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Calibration standards and phantoms for fluorescence optical measurements
Medical Laser Application (2011) 26, 101—108
available at www.sciencedirect.com
journal homepage: www.elsevier.de/mla
Calibration standards and phantoms for fluorescence optical measurements Uwe J. Netz ∗, Jan Toelsner , Uwe Bindig Laser- und Medizin-Technologie GmbH, Berlin (LMTB), Fabeckstr. 60—62, 14195 Berlin, Germany Received 4 May 2011; accepted 12 May 2011
KEYWORDS Phantoms; Fluorescence; Indocyanine green; Epoxy resin; Titanium dioxide; Optical properties; Breast; Skin
Summary We report on the development and production of tissue-like optical phantoms for detection and characterization of fluorescent objects close to the tissue surface. The phantoms mimic bulk optical properties such as light scattering and absorption of female breast tissue and human skin. Fluorescence sources are embedded for a given well-defined localization and volume to simulate pathologic lesions that are marked by specific molecular substances labeled with a fluorescent dye. The phantoms were made of epoxy resin with added titanium dioxide and black epoxy color paste for adjustment of the tissue-like scattering and absorbing properties. Indocyanine green was used as a fluorescent dye. The phantoms were evaluated with an experimental set-up for a new method for in vivo laser fluorescence tomography in the frequency domain. © 2011 Published by Elsevier GmbH.
Introduction There are many of methods and devices available in biology and medicine that are based on optical procedures, e.g. pulse oximeter or microtiter plate reader. From the research and development phase for new systems through to the calibration and quality control of existing devices, tissue-simulating objects are required for defined testing conditions. These so-called ‘phantoms’ or standards are stable measuring objects that exhibit defined characteristics
∗ Corresponding author. Tel.: +49 30 844 923 40; fax: +49 30 844 923 99. E-mail address: [email protected] (U.J. Netz).
1615-1615/$ – see front matter © 2011 Published by Elsevier GmbH. doi:10.1016/j.mla.2011.05.002
within the dimensions to be measured and always give the same result. With just a simple measurement it is possible to calibrate individual devices to give the same result. Phantoms used for optical measurements are required to exhibit defined and optimally adjusted optical properties such as absorption of light, scattering and emission of fluorescence. This means that the optical properties of the model tissue to be imitated by the phantom must already be known. The LMTB has an extensive data bank of optical characteristics of different human tissue types together with literature relating to these characteristics [1]. If the optical characteristics of the tissue are unknown, then they must be determined. At the LMTB the method of choice is the integrating-sphere spectroscopy combined with an inverse Monte Carlo simulation [2]. Other methods are the use of time-resolved measurement techniques by
102 analyzing the deformation of short laser pulses when they pass through a tissue sample [3] or the exact phase detection and calculation of high-frequency intensity-modulated light [4,5]. Another approach is the spatially resolved reflectance spectroscopy where the laterally backscattered light from within the tissue is measured and analyzed [6,7]. Once the optical characteristics of the biological tissue are known, the next step is the selection of suitable materials. A phantom is usually made up of a bulk material or matrix that is clear and transparent, itself contributing little or nothing to the optical characteristics. Selected components are added to this bulk to adjust the absorption, scattering and fluorescent properties to those found in the original biological material. If a phantom is required to mimic the tissue optical properties over a greater spectral area, finding substances that exhibit a similar spectral pattern for scattering and absorption or fluorescence can be difficult and a mixture of several substances may be necessary to give a satisfactory result. Further problems maybe encountered with fluorescence phantoms because the materials used for the matrix, absorption and scattering should have minimal or no autofluorescence. The maximum tolerable level is that of the target tissue. If special biomolecules or fluorescent dyes are to be embedded in a phantom, care should be taken due to possible differences in the chemical bonds compared to the usual solvent medium which can lead to quite different spectral properties. An excellent review of commonly used phantom materials is given by Pogue and Patterson [8]. Usually phantoms are required to have a particular geometrical form which reflect normal measuring conditions. This can be achieved with liquid phantoms using suitably shaped containers and with solid materials provided that they can be mechanically shaped. Liquid phantoms usually have an aqueous based matrix. Intralipid is an emulsion of soya oil in water and is often used for liquid scattering phantoms with the lipid drops serving as the scattering objects [9—11]. Addition of a dye or an ink allows the absorption to be adjusted so that the solution absorbs in the wavelength region of interest, e.g. black India ink [9,12,13], the ProJET dye family [14—17], or biological dyes such as hemoglobin or methemoglobin [11]. For simulations of fluorescence sources in a defined position for a defined volume, a suitable fluorescent dye must be embedded in the phantom [18]. Simple solid phantoms in a rough approximation to human tissue can be made by machining solid white material that has low absorption and volume scattering, e.g. white polyoxymethylene (POM or Delrin® ) [19] or polytetrafluorethylene (PTFE or Teflon® ), however the optical properties of these materials cannot be changed. In order to produce a solid phantom with defined and adjustable optical properties, it is necessary to use a molding method which allows other components to be mixed into the matrix material during the liquid phase before the phantom becomes solid. Absorbing dyes or inks as mentioned above are added to adjust the absorbing properties. The scattering properties of tissue can be attained by adding scattering particles, such as titanium dioxide (TiO2 ) powder or silica microspheres [11,16,17]. If phantoms are required for a limited time, i.e. days or weeks, they can be made from gelatin [18] or agar [10,13].
