1.

Introduction

Optical spectroscopy of tissue is a powerful technique that provides a variety of diagnostically useful information about tissue optical properties and constituents.^1,2 Reflectance spectroscopy contains information about the presence and concentration of tissue chromophores as well as ultrastructural information related to scattering.² Meanwhile, fluorescence spectroscopy has the ability to detect endogenous molecules such as NADH and collagen as well as exogenous fluorescent markers or drugs.³ Optical spectra acquired from tissue contain the competing effects of all tissue optical properties and are also highly dependent on illumination and detection geometry. As a result, the isolation and quantitative measurement of individual tissue optical properties and tissue constituents present a major challenge in optical spectroscopy.

Single-fiber reflectance (SFR) spectroscopy, in which the illumination and detection are performed by the same optical fiber, can be used to address this challenge.In this geometry, the measurement volume is confined to shallow depths, which, while dependent on the optical properties, are on the order of the fiber diameter.^4,5Additionally, the measurement is sensitive to the scattering phase function.⁶ As such, SFR spectroscopy may be well suited for detection of localized changes to the tissue microstructure that are expected to accompany early onset of disease. Additionally, the compact and simple probe design allows easy incorporation of small-diameter SFR probes into many clinical tools, such as endoscopic catheters^7,8 and FNA needles.^9,10

Recently, our group has shown that the tissue absorption coefficient, ${\mathit{\mu }}_{\mathrm{a}}[{\mathrm{mm}}^{-1}]$ (in this article, all wavelength-dependent variables are presented in boldface), can be accurately quantified without prior knowledge of the tissue scattering properties from an SFR measurement through the use of empirical models for the average photon path length and the collected SFR in the absence of absorption.¹¹ Decomposition of ${\mathit{\mu }}_{\mathrm{a}}$ into the constituent absorption spectra of known tissue chromophores enables accurate measurement of chromophore concentrations and microvascular parameters such as local blood oxygen saturation, blood volume fraction, and mean vessel diameter, which can be used for differentiating between healthy and cancerous tissue.¹⁰ Due to the sensitivity of the SFR geometry to the scattering phase function, we have shown that acquiring at least two successive SFR measurements with different fiber diameters enables quantification of the reduced scattering coefficient, ${\mathit{\mu }}_{\mathrm{s}}^{\prime }=(1-{\mathit{g}}_1){\mathit{\mu }}_{\mathrm{s}}[{\mathrm{mm}}^{-1}]$, and the phase function parameter $\mathit{\gamma }=(1-{\mathit{g}}_2)/(1-{\mathit{g}}_1)$[-] as well.^12,13 These scattering parameters are functions of the first- and second-Legendre moments of the scattering phase function, ${\mathit{g}}_1$[-] and ${\mathit{g}}_2$[-], where $\mathit{\gamma }$ represents the likelihood of large-angle backscattering events. Specifically, an increase in $\mathit{\gamma }$ indicates a decrease in large-angle backscattering. The scattering phase function is directly related to the tissue refractive index correlation function through a Fourier transform relationship;^14,15 thus, quantitative measurement of ${\mathit{\mu }}_{\mathrm{s}}^{\prime }$ and $\mathit{\gamma }$ can provide insight into the tissue microstructure, which is useful in diagnosing early onset of disease. The MDSFR spectroscopy technique has been validated in both Monte Carlo simulations^12,16 and tissue-mimicking liquid optical phantoms,¹³ and has recently been used in vivo to quantify optical properties in a murine cancer model.¹⁷

The tissue optical properties measured with the multidiameter single-fiber reflectance (MDSFR) technique can be used for correction of fluorescence spectroscopy, where in situ quantification of fluorophore concentrations from the fluorescence spectra is complicated by the effects of tissue optical properties on the excitation and emission of light. We have recently developed a semi-empirical model for single-fiber fluorescence (SFF) spectroscopy that corrects for the effects of tissue optical properties at both the excitation and emission wavelengths to enable accurate quantification of intrinsic fluorescence,¹⁸ given as the product of the tissue fluorophore absorption coefficient at the excitation wavelength, ${\mathit{\mu }}_{\mathrm{a},x}^{\mathrm{f}}[{\mathrm{mm}}^{-1}]$, and the quantum efficiency across the emission spectrum, $\mathit{Q}$[-]. Section 2.1 provides further details regarding the MDSFR and SFF models and their use.

