3.1. Experimental Demonstration of Anti-Stokes Fluorescence Emission of RhB Dye at the Droplets Interface
Figure 2A shows emission spectra of RhB in both water and HFE-7500 fluorocarbon carrier oil (2% wt. of Krytox), with maximum intensities around 582 nm and 575 nm, respectively.
Figure 2B shows the fluorescence exponential decay of RhB in HFE oil vs. time, which was recorded on another dedicated setup described elsewhere [
22,
23]. The setup includes a 510 nm CW laser excitation and a notch filter cutting off wavelengths between 500 nm and 520 nm (approximately). The red curve (trace) in
Figure 2B corresponds to the decline of fluorescence emission for wavelengths
> 520 nm, whereas the blue trace corresponds to the decline of blue-shifted emission (
< 510 nm). One can deduce from these measurements a fluorescence lifetime of about 4 ns for RhB dye in HFE oil, which is of the same order as the value found for RhB in water (1.7 ns) and corresponds indeed to a fluorescence emission [
24]. The observed 7 nm shift between the two maxima should be attributed to the solvatochromism effect exhibited by many fluorescent dyes when dissolved in different solvents.
We present in
Figure 2C the fluorescence intensity vs. time of RhB droplets (0.5 mM), with a mean diameter and velocity of approximately 33
m and 6.3 cm/s, respectively. Droplets were carried by HFE-7500 fluorocarbon oil in a 30
m × 30
m microfluidic channel. We observe two types of photoluminescence signals. The first one (green) corresponds to the standard fluorescence signal of RhB, i.e., Stokes shifted emission, recorded for wavelengths above 540 nm, which are at higher wavelengths than the excitation’s. The second type of photoluminescence (red signal), detected in the range 500–520 nm, i.e., at smaller wavelengths than the excitation wavelength, corresponds to anti-Stokes emission of fluorescence of these droplets. Moreover, as shown in
Figure 2C, the observed anti-Stokes photoluminescence from droplets is mainly localized at the interface of the droplets, whereas the standard (Stokes) fluorescence signal does not show any significant difference between the droplet interface and its bulk. This result suggests that anti-Stokes photoluminescence should be enhanced by the surrounding molecules of RhB molecules at this interface, i.e., either fluorocarbon oil molecules or/and krytox surfactant molecules.
In order to better understand the origin of such an effect, we recorded photoluminescence signals from droplets of RhB in water carried by HFE-7500 fluorocarbon oil alone (without krytox surfactant) and from droplets made of RhB in benzyl alcohol (BA). Results are shown in
Figure 2D and
Figure 2E, respectively. We notice an absence of a blue-shifted signal (red) in both cases. This result shows first that the detected blue-shifted photoluminescence (BSPL) signal corresponds definitely to a genuine anti-Stokes laser-induced fluorescence and not to an experimental artifact, e.g., an external noise in the 500–520 nm channel. It also shows that this phenomenon is enabled by the presence of krytox surfactant or/and the presence of RhB molecules inside the fluorocarbon oil phase. It’s worth noting that RhB molecules are highly soluble in Benzyl alcohol, which hinders the diffusion of RhB molecules into fluorocarbon oil. The size and droplet velocities were 103
m and 1.9 cm/s, respectively, where other experimental conditions, such as surfactant concentration and excitation wavelength, were kept constant [
14].
3.2. Effect of the Surfactant Droplets on Anti-Stokes Fluorescence Emission
Figure 3 shows the intensity of ASF of large droplets (ø130
m) made of RhB in water and flowing in HFE-7500 fluorocarbon oil with krytox surfactant and in HFE-7500 fluorocarbon oil with KryJeffa surfactant. In contrast with krytox surfactant, KryJeffa surfactant is made of a fluorocarbon polymer block linked to a hydrophilic PEG block and does not carry any electrical charge like krytox does.