U.J. Netz et al. Epoxy or polyester resin [14—16], polyurethane [17] or silicone [20,21] are materials that are suitable for the production of long-term, stable phantoms. The main advantage of such materials is that the resin or polyurethane can be easily machined after solidification, and it is easy to remove complex geometric shapes made of silicone from molds. This paper reports on the production and evaluation of epoxy resin phantoms. The aim was to introduce defined fluorescent sources with different geometries and concentrations at different depths under the surface of phantoms which had similar optical properties to the female breast and skin tissue. The phantoms were made for the evaluation of a fluorescence tomographic screening method to be used on tissue surfaces.
Material and methods Materials used As a matrix for the phantoms we used a two-component epoxy resin (TOOLCRAFT Epoxydharz L; Conrad Electronics SE, Hirschau, Germany). The tissue-like scattering was effected by addition of TiO2 (P25; Degussa-Hüls, Frankfurt/Main, Germany) in powder form, and the absorption by addition of a black color paste (TOOLCRAFT Epoxydfarbpaste Schwarz; Conrad Electronics SE, Hirschau, Germany). The phantoms were produced to simulate mammary (fatty) tissue and skin tissue, the latter being differentiated into epidermis and dermis. Indocyanine green (ICG; PULSION Medical Systems AG, München, Germany) was used as the fluorescent dye. This dye is currently the only clinically approved fluorescent dye in the near infrared (NIR) spectral region. Here it represents a class of molecular marker substances currently under development that bind specifically to specific target molecules in pathologically changed tissue and are labeled with a fluorescent dye.
Production of the phantoms The optical properties of the individual substances were determined as a first step. The scattering properties of the TiO2 powder embedded in epoxy resin were determined using the integrating-sphere spectroscopy method in combination with the inverse Monte Carlo simulation [2]. The absorption properties of epoxy resin and black color paste were determined in a classic spectrometer (Spectrophotometer Lambda 2; PerkinElmer LAS Inc., Germany). The resulting optical spectra of the individual substances were used to calculate a formula for different phantoms and target parameters. In order to test whether the phantom demonstrated the desired target parameters, an additional thin sample was made from the phantom material and measured spectroscopically. The method chosen to determine the optical parameters absorption and scattering was the integrating sphere method. The total and diffuse transmission, and then the diffuse remission of the sample were determined for the thin phantom sample using the integrating sphere. The optical parameters of the sample were determined from the proportion of the measured scattered light using the inverse Monte Carlo simulation to calculate the radiation transfer through the sample.
Calibration standards and phantoms for fluorescence optical measurements Indocyanine green (ICG) was initially dissolved in a small amount of ethanol and then added to the epoxy resin in the liquid phase. The resulting fluorescence properties were checked using absorption, extinction and emission spectroscopy (Spectrophotometer Lambda 2 and Luminescence spectrometer LS-50B; PerkinElmer LAS Inc., Germany). Determination of the quantum efficiency is problematic. However if the quantum efficiency is known in a solvent, the quantum efficiency can then be estimated for the intended medium by comparative spectroscopic measurements. The phantoms were made by pouring liquid epoxy resin into molds of silicone and left to cure. For the fatty tissue phantom, a cube-shaped phantom was constructed with side lengths of 40 mm. The skin phantom was made in a mold with the dimensions 50 mm × 20 mm × 100 mm. The outer form of the phantom is determined by geometrical and measurement requirements, not by the anatomical model. After demolding, the surfaces were machined to obtain flat surfaces. Holes were drilled on four sides of the fatty tissue phantoms, parallel to the surface, and at varying distances and diameters, to facilitate the fluorescence measurements. Repeat casts with exactly the same epoxy resin formula were mixed with defined concentrations of ICG dissolved in ethanol for simulation of fluorescence sources at different depths, geometry and concentration. A core drill was used to drill rods from this material with different diameters (1.5 and 2 mm) to fit into the holes which had been drilled in the fatty tissue phantom. Larger rod diameters (6 mm) were made using special molds. The skin phantom was set up in a lateral position made up of three layers to simulate the epidermis, dermis and subcutaneous (fatty) tissue. Cylinders composed of the phantom
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material for fatty tissue plus ICG with different depth extensions and diameters were cast in the dermis.
