The detailed mechanisms associated with the influence of scattering and absorption properties on the fluorescence intensity sampled by a single optical fiber have recently been elucidated based on Monte Carlo simulated data. Here we develop an experimental single fiber fluorescence (SFF) spectroscopy setup and validate the Monte Carlo data and semi-empirical model equation that describes the SFF signal as a function of scattering. We present a calibration procedure that corrects the SFF signal for all system-related, wavelength dependent transmission efficiencies to yield an absolute value of intrinsic fluorescence. The validity of the Monte Carlo data and semi-empirical model is demonstrated using a set of fluorescent phantoms with varying concentrations of Intralipid to vary the scattering properties, yielding a wide range of reduced scattering coefficients (μ′s = 0–7 mm−1). We also introduce a small modification to the model to account for the case of μ′s = 0 mm−1 and show its relation to the experimental, simulated and theoretically calculated value of SFF intensity in the absence of scattering. Finally, we show that our method is also accurate in the presence of absorbers by performing measurements on phantoms containing red blood cells and correcting for their absorption properties.
© 2014 Optical Society of America
Fluorescence detection and quantification is important for many biomedical applications. For example, the detection of endogenous fluorophores such as NADH and FAD can indicate pathological alterations in tissues [1–3], while the quantification of exogenous compounds such as photosensitizers or chemotherapeutic agents that are fluorescent allows monitoring of drug distribution during photodynamic therapy (PDT) and chemotherapy [4–6].
In biomedical applications, the dependence of the detected fluorescence signal on the absorption and scattering properties of the tissue complicates its quantification. In order to quantitatively analyze fluorescence in tissue, it is important to obtain an intrinsic fluorescence signal that is independent of these optical property effects. Previously developed methods to extract intrinsic fluorescence spectra involve the acquisition of a paired measurement of fluorescence and white-light reflectance, where the latter is used to inform a correction of the influence of optical properties on fluorescence. This general approach has been extensively investigated for multi-fiber fluorescence probes, with separate source(s) and detectors [7–15]. These probes collect multiply scattered, or diffuse, light and sample volumes of tissue on the orders of several mm3. An alternative approach for fluorescence measurements is to use small fiber optic probes that utilize a single optical fiber to both deliver excitation light and collect emitted fluorescence [16–20]; such a measurement results in a localized sampling volume, with the majority of the collected signal originating very close to the probe face .
Our group has recently published on the detailed mechanisms associated with the influence of scattering and absorption properties on the fluorescence intensity sampled by a single optical fiber [21, 22]. We showed, based on an extensive Monte Carlo simulated data set, that for a single fiber i) the measured fluorescence is proportional to the fiber diameter, and ii) the fluorescence as a function of scattering shows a bi-phasic effect where in the first phase fluorescence decreases with increased scattering and in the second phase fluorescence increases with increased scattering. In Fig. 1 Monte Carlo simulated data of single fiber fluorescence (SFF) intensity divided by the effective fiber diameter ( ) is plotted as a function of the product of the reduced scattering coefficient and the fiber diameter (μ′sdfib). The observed bi-phasic effect was demonstrated to be the result of the combined effects of effective sampling volume, effective excitation fluence within the effective sampling volume and the collection probability of emitted photons from the effective sampling volume . Furthermore, we showed that these combined effects can be mathematically described by an empirical model equation, cf. Eq. (5).
The aim of the current study is to develop an experimental SFF spectroscopy setup and validate the Monte Carlo data and semi-empirical model equation. To this end we will present a calibration procedure that corrects the SFF signal for all system-related, wavelength dependent transmission efficiencies to yield an absolute value of intrinsic fluorescence. We demonstrate the validity of the Monte Carlo data and semi-empirical model using a set of fluorescent phantoms with varying concentrations of Intralipid to vary the scattering properties, yielding a wide range of reduced scattering coefficients (μ′s = 0–7 mm−1). We also introduce a small modification to the model to account for the case of μ′s = 0 mm−1 and show its relation to the experimental, simulated and analytically calculated value of SFF intensity in the absence of scattering. Finally, we demonstrate the accuracy of the method in the presence of absorption by performing measurements on phantoms which are fluorescing, scattering and absorbing.
