure 1A. The B value was then calculated using eq. (1) for each molecule.
18 19
Figure 1
Fluorescence images of a PMMA matrix layer with DXP molecule imbedded (A) 7
1 2 3 4
and a blank sample of PMMA matrix layer without DXP molecule (B). The fluorescence spots at the image B belong to impurities. The averaged signal in the area between the solid circle and the dashed circle was used as the background for the selected molecule. Average excitation power density of ~ 1 W/cm2 and an exposure time of 10 s were used for the measurements.
5 6
3
7
3.1 Brightness of DXP molecules
8 9 10 11
Results and Discussion
Figure 2 Brightness histograms for DXP molecules (gray) and the impurities (black) in PMMA matrix on glass substrates. Average excitation power density of ~ 1 W/cm2 and an exposure time of 10 s were used for the measurements.
12 13
The brightness histogram obtained from thousands of dye molecules dispersed in a PMMA
14
film on glass cover slips is shown by gray color in Figure 2. The average B value is 74 cm2W-1s-1.
15
Though a great attention was paid to avoid luminescent impurities, still bright spots were observed
16
in the blank sample. The average B of the impurities on the blank sample was 63 cm2W-1s-1 and the
17
histogram is shown in black color in Figure 2. Note that in our case the average B of the impurities
18
is quite similar to that of DXP. However, the concentration of impurities is much lower, which is
19
obviously the necessary condition for a single-molecule experiment. Assuming that the impurities´
20
concentration is the same in the blank sample and the sample with the dye, the real B distribution
8
1
for the dye can be obtained by subtracting the histograms of the dye and blank samples obtained
2
from the same number of images (See Figure S2 in SI). The real averaged B for the dye molecules
3
can be thus calculated:
4
Bdye
N sample Bsample Nimpurity Bimpurity
(3)
N sample Nimpurity
5
In which , , are the real average B value of the dye, experimental B value
6
for the sample (DXP in PMMA) and experimental B value for impurities (PMMA matrix only),
7
respectively; Nsample and Nimpurity are the number of spots per image detected of the sample and the
8
blank.
9
The average B value of DXP on glass substrate was obtained to be 78 cm2W-1s-1, which is in a good
10
agreement with the calculated value. All the B values used hereafter are corrected by subtraction of
11
the luminescent impurities contribution.
12
3.2 Effect of the power density
13
It is well known that optical spectroscopy often uses too high excitation power affecting the
14
excited state processes and inducing photochemistry in the studied materials[32,33]. Transition
15
saturation and chemical photodegradation can influence the fluorescence intensity, which
16
especially concerns SMS at room temperature where high excitation power is necessary to see the
17
molecules at all. For multichromophoric systems such as conjugated polymer, singlet-singlet and
18
singlet-triplet annihilation has also to be taken into account[34–38]. Therefore, it is important to
19
consider the effect of the excitation power density on B.
20
It is very difficult to measure B of a single dye molecule at very low or very high excitation
21
power due to the light detection limitations and photo-stability of the dye. Therefore we measured
9
1
relative ensemble averaged brightness of DXP with ~100 times higher concentration in the host
2
PMMA matrix film. Even at such concentration the dye molecules are still well isolated from each
3
other without any interactions e.g. Förster resonance energy transfer etc. So the molecules behave
4
the same as they do in the single molecule measurements and the fluorescence intensity of such a
5
film is the sum of the intensities from many isolated molecules. The ensemble averaged
6
“brightness” (Bf) of such a film was calculated as the average fluorescence intensity (photons/s)
7
coming from the excited area of the sample divided by the excitation power. Bf is thus proportional
8
to the averaged B of individual dye molecules. The power dependence of Bf thus represents the
9
power dependence of an average individual molecule. The solid squares in Figure 3 show that the
10
Bf of DXP decreases with the increasing of the excitation power density above 0.5 W·cm-2.
11
The excitation power can affect the Bf value in two different ways. Firstly, it is known that
12
light irradiation can generate fluorescence quenchers especially for conjugated polymers[12,13].
13
These quenchers decrease the Bf value due to static or/and dynamical fluorescence quenching.
14
Secondly, the molecule can be excited to a triplet state or other long-lived dark states. Both of the
15
described effects can reveal themselves as fluorescence blinking behavior of the single molecules
16
at different timescales [14,36,39]. Increasing the power density increases the production of these
17
dark states and thus decreases the fluorescence intensity. Since the power dependence reflects properties of the particular system, it is totally different
18 19
for
different
materials.