We notice for large droplets stabilized with krytox that the maxima of both green and red signals are significantly shifted towards the left side of the peaks, i.e., towards the rear part of the droplet interface, giving hence to the photoluminescence signals a sawtooth-like shape (
Figure 3A). Such an effect is usually observed in droplet microfluidics, and it is attributed to the well-known tip-streaming effect [
25,
26]. Moreover, when the used surfactant carries a negative electric charge, like krytox, the higher density of surfactant molecules at the droplet back interface (induced by the tip-streaming effect) leads, in turn, to an increase in the density of positively charged RhB molecules (at pH = 3) at this same area, driven by electrical interactions. This effect enables, hence, the probing of the distribution of RhB molecules inside the droplet and at its interface optically. We may notice also that the increase in photoluminescence signal at the rear part of the droplet interface is much more enhanced for anti-Stokes fluorescence (red signal) than for Stokes fluorescence (green signal). Interestingly, when using the non-ionic KryJeffa surfactant (
Figure 3B), though the tip-streaming effect should still be active, we do not observe the sawtooth-like shape on the fluorescence (green) signal of RhB anymore. The missing of any noticeable asymmetry between the fluorescence intensity at the rear and the front parts of the droplets does not mean that there should be no difference between them when using Kryjeffa surfactant. It means only that there is no difference between the density of rhodamine molecules at the front and at the back of the droplet. This effect may be explained by the absence of electrostatic interaction between rhodamine B molecules and Kryjeffa surfactant molecules [
14,
25,
26,
27].
More interesting is the absence of the anti-Stokes (red) photoluminescence when KryJeffa surfactant is used. Furthermore, one may notice the approximately rectangular shape of the fluorescence intensity green peaks in
Figure 3A, which indicates that droplets are more likely to have a rigid interface, which does not deform easily in the viscous flow. In contrast, without surfactant, as shown in
Figure 3B, the shape of droplets’ fluorescence intensity peaks shows an approximate bell shape. This indicates that droplets, in this case, are more likely to adopt a soft interface, which deforms easily in the viscous flow. This may be explained by the increase in fluorescence intensity as the curved interface of the droplet moves more and more across the (still) laser spot before reaching a maximum value when the overlap between the laser footprint and the droplet is at its maximum. It’s worth noting that KryJeffa surfactant is a much more efficient surfactant than krytox surfactant due to the presence of the PEG hydrophilic block, which may also hinder the diffusion of RhB molecules to the HFE-7500 fluorocarbon oil phase. Based on these observations, it appears that anti-Stokes fluorescence is enabled when RhB molecules are rather in contact or surrounded by fluorocarbon oil molecules.
Finally, we present in
Figure 4 the result of an experiment where we used another surfactant, namely TBA-Krytox, for which the ionic charge of krytox molecules was neutralized by the addition of a strong base, which consists of tri-benzyl ammonium (TBA). It’s worth noting that when using such a surfactant, fluorocarbon oil turns pink-colored, indicating that rhodamine molecules diffuse easily from droplets to the continuous oil phase in the presence of this surfactant. The fluorescence pattern from this experiment is quite different from the previous ones. In particular, one observes that during the passage of the fluorocarbon oil plugs across the laser beam, a relatively intense ASF emission is also detected. This result confirms that the origin of the ASF signal should be tightly linked to the surrounding RhB molecules by HFE-7500 fluorocarbon oil molecules.
3.3. Effect of the Incident Laser Beam Power
Inspired by the results we obtained with RhB droplets and TB-krytox surfactant, we performed an experiment where RhB was solubilized directly in fluorocarbon oil, using 2% (wt.) of krytox in a single-phase microfluidic flow (no droplets), while the power of the excitation laser beam was varied from 0.5 mW to 3.5 mW, approximately.
Results presented in
Figure 5 show the intensity of both Stokes and anti-Stokes fluorescence vs. the power of the pumping laser. One can notice that both Stokes and anti-Stokes fluorescence signals increase linearly as the power excitation intensity increases, with practically the same normalized rate (quantum yield), that is, 5.7 ×
/mW and 6.0 ×
/mW, respectively. Such a linear variation confirms a single photon absorption process which should be at the origin of the observed anti-Stokes and Stokes emission. Basically, such results may be easily understood according to simple theoretical considerations. Indeed, let us first consider a dye solution,
an elementary volume, where
represents its “thickness” along the x-direction parallel to an incident light beam, and
its cross section. Let also
be the intensity (per unit surface) of the incident light beam when it enters the elementary volume
. According to the Beer–Lambert law, the change in
through the thickness
may be expressed as
where
represents the extinction coefficient (or absorption coefficient) and
C the concentration of the dye solution (assumed to be homogeneous). Integration of the previous differential equation leads to the expression of the intensity of the light beam across
, which is
.