Conducting MDSFR spectroscopy by sequential placement of multiple optical fibers is time consuming and sensitive to errors in probe placement, making this approach clinically impractical. We have previously demonstrated a means of simulating a single fiber with a variable diameter using a coherent fiber bundle and a series of pinholes to control the effective fiber diameter.¹⁹ While successful, this technique is still time consuming and limited by back reflections.

Here, we present the development and characterization of an MDSFR/SFF system which uses a 19-core fiber bundle and eliminates free-space optical components for improved robustness, signal-to-noise ratio (SNR), and acquisition time. In this article, we summarize the MDSFR and SFF models, the critical aspects of the system design, calibration and measurement algorithms, and the characterization and validation of the system using liquid optical phantoms.

2.

Methods and Materials

2.2.

Design of the MDSFR/SFF System

The MDSFR/SFF system is based upon a custom-built fiber bundle that consists of 19 optical fibers with core sizes of 200 μm (CeramOptec, Germany). At the distal end, the fibers are bundled into three concentric groups comprised of one, six, and 12 fibers, as shown in Fig. 1. Using a CCD camera, the outer diameter of the middle and outer rings of fiber cores were found to be 650 and 1060 μm, respectively. At the opposite end, the fibers are individually terminated to allow direct fiber optic coupling to individual cores, as seen in Fig. 1(c). Each fiber in the bundle is trifurcated to be connected to (1) a fiber delivering light from a halogen lamp (HL-2000-FHSA, Ocean Optics, The Netherlands), (2) a fiber delivering light from a 365-nm LED (NC4U133A, Nichia, Japan) or a 780-nm LED (L780-06-55, Marubeni, USA), and (3) a fiber collecting light returning from the sample and delivering it to one of the three spectrometers. A series of fiber optic interconnects, along with three computer-controlled shutters, enables illumination and spectroscopic detection of the center fiber, the middle ring, and the outer ring of fibers, independently. The optical pathways are shown schematically in Figs. 1(c) and 1(d). Homogeneous illumination of each fiber in the bundle was verified to be within a standard deviation of 7.6% using a CCD camera.

Fig. 1

The MDSFR/SFF system: (a) CCD image of the distal end of the fiber bundle probe showing the illumination uniformity between fiber cores. Scale bar is 200 μm. (b) Schematic representation of (a) with numbering of the fiber cores. (c) Detail schematic of the trifurcation at the proximal end of each fiber in the bundle. (d) Schematic of the fiber tree. (e) Photograph of the fiber tree used in the MDSFR/SFF system.

For detection, spectra are acquired from the three groups of fibers using three spectrometers (two S2000s, one USB2000+, Ocean Optics, The Netherlands) with an overlapping spectral range of 350 to 1000 nm. Each spectrometer is filtered by a long-pass filter with a 385 nm cut-off wavelength (GL-GG385-12, Avantes, The Netherlands) to remove fluorescence excitation light. For SFF, all fibers in the bundle are illuminated simultaneously by the 365-nm LED while detecting on all spectrometers, thus providing an SFF measurement using the largest effective fiber diameter of 1.0 mm.

Lastly, a laptop running LabView coordinates fiber illumination, merging of the three spectrometer channels, calibration, and display of calibrated MDSFR and SFF spectra. A foot pedal is used to initiate each combined MDSFR/SFF measurement, which requires $<8\mathrm{s}$ to complete in the current configuration.

3.

Results

3.1.

Determination of Single-Fiber Equivalence and Effective Fiber Diameter

Analyzing the effective photon path length in the Intralipid-based phantoms using Eq. (17), $\langle {\mathit{L}}_{\mathrm{SFR}}\rangle$ at 611 nm is observed to correlate well between the fiber bundle and the solid-core fibers with an overall Pearson correlation coefficient of $r=0.987$ for the three effective diameters, as shown in Fig. 2(a). The RMS residual errors for the merged channels, $d_{\mathrm{f}}=1.0$ and $0.6\mathrm{mm}$, are 3.93% and 5.93%, respectively. The measurements for $d_{\mathrm{f}}=0.2\mathrm{mm}$ represent comparisons between two solid-core fibers because this channel consists of only one fiber core in the MDSFR system, and the increased scatter observed in some of these measurements arises from the reduction in signal from SFR measurements with decreasing dimensionless scattering coefficient, ${\mathit{\mu }}_{\mathrm{s}}^{\prime }{d}_{\mathrm{f}}$, as seen in Eq. (3).