2. Materials and methods
2.1.1. The SFF model
The intrinsic fluorescence (Fi(λm) [nm−1 mm−1]) is defined as the absolute fluorescence spectrum, independent of the optical properties of the medium, and is given by the product of the absorption coefficient of the fluorophore at the excitation wavelength ( ) and the fluorescence quantum yield of the fluorophore (Qf (λm)):22]:
2.1.2. The case of μ′s =0, μa=0
In the case of μ′s =0 mm−1 and μa=0 mm−1, fluorescence is unaltered by scattering and absorption and the total fluorescence intensity collected by the fiber ( [# photons]) depends on the fiber collection efficiency η, Qf, and the excitation power (Plaser [# photons]), and is given by:23, 24]: Fig. 2), i.e. , where b is the apparent point of origin of excitation light within the fiber: Eqs. (7) and (8) in Eq. (9), assuming the attenuation of the excitation light due to absorption by the fluorophores to be negligible (i.e. assuming is very small), and rearranging leads to Eq. (5) can be adapted to yield a value of 0.041 at μ′s =0 mm−1: Eq. (10) to (11) we have reintroduced the νn term to match the Monte Carlo results of our previous paper .
2.1.3. SFF calibration
The SFF-model was developed based on Monte Carlo simulations where the single fiber fluorescence signal was defined as the ratio of collected fluorescence photons (TMCP) and excitation photons launched (TXPL).
In the second calibration step, a light source with a narrow bandwidth (e.g. laser or LED) is used. The wavelength of the light source is only of secondary importance; it should preferably be in a range of high system transmission efficiency to provide a good signal-to-noise ratio. In our case we have used an LED of 780 nm which is coupled into the system as shown in Fig. 3. Light from the LED is guided through the single fiber and exits the probe at its distal end. The wavelength-integrated power out of the fiber is measured with an integrating sphere ( [mW]) and a reflectance spectrum is recorded by submerging the probe tip into a calibration phantom with a known reflectance (RIL(λ) [−]). Here we use a phantom that contains Intralipid as scatterer with a μ′s (600nm)= 1.8 mm−1 . Its absolute single fiber reflectance RIL(λ) for all used fiber diameters was previously determined through Monte Carlo simulations.
The collected spectrum ILED(λ) [counts s−1 nm−1] is given by:Eq. (16) has to be integrated over wavelength: Eqs. (13) – (17) can be combined to yield :
2.2. Experimental methods
2.2.1. Single fiber fluorescence setup
A schematic diagram of the single fiber fluorescence setup is shown in Fig. 3. It consists of a spectrometer (QE-4000, Ocean Optics, Duiven, The Netherlands), a 405 nm diode laser (PPMT-45(405–60), Power Technology, Little Rock, USA) for excitation, a 780 nm LED for calibration and a solid core multi-mode fiber which is connected to the other components through a trifurcated optical fiber. During measurements, excitation light is emitted from the laser and travels to the distal end of the single fiber from where it enters the medium. Inside the medium fluorescence is generated and a fraction of it reenters the single fiber and is detected by the spectrometer. Reflected excitation light is filtered out by a 430 nm high pass filter in front of the spectrometer.
2.2.2. Fluorescent phantom measurements
We prepared scattering phantoms by mixing different concentrations of Intralipid 20% (Fresenius Kabi, s-Hertogenbosch, Netherlands) with PBS (J.T.Baker, Deventer, Netherlands), with resulting reduced scattering coefficients of μ′s(405nm)=[0, 0.005, 0.01, 0.05, 0.11, 0.16, 0.22, 0.33, 0.55, 0.83, 1.1, 2.19, 3.3, 4.38, 5.46, 6.58] mm−1 . To add fluorescence to the phantoms, fluorescein (Sigma Aldrich, Zwijndrecht, The Netherlands) was dissolved in PBS and mixed into the phantoms, yielding a final concentration of 1 μM. The absorption coefficient of fluorescein was measured in a spectrophotometer (UV2101PC, Shimadzu,’s-Hertogenbosch, The Netherlands) and determined to be for a 1 μM solution.
To show that the SFF signal can be corrected for absorption, we prepared phantoms with 1 μM fluorescein dissolved in PBS, Intralipid (μ′s(405nm) =0.83 mm−1) and added isolated red blood cells (RBCs) as absorber. The RBCs were isolated from the plasma by three cycles of centrrifugation (7 min, 2000 rpm, 21 degrees) and subsequent resuspension of the RBC pellet in PBS. Four phantoms were prepared with 0, 0.25, 0.5 and 1 % of RBCs, which led to absorption coefficient of . Absorption coefficients were determined from single fiber reflectance (SFR) measurements . One last phantom was prepared with Intralipid (μ′s(405nm)=0.83 mm−1), RBCs ( ) and 1 μM fluorescein which was incubated in 2 M of BSA for half an hour prior to mixing with RBCs and Intralipid to avoid binding of the fluorescein with the RBCs. Combined SFF and SFR measurements were performed with a single fiber probe with the diameter dfib=0.6 mm.