Phantom evaluation The fluorescence tomographical method to be evaluated by phantom measurements is based on intensity-modulated NIR light in the radio frequency range that illuminates a surface by raster scanning and two-dimensional imaging and demodulation of the emitted fluorescence light at the surface. This two-dimensional homodyne detection method is described in detail in [22]; the set-up used can be seen in Fig. 1. In brief, the main components of the imaging system are an intensified CCD camera (PicoStar HR 12; LaVision GmbH, Göttingen, Germany) with an imaging lens 50 mm f/1.8 (Nikon), a modulated diode laser at 755 nm, 8 mW (LDH-M-C-760; PicoQuant GmbH, Berlin, Germany), and a galvanic mirror scanner (General Scanning Inc., Watertown, MA, USA). Laser light intensity and the amplification of the intensifier are both modulated in the MHz range at the same frequency. By shifting the phase between the laser and the intensifier, the modulation is analyzed and amplitude and phase are derived by fast Fourier transform. The bandwidth of the laser emission is limited by an excitation filter 769/41 nm. A beam splitting mirror is used for deflecting the excitation light to the sample. The mirror acts as a fluorescence filter with 95% transmission for the fluorescent light and below 0.1% transmission for excitation. This amount of excitation light is sufficient for reference measurement. The fluorescence signal was measured with an additional fluorescence filter 832/37 nm. The mirror scanner moves the laser spot over the tissue surface for tomographic imaging.
Figure 1 Experimental set-up for excitation of fluorescence light in the frequency domain with high-frequency laser light modulation and two-dimensional detection by intensified CCD camera. The high rate imager supplies the intensifier with high voltage and the photocathode with the modulation frequency.
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Figure 2 Fluorescence spectra of epoxy resin and ICG in resin at various concentrations, ICG spectra are backgroundcorrected by the resin spectrum, excitation wavelength is 755 nm.
For the evaluation of phantom homogeneity, measurements were made on the fatty tissue phantom where (a) the source location was varied on one side of one phantom, (b) on all sides of the same phantom, and finally (c) on one side of each of several phantoms with identical formulation. The measurand was the re-emitted excitation light at the source location. In the images of amplitude and phase a region of interest of 6 × 6 pixels was defined around the maximum of the Gaussian-like amplitude distribution, from which the mean value of the amplitude and the phase was calculated. The full width at half maximum of the Gaussian distribution was about 10 pixels. The fluorescence measurements were performed at the fatty phantom by placing ICG-containing rods at increasing depth. The fluorescence signal was detected and analyzed over a frequency range from 100 MHz to 700 MHz.
Results Optical parameters of phantom components Absorption of epoxy resin in the NIR is negligible. It exhibits some autofluorescence; however, the intensity of fluorescence emission is less than the equivalent autofluorescence of tissue (Fig. 2) [23]. Introduction of small concentrations of ICG dissolved (<10−4 Mol/L) in ethanol into the epoxy resin does not cause a problem. However, at a higher concentration of ethanol the curing process is prolonged and the material shrinks. The emission maximum of ICG fluorescence is above 800 nm, but shifts more to the infrared with increasing concentration. Comparative measurements show that the quantum yield of ICG in epoxy resin is about 70% that of phosphate buffered saline.
Figure 3 Absorption and scattering coefficient of tissue and phantom material for mammary tissue and skin, anisotropy factor g for phantom material and mammary tissue. EP stands for the phantom formulation for mammary tissue, HE for epidermal tissue, and HD for dermal tissue.
Fat and skin phantoms It was possible, using black epoxy color paste and TiO2 , to adjust the optical properties of the phantom to imitate those of mammary tissue (Fig. 3). The black epoxy paste used
exhibits an even absorption over a wide spectrum without distinct absorption bands. The anisotropy factor of TiO2 in epoxy resin is similar to that of mammary tissue and is almost constant over the wavelength region of interest, being at
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Figure 4 Relative error of absorption (mua = a ), scattering coefficient (red mus = s ) and anisotropy factor (g) from standard deviation when matrix and components are mixed together four times.