In principle, only a fraction of the light beam energy (absorbed locally by the dye) will be emitted as laser-induced fluorescence (LIF) in the related emission band. The ratio,
, of LIF intensity with respect to the absorbed light intensity is defined as the quantum yield (or efficiency) of the dye. Furthermore, the fluorescence quantum yield depends on many parameters such as the polarity of the solvent, dye concentration, temperature, or pH. For our study, RhB exhibits a high fluorescence quantum yield (
) at low concentrations (
to
M) [
28,
29,
30], whereas at higher concentrations (>10
M), the quantum yield decreases rapidly to less than 0.1 [
28,
29,
31]. Currently, it is widely accepted that at high concentrations in aqueous solutions, RhB molecules form dimers (due to strong electrostatic and dispersion interactions) with an equilibrium constant of about 2100 mol
(at 20
C) [
29]. Because dimers of RhB can only make a weak contribution to fluorescence (whereas they are capable of strong optical absorption), the fluorescence quantum yield of high-concentration aqueous RhB solutions can be strongly affected by dimerization. Within the frame of a single photon absorption process, the absorbed light intensity
by the dye molecules confined in the volume
should correspond to an emitted fluorescence intensity
, which may be expressed as
Combining Equations (
1) and (
2) leads to
Hence, the intensity (per unit volume) of the fluorescence emitted by a dye solution increases linearly with (i) the intensity
I of the light excitation, (ii) the concentration
C of the dye solution, and (iii) the quantum yield
. Furthermore, applying Equation (
3) and assuming that the absorption coefficient,
, to be the same for both photoluminescence processes, one can conclude that the quantum yield should be practically the same for both Stokes emission and anti-Stokes emission,
and
, respectively.
3.4. Dependence of Stokes and Anti-Stokes Fluorescence vs. Temperature in a Microfluidic Chamber
Rhodamine B dye has been extensively studied for its highly sensitive fluorescence emission to temperature. We investigated in this work the dependence of anti-Stokes fluorescence of RhB in fluorocarbon oil, in the 20
C to 50
C temperature range, for two excitation power values: 3.5 mW and 0.5 mW. Preliminary results are presented in
Figure 6 and
Figure 7, respectively. As one can notice, both Stokes and anti-Stokes fluorescence intensities decrease as temperature increases, but with a noticeable difference with the regular linear decrease observed for Stokes fluorescence intensity vs. temperature [
15,
16]. In particular, one may remark, for instance, that for 3.5 mW power illumination, we observe two temperature intervals: (i) below 25
C, for which the fluorescence intensity is more or less constant, and (ii) above 25
C, for which the fluorescence intensity decreases linearly vs. temperature with a temperature sensitivity of about
C for Stokes fluorescence and
C for anti-Stokes fluorescence, as shown in
Figure 6. One may also remark that the Stokes fluorescence linear decrease in the thermal sensitivity is approximately five times smaller than the reported literature data [
15,
16].
More interestingly, with a lower pumping laser power of 0.5 mW, we observe another transition for both Stokes and anti-Stokes of fluorescence intensities at a temperature of 36
C, as shown in
Figure 7. Below this temperature, both Stokes and anti-Stokes fluorescence intensities are practically constant, whereas above this temperature, fluorescence intensities start to decrease linearly vs. temperature with a thermal sensitivity
C.
3.5. Suggested Origin of the Anti-Stokes Photoluminescence in Oil Phase: Low Thermal Conductivity of Surrounding Fluorocarbon Oil
Let us first summarize the main features of the fluorescence of RhB in fluorocarbon HFE-7500 oil.
The intensity of anti-Stokes fluorescence may be relatively high and may represent more than 60% of the intensity of Stokes fluorescence, whereas in water solution, anti-Stokes fluorescence is very weak and represents less than 5% of the Stokes fluorescence;
The wavelength of the observed anti-Stock fluorescence is smaller than the excitation wavelength, which means that excess energy of the emitted photons by this process should be provided by the thermal bath;
When the temperature is increased, both Stokes and anti-Stokes Fluorescence intensities remain practically constant before a transition temperature appears, above which fluorescence starts to decrease linearly with a thermal sensitivity of about C (Stokes) or C (anti-Stokes);
The transition temperature value reveals that to depend on the power of the incident excitation light, for an excitation power of 3.5 mW, the transition temperature is found to be about 25 C, whereas for a smaller excitation power (0.5 mW), the transition temperature was found to be about 36 C.