Fig. 2

Comparison of SFR measurements in Intralipid phantoms between the fiber bundle system and solid-core fibers. (a) Comparison of $\langle {\mathit{L}}_{\mathrm{SFR}}\rangle$ at 611 nm. Data correspond to ${\mathit{\mu }}_{\mathrm{a}}$(611 nm) of 0.5 (diamond), 1 (square), 2 (upright triangle), and 3 (downward triangle) ${\mathrm{mm}}^{-1}$, respectively. (b) Comparison of ${\mathit{R}}_{\mathrm{SF}}^0$ at 425 (diamond), 525 (square), 625 (upright triangle), 725 (downward triangle), and 825 (circle) nm, respectively. In (a) and (b), the blue, red, and black data correspond to fiber diameters of 1.0, 0.6, and 0.2 mm, respectively. (c) The plot of representative SFR spectra measured with the fiber bundle system (green) and solid-core fibers (blue) for ${\mathit{\mu }}_s^{\prime }(611\mathrm{nm})=1.8{\mathrm{mm}}^{-1}$, ${\mathit{\mu }}_{\mathrm{a}}(611\mathrm{nm})=0$ and $0.5{\mathrm{mm}}^{-1}$.

The path-length analysis above relies only on the measured reflectance at one wavelength. To determine if the reconstructed spectra in the MDSFR system are equivalent to SFR spectra from solid-core fibers across the entire spectrum, the measured ${\mathit{R}}_{\mathrm{SF}}^0$ for both systems was compared. Figure 2(b) displays the correlation between the ${\mathit{R}}_{\mathrm{SF}}^0$ measured with the fiber bundle and the ${\mathit{R}}_{\mathrm{SF}}^0$ measured by the solid-core fiber for a range of wavelengths. The ${\mathit{R}}_{\mathrm{SF}}^0$ measurements are strongly correlated ($r=1.000$) and RMS residual errors for the merged channels, $d_{\mathrm{f}}=1.0$ and $0.6\mathrm{mm}$, are 3.06% and 3.83%, respectively. Figure 2(c) displays an overlay of representative SFR spectra measured by the fiber bundle and by the solid-core fibers.

3.2.

Validation of Extracted Optical Properties by MDSFR

The MDSFR spectra acquired from the Polybead phantom using both the MDSFR/SFF system and individual solid-core fibers are shown in Fig. 3(a). As observed in the Intralipid phantoms, the reflectance spectra acquired from the Polybead phantoms using the MDSFR/SFF system are in good agreement with those of the 0.2-, 0.6-, and 1.0-mm diameter solid-core fibers. The extracted scattering properties of ${\mathit{\mu }}_{\mathrm{s}}^{\prime }$ and $\mathit{\gamma }$, shown in Fig. 3(b), also agree well with the solid-core fiber measurements, with overlapping 95% confidence intervals across the measured spectrum. In comparison with the scattering properties predicted by Mie theory, the predicted ${\mathit{\mu }}_{\mathrm{s}}^{\prime }$ lies within the 95% confidence interval of the values extracted by the MDSFR/SFF system over the entire spectral range. The extracted $\mathit{\gamma }$ is slightly higher than the $\mathit{\gamma }$ predicted by theory, which has been attributed to the differences in higher-order moments between the modified Henyey–Greenstein phase function used to create the MDSFR model and the Mie phase function.^13,23

Fig. 3

Comparison of SFR measurements in Polybead phantoms between the fiber bundle system (green) and solid-core fibers (blue). (a) Plot of representative SFR spectra measured with the fiber bundle system and solid-core fibers. (b) Comparison of the extracted $\mathit{\gamma }$ and (c) ${\mathit{\mu }}_{\mathrm{s}}^{\prime }$. Dashed lines represent values predicted by Mie theory. Shaded bands in (b) and (c) represent the 95% confidence interval for each measurement.

Interestingly, the scattering properties measured by the MDSFR/SFF system are slightly closer to those predicted by Mie theory and our previous measurements¹³ than the properties extracted from the solid-core fiber measurements presented here. This is likely due to the independent calibration and measurement of each solid-core fiber, which could allow small relative changes in the calibrated reflectance between fiber diameters. Because the calibration and measurement of each diameter in the MDSFR/SFF system occur in one step, the potential for differences in measurement condition between diameters is greatly reduced.

3.3.