0.86 a little below the anisotropy factor for mammary tissue at 0.91. Other mixtures with the same formula gave slight variations of the optical parameters (Fig. 4). Of these the anisotropy factor showed the least variation; the reduced scattering coefficient showed a variation of about 5% and the absorption coefficient of about 20%. Phantoms were produced with drill holes of varying diameter for placing ICG rods at different levels in the sample (Fig. 5). The fluorescence emission of the ICG cores remained stable for the
Figure 6 Skin phantom with three layers: epidermis 200 m, dermis 2 mm, fatty tissue; containing lesions with ICG in different depth extensions (1, 2, 3, and 4 mm) and diameters (3, and 5 mm).
entire period of investigation (approx. 1 year). If the ICG cores were directly irradiated with the focused excitation laser radiation, the fluorescence was irreversibly bleached within a few minutes. However, when an ICG core was irradiated within the fatty tissue phantom, at less than a millimeter under the surface, no bleaching was observed. For the skin phantom, the individual layers were adjusted so that their optical characteristics corresponded to the known characteristics of epidermis and dermis [24,25] and those of fatty tissue were used to mimic subcutaneous fat. The optical characteristics remained stable for the entire investigation time (approx. 1 year). The phantom was made up of a 15 mm thick layer of subcutaneous fatty tissue, a 2 mm thick layer of dermis and 200 m thick layer of epidermis (Fig. 6).
Evaluation of the phantoms Figure 5 Cube phantom with tissue-like optical properties of mammary tissue and rods for simulation of lymph vessels or lymph nodes at different depths beneath the surface with different concentrations of ICG and different diameters. The dimension of the cube is 40 mm × 40 mm × 40 mm, the length of rods is 2 mm (not shown), 10 mm, and 30 mm, the diameters are 1.0, 1.5, 2, and 6 mm.
The remission measurement of the excitation light at different sites on a phantom shows that the value of the amplitude has a relative error of 6% compared to 0.3% for repeated measurements on the same site. Measurements made on different positions on four phantoms with the same formula resulted in a relative total error of 13% (Table 1). The
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Table 1 Error from measurement of remitted excitation light at different phantom positions.
Same position Different positions Different positions on multiple phantoms
Relative error amplitude (%)
Error phase (◦ )
0.3 6.0 13.0
0.6 0.6 0.8
phase was less susceptible to the position and the phantom variation. Considering the mammary tissue phantom (Fig. 7a), if an increasing depth of the fluorescence source was used, the modulation amplitude decreased exponentially and phase increased linearly (Fig. 7b). It was possible to detect amplitude and phase of fluorescence light from a depth of up to 6.5 mm below the surface at a modulation frequency of 625 MHz for rods with a diameter of 1.5 mm and an ICG content of 10−4 Mol/L. Rods with a lower ICG concentra-
Figure 7 (a) Amplitude and phase of fluorescence images (pseudo color) from a mammary tissue phantom with a fluorescence rod of 1.5 mm diameter at depth of 3 mm in horizontal plane at 625 MHz and signals derived from the acquired fluorescence images depending on the (b) depth of fluorescent inclusion and (c) the modulation frequency.
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Acknowledgment
tion led to a linear decrease in the fluorescence signal. As a result, a concentration of 10−5 Mol/L was detectable to a depth of about 5.0 mm. At increasing modulation frequencies the amplitude was damped and the phase increased (Fig. 7c).
This work was co-financed by the Senate of Berlin and the European Union (European funds for regional development) (FKZ 10138596).
Discussion
Zusammenfassung
The relative errors caused by repeated mixes of the same phantom formula should be examined more closely. The anisotropy factor is a property of the material and is independent of the scatterer concentration over a wide range. Therefore the relative error of the anisotropy factor reflects the error of the measurement and calculation of the optical parameters without being affected by the weighing procedure. The scattering and absorption coefficient error depends on the accuracy of the weighing procedure, particularly when weighing out small amounts of the constituents. Regarding absorption, the larger error of about 20% is caused by the higher intrinsic absorption of the color paste. Only small amounts are weighed out, i.e. 15 mg color paste in 100 g epoxy resin which means that the accuracy of the weighing procedure has a significant influence on the error. Measurement of the reflected light at different excitation light positions using several phantoms with the same formula shows that the amplitude is more prone to error than the phase. The phase is the measure of the path length of light, whereas the amplitude is a measure of the attenuation of the modulation of light. Small differences in the phases indicate a high level of homogeneity and reproducibility. The more intensively fluctuating amplitude values are probably caused by inconsistencies in the surface of the phantom, resulting from the mechanical working of the phantom after demolding. It can lead to differences in the radiation profile properties of the sample and can be improved by additional polishing of the sample. The amplitude and phase of the fluorescence signal of the mammary tissue phantom exhibits behavior which is dependent on the depth of the fluorescence source and the modulation frequency. The stable fluorescence characteristics of the ICG core show that ICG in epoxy resin is photostable when used for measurements with physiologically compatible or slightly raised excitation intensities, and is therefore suitable for simulation of fluorescence sources in permanent and stabile phantoms.