For
example,
20
poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene] (MEH-PPV), the deviation of Bf
21
starts already at excitation power density as low as 0.03 W·cm-2 (solid circles in Figure 3) and
22
decreases further with increasing the power density. The contribution of the irreversible photo 10
for
the
conjugated
polymer
1
bleaching was negligible as shown in Figure S3 in SI. Thus, one needs to be aware that high
2
excitation power can substantially decrease the experimental B values. In order to avoid the power
3
effect, the brightness measurement should be performed under low enough excitation power and as
4
short as possible exposure time. A detailed study on the excitation power dependence of
5
fluorescence quantum yield of isolated conjugated polymer chains will be presented in a
6
forthcoming publication.
7 8 9 10 11
Figure 3 Excitation power density dependence of the ensemble-averaged fluorescence ability of DXP and MEH-PPV imbedded in PMMA (see text for details). At each power density the lowest possible exposure time (for the sake of minimizing photobleaching and still having acceptable single to noise ration) were used.
12
3.3 Effect of the excitation light polarization
13
As already mentioned above, the B of a single molecule strongly depends on the orientation of
14
the absorption transition dipole moment relative to the electric field of the excitation light, and on
15
the orientation of the emission transition dipole moment relative to the microscope optical axis.
16
Thus, the excitation light polarization seems to be very important for correct brightness
17
measurements.
18
As shown in Figure 4, in practice the excitation light polarization indeed influences the shape
19
of the B distributions and the averaged B value for DXP. When excited by the laser with the same 11
1
power density, the average B value for linearly polarized excitation (=72) is higher than that
2
for almost circularly polarized excitation (=58). The distribution is also broader for the
3
linearly polarized excitation. This can be qualitatively explained by taking into account the cosine
4
square dependence of the excitation probability on the angle between the transition dipole moment
5
and the excitation light electric field. With linearly polarized excitation, molecules whose dipole is
6
parallel to the electric field of the excitation light will be strongly excited giving higher B values,
7
while molecules with dipoles perpendicular to the exciting electric field are not excited. Contrarily,
8
for circularly polarized excitation, all the molecules will be excited equally resulting in a narrower
9
B distribution with main peak around 50 (measured with elliptically polarized excitation 1.6:1).
10
Here we only consider the orientation of the molecules in the sample plane (or projection on the
11
sample plane). It is also worth to note that more molecules are detected using circularly polarized
12
excitation than using linearly polarized excitation.
13 12
1 2 3
Figure 4 Brightness histogram of DXP molecules measured on glass substrates using circularly (A) and linearly (B) polarized excitation light. Average excitation power density of ~ 1 W/cm2 and an exposure time of 10 s were used for the measurements.
4
We tried to simulate approximately the brightness histogram for linearly and circularly
5
polarized excitation (Figure S4 in SI). The simulation was performed by assuming that the
6
molecules are free dipoles randomly orientated in 3D in the matrix layer without considering any
7
interface effect. The excitation efficiency and collection efficiency for each molecule were
8
calculated based on our experimental conditions (total collection efficiency of 0.36 and NA=1.25).
9
Even though the simulation does not perfectly fit with the experimental data, it shows qualitatively
10
the same trend as the experimental results. Obviously more factors like the interface effect, the
11
distance from the dye to the interface and the orientation distribution of the dye molecules etc.
12
have to be considered[40,41]. In addition, orientation distribution of the dye molecules in the
13
matrix film can be not random [Vacha : Phys. Chem. Chem. Phys., 2011,13, 6970–6976].
14 15
3.4 Effect of the substrates
16
Glass cover slips usually give quite high luminescence background (originated from defect
17
luminescence and luminescece of impurities in the glass) when measured by fluorescence
18
microscopy, especially when low NA objective lens and short excitation wavelength are used. For
19
example, it is practically impossible to see single dye molecules on a glass substrate when a dry
20
objective lens with NA=0.6 is used. With high NA oil objective lens, the glass luminescence
21
contribution is substantially smaller.
22
The background luminescence can be avoided by using fused silica (quartz) substrates instead.
23
However, experimentally we found that the average B values are very different for the same dye on
24
glass and quartz substrates. The average B value of DXP in PMMA on quartz was found to be 30 13
1
which is 2.6 times lower than that on glass cover slips (as shown in Figure 5). Note that the
2
samples for both experiments were prepared in exactly the same way and no differences in other
3
photophysical properties like fluorescence spectra and lifetime were detected. It is clear that the
4
decrease of is not related to the photophysical properties of the molecules themselves. So the
5
difference can only come from the light collection efficiency and data analysis.