Validation of SFF Through Extraction of $\mathit{Q}{\mathit{\mu }}_{\mathrm{a},x}^{\mathrm{f}}$

The extraction of the intrinsic fluorescence of fluorescein in Intralipid scattering phantoms was used to validate quantitative fluorescence spectroscopy with the MDSFR/SFF device. In these measurements, the scattering properties of Intralipid-fluorescein phantoms were extracted from the MDSFR measurement and used to correct the SFF measurement for the effects of the optical properties. The intrinsic fluorescence $\mathit{Q}{\mathit{\mu }}_{\mathrm{a},\mathrm{x}}^{\mathrm{f}}$ was then extracted by fitting the data to Eq. (6) by nonlinear regression. The resulting $\mathit{Q}{\mathit{\mu }}_{\mathrm{a},\mathrm{x}}^{\mathrm{f}}$ was found to be $7.0\pm 0.3{(10)}^{-3}{\mathrm{mm}}^{-1}$, which yields a $\mathit{Q}$ of $0.92\pm 0.04$ for the measured ${\mathit{\mu }}_{\mathrm{a},\mathrm{x}}^{\mathrm{f}}$ of $7.6\pm 0.1{(10)}^{-3}{\mathrm{mm}}^{-1}$. The measured $\mathit{Q}$ is in good agreement with the published value for fluorescein, $\mathit{Q}=0.88$, at the measured pH of 7.4.²⁴ Using the extracted $\mathit{Q}{\mathit{\mu }}_{\mathrm{a},\mathrm{x}}^{\mathrm{f}}$ of $7.0{(10)}^{-3}{\mathrm{mm}}^{-1}$, the dimensionless SFF, ${\mathit{F}}_{\mathrm{SF}}/d_{\mathrm{f}}$, was found to be in good agreement with Eq. (7) throughout the investigated range of dimensionless scattering coefficient, ${\mathit{\mu }}_{\mathrm{s}}^{\prime }{d}_{\mathrm{f}}$, as seen in Fig. 4.

Fig. 4

The nondimensionalized SFF corresponding to the best fit of $\mathit{Q}{\mathit{\mu }}_{\mathrm{a},\mathrm{x}}^{\mathrm{f}}$ to the data (black data points) and the predicted dependence on ${\overline{\mathit{\mu }}}_s^{\prime }{d}_{\mathrm{f}}$ (gray-dashed line) on (a) linear scale and (b) a semi-log scale.

4.

Discussion

Based on the similarity observed in Figs. 2(c) and 3(a) between the SFR spectra measured by the fiber bundle system and solid-core fibers, we conclude that the merged spectra from the fiber bundle system do accurately represent spectra from single solid-core fibers across the entire measured spectral range. The consistency between the optical properties ${\mathit{\mu }}_{\mathrm{a}}$, ${\mathit{\mu }}_{\mathrm{s}}^{\prime }$, $\mathit{\gamma }$, and $\mathit{Q}{\mathit{\mu }}_{\mathrm{a},\mathrm{x}}^{\mathrm{f}}$ measured by the fiber bundle system and the predicted values in both the Polybead and fluorescein phantoms further supports that the fiber bundle system accurately simulates single fibers with $d_{\mathrm{f}}=[0.2,0.6,1.0]\mathrm{mm}$. Significantly, the scattering properties recovered from the fiber bundle system were found to be slightly closer to their predicted values than those recovered by separate single-fiber measurements, which illustrates the potential for small measurement errors when calibrating each fiber independently and demonstrates the sensitivity of the scattering property measurement to such errors.

As observed in our previous study,¹³ the measured $\mathit{\gamma }$ consistently over-predicted the true value for both the fiber bundle and single-fiber systems. This discrepancy has been predicted by simulations and attributed to subtle differences between the modeled modified Henyey–Greenstein phase function and the true Mie phase function. However, it is also important to note that the MDSFR technique relies upon accurate Monte Carlo simulation of Intralipid scattering and the use of Intralipid as a calibration reference. Therefore, the accuracy of the extracted optical properties depends on accurate knowledge of the Intralipid scattering coefficient and phase function. Additionally, the batch-to-batch variation of the Intralipid properties must be minimal. The optical properties of Intralipid have been extensively studied,²² and observed batch-to-batch variations in scattering were found to be near 2% over a period of 7 years;²⁵ however, the sensitivity of our technique to Intralipid scattering could potentially play a role in the discrepancy between the measured and predicted $\mathit{\gamma }$ in the Polybead phantoms.