Kalibrierstandards und Phantome für fluoreszenzoptische Messungen
Conclusion We were able to show that it is possible to build solid phantoms from epoxy resin with a well-defined shape and optical properties for the purpose of testing optical fluorescence measurement devices. A fluorescent dye was included in the epoxy matrix either permanently or in the form of exchangeable rods for simulating sources of fluorescence with different volumes and concentrations at different depths. The phantoms showed similar optical properties to the target tissue as well as a high degree of homogeneity and long-time stability.
Wir berichten in diesem Artikel über die Entwicklung und Herstellung von Phantomen mit gewebeähnlichen optischen Eigenschaften für die Detektion und Charakterisierung von oberflächennahen Fluoreszenzquellen. Die Phantome simulieren die optischen Parameter bezügliche Lichtstreuung und —absorption von weiblichem Brustgewebe bzw. Hautgewebe. Zur Simulation von pathologischen Gewebearealen, die mit einem spezifischen molekularen Marker belegt sind, an den ein Fluoreszenzfarbstoff gekoppelt ist, wurden in die Phantome Fluoreszenzquellen mit definierter Position und Ausdehnung integriert. Die Phantome bestehen aus Epoxidharz sowie Titandioxid und einer schwarzen Epoxid-Farbpaste zum Einstellen der gewebeähnlichen Streu- und Absorptionseigenschaften. Als Fluoreszenzfarbstoff wurde Indocyaningrün verwendet. Die Phantome wurden mit einem Experimentalaufbau für ein neues Verfahren einer In-vivo-Laserfluoreszenztomografie in der Frequency-Domain evaluiert. Schlüsselwörter: Phantome; Fluoreszenz; Indocyaningrün; Epoxidharz; Titandioxid; Optische Eigenschaften; Brust; Haut
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108 [8] Pogue BW, Patterson MS. Review of tissue simulating phantoms for optical spectroscopy, imaging and dosimetry. J Biomed Opt 2006;11(4):041102. [9] Flock ST, Jacques SL, Wilson BC, Star WM, van Gemert MJ. Optical properties of Intralipid: a phantom medium for light propagation studies. Lasers Surg Med 1992;12(5): 510—9. [10] Wagnières G, Cheng S, Zellweger M, Utke N, Braichotte D, Ballini JP, et al. An optical phantom with tissue-like properties in the visible for use in PDT and fluorescence spectroscopy. Phys Med Biol 1997;42(7):1415—26. [11] Soyemi OO, Landry MR, Yang Y, Idwasi PO, Soller BR. Skin color correction for tissue spectroscopy: demonstration of a novel approach with tissue-mimicking phantoms. Appl Spectrosc 2005;59(2):237—44. [12] Madsen SJ, Patterson MS, Wilson BC. The use of India ink as an optical absorber in tissue-simulating phantoms. Phys Med Biol 1992;37(4):985—93. [13] Cubeddu R, Pifferi A, Taroni P, Torricelli A, Valentini G. A solid tissue phantom for photon migration studies. Phys Med Biol 1997;42(10):1971—9. [14] Firbank M, Oda M, Delpy DT. An improved design for a stable and reproducible phantom material for use in nearinfrared spectroscopy and imaging. Phys Med Biol 1995;40(5): 955—61. [15] Boas DA. Diffuse photon probes of structural and dynamical properties of turbid media: theory and biomedical applications. Dissertation. University of Pennsylvania, Philadelphia, PA, USA; 1996. [16] Sukowski U, Schubert F, Grosenick D, Rinneberg H. Preparation of solid phantoms with defined scattering and absorption properties for optical tomography. Phys Med Biol 1996;41(9):1823—44.