It’s worth noting that the thermal conductivity of HFE-7500 fluorocarbon oil (at 25
C) is approximately one order smaller than the water’s thermal conductivity: 0.065 W/m·K for fluorocarbon oil and 0.614 W/m·K for water. We suggest that this large difference in thermal conductivities of the fluorocarbon carrier oil and of the water should be at the origin of the difference in fluorescence properties of RhB in the two solvents. In particular, the small value of thermal conductivity of HFE-7500 should enhance a local accumulation of heat and an increased temperature under the illuminated volume
of the solution sample by the highly focused excitation laser beam. When a RhB molecule absorbs an incident photon, the excited electronic state (
) may interact and exchange energy with the surrounding medium phonons. After a number of interactions, the excited electronic state will re-emit a photon, which can possess higher energy than that of the excitation photon. In this type of process, the additional energy of the emitted photon by anti-Stokes fluorescence is provided by the heat of the surrounding medium, while the excitation light, which irradiates the dye molecules, has a longer wavelength (lower energy) than that of the maximum emission, molecules located at a higher vibration energy level, also termed as ’hot band’, can absorb these photons and reach the excited state. Coupling between excited state electron and thermal energy can occur also. Finally, the excited state can decay back to the ground state (
) and generate a fluorescence emission whose wavelength is shorter than that of the excitation light. Since the anti-Stokes luminescence process occurs at higher vibrational levels of the singlet ground state, it may prefer molecules with rich vibrational energy levels and is strongly temperature dependent due to the fact that the populations of higher vibrational energy levels are determined by Boltzmann distribution. Additionally, populations of higher vibrational energy levels are very limited, so effective anti-Stokes luminescence is usually observed in dye molecules with high quantum yield and molar extinction coefficient, such as Rhodamine B. We believe that hot-band absorption materials are very promising optical probes for diagnosis and detection in vivo [
7]. Nevertheless, one important problem to be addressed is the relatively low luminescence efficiency of hot-band absorption materials. Droplet microfluidics can be used to address such a problem, as light and heat may be both confined inside droplets by total internal reflection (TIR) on the droplet interface. Hence, we think there is plenty of room for improvement and advancement in this domain.
As mentioned in the introduction section, the temperature sensitivity of fluorescence emission of Rhodamine B may be explained by the existence of a photoinduced reaction leading to the formation of a highly polar excited conformer called TICT [
17,
18,
19]. In the case of rhodamine B, it is the xanthene group that rotates during isomerisation process [
17,
18], and since such a rearrangement is thermally activated, the rate of formation of the TICT state increases with temperature.
Finally, based on our results related to the observed transition temperatures vs. the power of pumping laser excitation, we think that though still preliminary, these results may give a new insight onto the coupling between hot-band absorption, anti-Stokes emission, and heat transport under different experimental conditions, such as flow velocity, viscosity, etc. We suggest that the temperature transitions observed at 25
C and 36
C should be related to the well-known thermal cooling effect [
9,
10], which is induced by anti-Stokes emission and is at the basis of optical refrigeration [
10]. In fact, when a substance absorbs a photon and emits another one of greater energy, this leads to the cooling down of the temperature of this substance by the pumping of its thermal energy. Therefore, when the temperature of the bath is increased (by actuation on the bath heating system), the final change in fluorescence intensity, i.e., increase or decrease, of dye molecules would depend on the respective rates of both (i) the decrease in local temperature vs. time due to anti-Stokes emission and (ii) the rate of increase in temperature driven by the heat of the bath and the heat brought by the pumping laser. Therefore, when using a high power pumping laser (3.5 mW), one may reach a transition temperature of around 25
C, which is smaller than the transition temperature observed when using a smaller power pumping laser (0.5 mW). This transition temperature should actually correspond to the temperature at which anti-Stokes optical cooling cannot compensate anymore for the increase in temperature due to the heat brought by both the bath heating system and the pumping laser.