It has been shown that the dianionic state of fluorescein is predominantly present in a solution with a pH of 7.4 . The fluorescence quantum yields of the dianion and the anion are 0.93 and 0.37, respectively, and with a 90% contribution of the dianion, the fluorescence quantum yield of our phantoms was therefore estimated to be Qf = 0.87.
SFF measurements were performed superficially with 5 different fiber diameters (dfib=[0.2, 0.4, 0.6, 0.8, 1] mm) by bringing the bare fiber probe just in contact with the liquid phantom. This was done by continuously monitoring white light emerging from the fiber tip and slowly bringing the phantom surface closer to the fiber using a labjack; a noticeable change in the white light output profile was visible as soon as the tip touched the liquid surface due to the change in medium refractive index. To correct for background signals and fluorescence caused by Intralipid itself, spectra of phantoms with the same Intralipid concentrations but without fluorescein were taken as well and subtracted from the spectra of the scattering and fluorescent phantoms. The system was calibrated as described above (Eq. (18)) and the resulting spectra were integrated over the emission wavelength. For comparison of the data with the semi-empirical model (Eq. (11)), the integrated signal was divided by .
3.1. SFF measurements on scattering phantoms
Fig. 4 shows the integrated of five different fiber diameters versus μ′s on linear and log scales. As mentioned previously, the bi-phasic behavior of the curves is caused by the combined effect of a decrease in sampling volume and an increase in excitation fluence and collection probability with increasing scattering. When the fluorescence is divided by its corresponding fiber diameter, as shown in Fig. 5, all five curves nearly collapse onto each other and when plotted as a function of dimensionless reduced scattering (μ′sdfib) the curve follows the Monte Carlo simulated data (black dots in Fig. 5) as well as the SFF model (blue line) quite well, proving the validity of the model and the experimental calibration procedure.
3.2. SFF measurements in the very low scattering regime
The SFF model was adapted to be valid in the case of μ′s = 0 mm−1 based on an analytical derivation. A very good correlation between the model, simulations and experimental SFF measurements of low scattering phantoms is demonstrated in Fig. 6.
In the case of μ′s = 0 mm−1 the calculated value of the collected fluorescence is not accounting for absorption of excitation light within the medium and assumed collection of photons up to infinity, which leads to a slight overestimation of collected fluorescence compared to our phantoms where and the distance from probe tip to the bottom of the container is roughly 15 mm. Numerical evaluation of Eq. (10) for this value yields for an integration depth up to 15 mm. The experimental value (average of 5 fiber diameters) was 0.033 ± 0.005 which is in agreement with the analytical value for the zero scattering case.
3.3. SFF measurements on scattering and absorbing phantoms
In order to demonstrate the validity of the absorption-correction which we presented in the theory part (Eq. 3 and 4), we created phantoms which contained fluorescein, Intralipid as scatterer and isolated red blood cells ( ) as absorbers. Fig. 7 a) displays how the fluorescence spectra of phantoms with different amounts of RBCs (red lines) are attenuated in comparison to the non-absorbing phantom which contains only fluorescein and Intralipid. When the three spectra with different amounts of RBCs are corrected for the effect of absorption, they collapse onto the same curve (blue lines in Fig. 7a). The spectral shapes of the corrected spectra are identical to the shape of the undistorted spectrum (Fig. 7b), but the amplitude of the corrected spectra is 15% smaller than of the undistorted spectrum. Since it is known that fluorescein tends to bind to RBCs , we hypothesize that this is the case in our phantom and that through the binding process the fluorescence is being quenched. Therefore we created one additional phantom with Intralipid as scatter and RBCs as absorber, but incubated the fluorescein in a 2M solution of BSA prior to adding the RBCs and Intralipid . In this way, fluorescein would bind to BSA, which does not quench the fluorescence and prevents that fluorescein binds to the RBCs. The result of this experiment is shown in Fig. 8. In this case, the difference between the corrected and undistorted spectrum is reduced to 7%.