U.J. Netz et al. [17] Vernon ML, Freàchette J, Painchaud Y, Caron S, Beaudry P. Fabrication and characterization of a solid polyurethane phantom for optical imaging through scattering media. Appl Opt 1999;38(19):4247—51. [18] Durkin AJ, Jaikumar S, Richards-Kortum R. Optically dilute, absorbing, and turbid phantoms for fluorescence spectroscopy of homogeneous and inhomogeneous samples. Appl Spectrosc 1993;47:2114—21. [19] Fantini S, Franceschini MA, Gaida G, Gratton E, Jess H, Mantulin WW, et al. Frequency-domain optical mammography: edge effect corrections. Med Phys 1996;23(1):149—57. [20] Lualdi M, Colombo A, Farina B, Tomatis S, Marchesini R. A phantom with tissue-like optical properties in the visible and near infrared for use in photomedicine. Lasers Surg Med 2001;28(3):237—43. [21] Andree S, Reble C, Helfmann J, Gersonde I, Illing G. Evaluation of a novel noncontact spectrally and spatially resolved reflectance setup with continuously variable sourcedetector separation using silicone phantoms. J Biomed Opt 2010;15(6):067009. [22] Netz UJ, Beuthan J, Hielscher AH. Multipixel system for gigahertz frequency-domain optical imaging of finger joints. Rev Sci Instrum 2008;79(3):034301. [23] De Grand AM, Lomnes SJ, Lee DS, Pietrzykowski M, Ohnishi S, Morgan TG, et al. Tissue-like phantoms for near-infrared fluorescence imaging system assessment and the training of surgeons. J Biomed Opt 2006;11(1):014007. [24] Simpson CR, Kohl M, Essenpreis M, Cope M. Near-infrared optical properties of ex vivo human skin and subcutaneous tissues measured using the Monte Carlo inversion technique. Phys Med Biol 1998;43(9):2465—78. [25] Jacques SL. Skin optics. Oregon Medical Laser Center News; 1998..
available at www.sciencedirect.com
journal homepage: www.elsevier.de/mla
Calibration standards and phantoms for fluorescence optical measurements Uwe J. Netz ∗, Jan Toelsner , Uwe Bindig Laser- und Medizin-Technologie GmbH, Berlin (LMTB), Fabeckstr. 60—62, 14195 Berlin, Germany Received 4 May 2011; accepted 12 May 2011
KEYWORDS Phantoms; Fluorescence; Indocyanine green; Epoxy resin; Titanium dioxide; Optical properties; Breast; Skin
Summary We report on the development and production of tissue-like optical phantoms for detection and characterization of fluorescent objects close to the tissue surface. The phantoms mimic bulk optical properties such as light scattering and absorption of female breast tissue and human skin. Fluorescence sources are embedded for a given well-defined localization and volume to simulate pathologic lesions that are marked by specific molecular substances labeled with a fluorescent dye. The phantoms were made of epoxy resin with added titanium dioxide and black epoxy color paste for adjustment of the tissue-like scattering and absorbing properties. Indocyanine green was used as a fluorescent dye. The phantoms were evaluated with an experimental set-up for a new method for in vivo laser fluorescence tomography in the frequency domain. © 2011 Published by Elsevier GmbH.
Introduction There are many of methods and devices available in biology and medicine that are based on optical procedures, e.g. pulse oximeter or microtiter plate reader. From the research and development phase for new systems through to the calibration and quality control of existing devices, tissue-simulating objects are required for defined testing conditions. These so-called ‘phantoms’ or standards are stable measuring objects that exhibit defined characteristics
∗ Corresponding author. Tel.: +49 30 844 923 40; fax: +49 30 844 923 99. E-mail address: [email protected] (U.J. Netz).