6
For samples prepared on glass substrates, all layers (PMMA matrix, substrate and the
7
immersion oil) have very similar refractive indexes (n=1.52) giving the maximum collection
8
efficiency with very small optical aberrations. However, the quartz (fused silica) substrate has a
9
lower refractive index (n=1.46)[42]. This causes the refractive index mismatch between the
10
PMMA layer, the substrate and the immersion oil which causes spherical aberration of the image
11
(as shown in Figure 5 insert) and low collection efficiency of the fluorescence. Both factors
12
decrease the experimental B value on quartz substrates.
13
14 15 16 17 18 19
Figure 5 Brightness histograms for DXP molecules (gray) and the impurities (black) in PMMA matrix on quartz substrates. Average excitation power density of ~ 1 W/cm2 and an exposure time of 10 s were used for the measurements. Insert is a cartoon of the fluorescence intensity profile of a single dye molecule on glass (A) and quartz (B) substrates, respectively. Due to the signal-to-noise ration, the low intensity in the meshed area was taken as the background in 14
1
the data analysis that decreased the B values.
2 3
3.5 Effect of the local environment
4
It is reported that fluorescent molecules can be used as probes at the nanometer scale due to
5
their sensitivity to the environment[43–46]. In the present work we studied the dye molecules
6
embedded in the PMMA matrix. The matrix isolates the molecule from the substrate surface,
7
provides relatively uniform and rigid environments and decreases the influence of atmospheric
8
oxygen. Usually, fluorophores are much less stable without isolation by the matrix.
9
Experimentally it is almost impossible to measure the correct B of individual molecules on bare
10
surfaces. One of the most important reasons is quenching effect from the substrates’ surface as
11
reported[47,48].
12
To illustrate this, we compared the fluorescence lifetimes of the relative ensemble samples
13
spin-casted on the substrates with and without matrix. Figure 6 shows the fluorescence dynamic
14
curves of DXP in different environments (solution, on bare glass surface and embedded in PMMA).
15
Significant dynamic quenching effect was observed when the sample was directly prepared on the
16
glass surface. Similar quenching effect from the surface was also observed for other dyes and
17
polymers which could due to the quenchers on the glass surface[47,48]. By fluorescence lifetime
18
measurements, we can only get information about the dynamic quenching. However, static
19
quenching could be also present.[25,26] In any case, the PMMA matrix provides very good
20
isolation between the surface and the molecules making the lifetime similar to that in solution.
15
1 2
Figure 6. Fluorescence decays of DXP in solution, PMMA film and on bare glass substrate.
3 4
Conclusion
5
Measurement of the single-molecule brightness using wide-field fluorescence microscope
6
was described in details. Good agreement between experimental brightness of single DXP dyes
7
and the theoretical estimations indicates that the average brightness value is a reliable parameter
8
and can be used to assess the fluorescence ability of single molecules and other objects.
9
Polarization and power of the excitation light have to be taken care of to get correct brightness
10
values. The refractive-index mismatch between the PMMA, substrate and immersion oil is the
11
reason for the reduction of the collection efficiency and spherical aberration when quartz
12
substrates are used for single-molecule experiments. Great care should be taken to avoid
13
quenching from the surface which can substantially reduce the brightness values. Using
14
exceptionally photostable perylenediimide dyes (e.g. DXP) as standards makes possible
15
comparison of brightness measurements carried out in different laboratories. Defocusing imaging
16
technique[Hofkens DOI: 10.1002/adma.200801873] should allow choosing the dye molecules
17
with in-plane orientation in the matrix film. This, in combination with circularly polarized
16
1
excitation, will remove uncertainty due to unknown 3D orientation of the molecules and improve
2
the standard even further. Quantitative measurement of brightness gives researchers a direct access
3
to investigating the formation of dark states at single molecule/particle level in complicated
4
heterogeneous materials[25–27] as well as quantitatively characterization of other systems[28,29].
5 6
Acknowledgements This study was financially supported by The Swedish Research Council, the Knut & Alice
7 8
Wallenberg Foundation, the Crafoord Foundation, and the Carl Trygger Foundation. J.H. and M.W.
9
thank Erasmus programme for the internship founding. D.S. thanks the Wenner-Gren foundation
10
for a postdoctoral scholarship.
11
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