4. Discussion and conclusion
We have developed a single fiber based spectroscopic method, that allows a full quantification of the intrinsic fluorescence in a turbid medium. We have shown, using an extensive set of phantoms, that the collected single fiber fluorescence indeed shows a bi-phasic behavior with increasing scattering, and proved the validity of the semi-empirical SFF model which was published previously (Eq. (5)).
Furthermore, we have presented a calibration procedure that corrects for system transmission properties, enabling measurement of the intrinsic fluorescence ( ) in a turbid medium. Note that calculation of the intrinsic fluorescence from the measured calibrated fluorescence requires knowledge of the reduced scattering coefficient of the medium at excitation and emission wavelengths; in case of phantoms such as used in this study these properties are well known. Moreover, we recently published a series of papers on the development of multi-diameter single fiber reflectance spectroscopy (MDSFR) which allows the determination of the reduced scattering coefficient of a turbid medium by measuring white-light reflectance spectra with multiple fiber diameters [30–32]. MDSFR and SFF can be easily combined in the same setup to perform subsequent white-light reflectance and fluorescence measurements , which allows the quantification of the intrinsic fluorescence even in media where the reduced scattering coefficient is not known a priori. Additionally, the MDSFR measurement does not only yield μ′s but also allows quantification of the absorption coefficient μa of endogenous and exogenous tissue absorbers, including of the fluorophore itself if it is sufficiently high. Independent measurement of through MDSFR allows, when combined with an SFF measurement of , the determination of the fluorescence quantum yield Qf in vivo, which, to the best of the authors knowledge, is not possible with other optical methods. Knowledge of the quantum yield in vivo could play an important role in the design of new photosensitizers that could be customized for specific tissues. Furthermore, if the pH dependence of the quantum yield of a specific fluorophore is known, which is the case for e.g. fluorescein, the combined SFF/MDSFR method could be used for in vivo pH sensing . Note that the fiber-bundle setup for MDSFR measurements  also easily facilitates reflectance measurements at a source-detector separation of 0.5 mm. The effective path-lengths of these photons are much longer than of the photons that contribute to the MDSFR signal, thereby increasing the sensitivity to absorption and allowing much smaller values to be measured compared to MDSFR alone.
The SFF model was successfully modified to meet the requirements for SFF measurements in a medium that contains little or no scattering. A small discrepancy was observed between the analytical and experimental fluorescence values at zero scattering; this is most likely related to the assumptions made in the analytical calculation, i.e. no attenuation of the excitation beam and collection of photons up to infinity. Both these assumptions are not met in the experimental situation, leading to a smaller experimental value compared to the analytical solution. Another small discrepancy between experimental data and Monte Carlo data is observed in the scattering case; while for the MC data the fluorescence scales perfectly with fiber diameter, some fiber-diameter dependent variations are observed experimentally (Fig. 5). These variations do not show a systematic trend, i.e. the 200 micron data are slightly lower while the 400 micron data are slightly higher than the fluorescence of the other fiber diameters, for all scattering coefficients. These deviations most likely originate from the experimental calibration procedure. Potential errors introduced in that step might be related to the assumption that α is wavelength independent to the Monte Carlo simulated reflectance RIL and to the power measurements of the calibration LED and the laser ( and Plaser). Finally, it should be noted that the MC simulations and thus the SFF model were designed for superficial measurements. Therefore, experimental measurements were also performed superficially. We did observe that the SFF signal is different in interstitial measurements, depending on scattering, fiber diameter and measurement depth; the behavior of the SFF signal in interstitial situations is a topic of future investigation.
We demonstrated that fluorescence measurements in presence of absorption can be corrected by applying a modified Lambert-Beer law. We have shown that the correction method worked for fluorescence measurements on phantoms containing Intralipid as scatterer and different concentrations of isolated red blood cells as absorbers. The fact, that the corrected fluorescence signal did not reach the height of the signal acquired from a non-absorbing phantom is likely to be caused by a quenching effect that occurs when fluorescein binds to RBCs. In order to test this hypothesis, we bound fluorescein to BSA before adding the RBCs and found that the corrected signal almost reached the signal of the phantom without absorber. The small difference between the amplitudes of the absorption-corrected and undistorted spectra of 7% may be caused by a smaller fraction of fluorescein molecules that were not bound or dissociated from their bond with BSA and were subsequently quenched through binding to the RBCs.
In conclusion, we have developed a fiber-optic method that allows the quantification of the intrinsic fluorescence in a turbid medium using a single optical fiber. We have demonstrated the feasibility of the method and the validity of the SFF model through measurements on a wide range of scattering and fluorescent phantoms.
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