1615-1615/$ – see front matter © 2011 Published by Elsevier GmbH. doi:10.1016/j.mla.2011.05.002
within the dimensions to be measured and always give the same result. With just a simple measurement it is possible to calibrate individual devices to give the same result. Phantoms used for optical measurements are required to exhibit defined and optimally adjusted optical properties such as absorption of light, scattering and emission of fluorescence. This means that the optical properties of the model tissue to be imitated by the phantom must already be known. The LMTB has an extensive data bank of optical characteristics of different human tissue types together with literature relating to these characteristics [1]. If the optical characteristics of the tissue are unknown, then they must be determined. At the LMTB the method of choice is the integrating-sphere spectroscopy combined with an inverse Monte Carlo simulation [2]. Other methods are the use of time-resolved measurement techniques by
102 analyzing the deformation of short laser pulses when they pass through a tissue sample [3] or the exact phase detection and calculation of high-frequency intensity-modulated light [4,5]. Another approach is the spatially resolved reflectance spectroscopy where the laterally backscattered light from within the tissue is measured and analyzed [6,7]. Once the optical characteristics of the biological tissue are known, the next step is the selection of suitable materials. A phantom is usually made up of a bulk material or matrix that is clear and transparent, itself contributing little or nothing to the optical characteristics. Selected components are added to this bulk to adjust the absorption, scattering and fluorescent properties to those found in the original biological material. If a phantom is required to mimic the tissue optical properties over a greater spectral area, finding substances that exhibit a similar spectral pattern for scattering and absorption or fluorescence can be difficult and a mixture of several substances may be necessary to give a satisfactory result. Further problems maybe encountered with fluorescence phantoms because the materials used for the matrix, absorption and scattering should have minimal or no autofluorescence. The maximum tolerable level is that of the target tissue. If special biomolecules or fluorescent dyes are to be embedded in a phantom, care should be taken due to possible differences in the chemical bonds compared to the usual solvent medium which can lead to quite different spectral properties. An excellent review of commonly used phantom materials is given by Pogue and Patterson [8]. Usually phantoms are required to have a particular geometrical form which reflect normal measuring conditions. This can be achieved with liquid phantoms using suitably shaped containers and with solid materials provided that they can be mechanically shaped. Liquid phantoms usually have an aqueous based matrix. Intralipid is an emulsion of soya oil in water and is often used for liquid scattering phantoms with the lipid drops serving as the scattering objects [9—11]. Addition of a dye or an ink allows the absorption to be adjusted so that the solution absorbs in the wavelength region of interest, e.g. black India ink [9,12,13], the ProJET dye family [14—17], or biological dyes such as hemoglobin or methemoglobin [11]. For simulations of fluorescence sources in a defined position for a defined volume, a suitable fluorescent dye must be embedded in the phantom [18]. Simple solid phantoms in a rough approximation to human tissue can be made by machining solid white material that has low absorption and volume scattering, e.g. white polyoxymethylene (POM or Delrin® ) [19] or polytetrafluorethylene (PTFE or Teflon® ), however the optical properties of these materials cannot be changed. In order to produce a solid phantom with defined and adjustable optical properties, it is necessary to use a molding method which allows other components to be mixed into the matrix material during the liquid phase before the phantom becomes solid. Absorbing dyes or inks as mentioned above are added to adjust the absorbing properties. The scattering properties of tissue can be attained by adding scattering particles, such as titanium dioxide (TiO2 ) powder or silica microspheres [11,16,17]. If phantoms are required for a limited time, i.e. days or weeks, they can be made from gelatin [18] or agar [10,13].
U.J. Netz et al. Epoxy or polyester resin [14—16], polyurethane [17] or silicone [20,21] are materials that are suitable for the production of long-term, stable phantoms. The main advantage of such materials is that the resin or polyurethane can be easily machined after solidification, and it is easy to remove complex geometric shapes made of silicone from molds. This paper reports on the production and evaluation of epoxy resin phantoms. The aim was to introduce defined fluorescent sources with different geometries and concentrations at different depths under the surface of phantoms which had similar optical properties to the female breast and skin tissue. The phantoms were made for the evaluation of a fluorescence tomographic screening method to be used on tissue surfaces.
Material and methods Materials used As a matrix for the phantoms we used a two-component epoxy resin (TOOLCRAFT Epoxydharz L; Conrad Electronics SE, Hirschau, Germany). The tissue-like scattering was effected by addition of TiO2 (P25; Degussa-Hüls, Frankfurt/Main, Germany) in powder form, and the absorption by addition of a black color paste (TOOLCRAFT Epoxydfarbpaste Schwarz; Conrad Electronics SE, Hirschau, Germany). The phantoms were produced to simulate mammary (fatty) tissue and skin tissue, the latter being differentiated into epidermis and dermis. Indocyanine green (ICG; PULSION Medical Systems AG, München, Germany) was used as the fluorescent dye. This dye is currently the only clinically approved fluorescent dye in the near infrared (NIR) spectral region. Here it represents a class of molecular marker substances currently under development that bind specifically to specific target molecules in pathologically changed tissue and are labeled with a fluorescent dye.
Production of the phantoms The optical properties of the individual substances were determined as a first step. The scattering properties of the TiO2 powder embedded in epoxy resin were determined using the integrating-sphere spectroscopy method in combination with the inverse Monte Carlo simulation [2]. The absorption properties of epoxy resin and black color paste were determined in a classic spectrometer (Spectrophotometer Lambda 2; PerkinElmer LAS Inc., Germany). The resulting optical spectra of the individual substances were used to calculate a formula for different phantoms and target parameters. In order to test whether the phantom demonstrated the desired target parameters, an additional thin sample was made from the phantom material and measured spectroscopically. The method chosen to determine the optical parameters absorption and scattering was the integrating sphere method. The total and diffuse transmission, and then the diffuse remission of the sample were determined for the thin phantom sample using the integrating sphere. The optical parameters of the sample were determined from the proportion of the measured scattered light using the inverse Monte Carlo simulation to calculate the radiation transfer through the sample.