The fiber bundle used in this study uses only a minimum number of fibers (7 or 19) to simulate a solid-core fiber, in contrast to our previous study in which each effective fiber diameter consists of thousands of small individual fibers. The successful simulation of solid-core fibers in both cases indicates that the coarseness of the fiber bundle has little impact on SFR measurements over the range of ${\mathit{\mu }}_{\mathrm{s}}^{\prime }{d}_{\mathrm{f}}$ investigated. This suggests that the use of a fiber bundle as a variable-diameter single fiber for MDSFR is broadly applicable to a range of fiber bundle geometries between these two extreme cases. As a result, alternative fiber bundle geometries can be considered to suit specific applications. For example, a probe with $d_{\mathrm{f}}=[0.2,0.4,0.6]$ mm might be considered for an application wishing to focus on skin epithelial properties, while an alternative probe with $d_{\mathrm{f}}=[0.8,1.2,1.5]$ mm could be employed to increase the sensitivity to deeper chromophores.⁴

The coarse bundle used in this study has several advantages for clinical use. The size and number of the individual fiber cores enable construction of the system with entirely fiber optic connections. This architecture eliminates nearly all back reflections and provides greater efficiency in light delivery and collection, all of which improve the signal-to-background ratio (SBR) and acquisition speed. For comparison, the previous MDSFR system based on a pinhole and free-space optics was limited by poor SBR for the 0.2-mm effective fiber diameter, with $\mathrm{SBR}=0.016$ for the ${\mathit{\mu }}_{\mathrm{s}}^{\prime }=3.6{\mathrm{mm}}^{-1}$ phantom. Using all the-fiber-optic design presented here, the 0.2-mm effective diameter displayed $\mathrm{SBR}=37$ for the same phantom conditions, demonstrating an over $2000\times$ improvement in SBR. In the current configuration, the fiber bundle system is able to complete a full measurement sequence capable of quantifying ${\mathit{\mu }}_{\mathrm{a}}$, ${\mathit{\mu }}_{\mathrm{s}}^{\prime }$, $\mathit{\gamma }$, and $\mathit{Q}{\mathit{\mu }}_{\mathrm{a},\mathrm{x}}^{\mathrm{f}}$ of a localized volume of tissue in $<8\mathrm{s}$. With an overall bundle diameter slightly over 1.0 mm, the bundle used in this study is capable of being delivered to hollow organs through an endoscope or used as a simple handheld probe for superficial tissues and open surgical sites. Additionally, the maximum effective fiber diameter of 1.0 mm provides measurement depth sufficient for probing the superficial vasculature lying beneath epithelial tissue and the longer photon path length increases the sensitivity to low concentrations of tissue chromophores.

While the liquid optical phantoms used in this study are well suited for validation and characterization of the system, they represent an idealized tissue environment in which the optical properties are spatially homogeneous. The different fiber diameters used in MDSFR measure over different tissue depths, but assume consistent scattering properties in the simultaneous solution of Eq. (3). Because the MDSFR system guarantees colocalization of the effective fiber diameter, the spatially averaged scattering properties sampled by each fiber diameter are expected to be quite similar in most tissues. However, the effect of layered tissue with stratified optical properties on MDSFR measurements has yet to be investigated. Similarly, the quantitative fluorescence model assumes homogeneous distribution of fluorophores as well as scatterers, and could be potentially confounded by unequal distributions of one or the other. Given the limited measurement volumes,⁴ any such heterogeneities are expected to be small; however, the effects of tissue optical property variations on the models will be the subject of future study.

5.

Conclusion

We have demonstrated a robust MDSFR/SFF system capable of quantifying ${\mathit{\mu }}_{\mathrm{a}}$, ${\mathit{\mu }}_{\mathrm{s}}^{\prime }$, $\mathit{\gamma }$, and $\mathit{Q}{\mathit{\mu }}_{\mathrm{a},\mathrm{x}}^{\mathrm{f}}$ from a small volume of tissue in one 8-s measurement, making it well suited for use in a clinical environment. The increased speed and robustness in comparison to the previous pinhole-based proof-of-concept system are a result of the elimination of free-space optics. While system calibration is critical to accurate optical property measurement, the daily calibration of the system requires only two simple measurements in liquid samples and an automated integrating sphere measurement, which are guided by the user interface for easy use by clinicians.

Using liquid optical phantoms, we have demonstrated that the system uses a fiber bundle to accurately simulate a variable-diameter solid-core fiber for both SFR and SFF spectroscopy. The effective path lengths and reflectances measured from the fiber bundle system match with those measured by the solid-core fibers and the extracted scattering properties ${\mathit{\mu }}_{\mathrm{s}}^{\prime }$ and $\mathit{\gamma }$ agree well with predicted values. Notably, we have used this system to demonstrate combined MDSFR/SFF spectroscopy, wherein the scattering and absorption properties are accurately quantified and then used to provide correction for quantitative fluorescence spectroscopy. Future work will investigate the use of this technique in stratified tissue optical properties and the integration of this system into clinical optical property measurements.