Calibration standards and phantoms for fluorescence optical measurements Indocyanine green (ICG) was initially dissolved in a small amount of ethanol and then added to the epoxy resin in the liquid phase. The resulting fluorescence properties were checked using absorption, extinction and emission spectroscopy (Spectrophotometer Lambda 2 and Luminescence spectrometer LS-50B; PerkinElmer LAS Inc., Germany). Determination of the quantum efficiency is problematic. However if the quantum efficiency is known in a solvent, the quantum efficiency can then be estimated for the intended medium by comparative spectroscopic measurements. The phantoms were made by pouring liquid epoxy resin into molds of silicone and left to cure. For the fatty tissue phantom, a cube-shaped phantom was constructed with side lengths of 40 mm. The skin phantom was made in a mold with the dimensions 50 mm × 20 mm × 100 mm. The outer form of the phantom is determined by geometrical and measurement requirements, not by the anatomical model. After demolding, the surfaces were machined to obtain flat surfaces. Holes were drilled on four sides of the fatty tissue phantoms, parallel to the surface, and at varying distances and diameters, to facilitate the fluorescence measurements. Repeat casts with exactly the same epoxy resin formula were mixed with defined concentrations of ICG dissolved in ethanol for simulation of fluorescence sources at different depths, geometry and concentration. A core drill was used to drill rods from this material with different diameters (1.5 and 2 mm) to fit into the holes which had been drilled in the fatty tissue phantom. Larger rod diameters (6 mm) were made using special molds. The skin phantom was set up in a lateral position made up of three layers to simulate the epidermis, dermis and subcutaneous (fatty) tissue. Cylinders composed of the phantom
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material for fatty tissue plus ICG with different depth extensions and diameters were cast in the dermis.
Phantom evaluation The fluorescence tomographical method to be evaluated by phantom measurements is based on intensity-modulated NIR light in the radio frequency range that illuminates a surface by raster scanning and two-dimensional imaging and demodulation of the emitted fluorescence light at the surface. This two-dimensional homodyne detection method is described in detail in [22]; the set-up used can be seen in Fig. 1. In brief, the main components of the imaging system are an intensified CCD camera (PicoStar HR 12; LaVision GmbH, Göttingen, Germany) with an imaging lens 50 mm f/1.8 (Nikon), a modulated diode laser at 755 nm, 8 mW (LDH-M-C-760; PicoQuant GmbH, Berlin, Germany), and a galvanic mirror scanner (General Scanning Inc., Watertown, MA, USA). Laser light intensity and the amplification of the intensifier are both modulated in the MHz range at the same frequency. By shifting the phase between the laser and the intensifier, the modulation is analyzed and amplitude and phase are derived by fast Fourier transform. The bandwidth of the laser emission is limited by an excitation filter 769/41 nm. A beam splitting mirror is used for deflecting the excitation light to the sample. The mirror acts as a fluorescence filter with 95% transmission for the fluorescent light and below 0.1% transmission for excitation. This amount of excitation light is sufficient for reference measurement. The fluorescence signal was measured with an additional fluorescence filter 832/37 nm. The mirror scanner moves the laser spot over the tissue surface for tomographic imaging.
Figure 1 Experimental set-up for excitation of fluorescence light in the frequency domain with high-frequency laser light modulation and two-dimensional detection by intensified CCD camera. The high rate imager supplies the intensifier with high voltage and the photocathode with the modulation frequency.
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Figure 2 Fluorescence spectra of epoxy resin and ICG in resin at various concentrations, ICG spectra are backgroundcorrected by the resin spectrum, excitation wavelength is 755 nm.
For the evaluation of phantom homogeneity, measurements were made on the fatty tissue phantom where (a) the source location was varied on one side of one phantom, (b) on all sides of the same phantom, and finally (c) on one side of each of several phantoms with identical formulation. The measurand was the re-emitted excitation light at the source location. In the images of amplitude and phase a region of interest of 6 × 6 pixels was defined around the maximum of the Gaussian-like amplitude distribution, from which the mean value of the amplitude and the phase was calculated. The full width at half maximum of the Gaussian distribution was about 10 pixels. The fluorescence measurements were performed at the fatty phantom by placing ICG-containing rods at increasing depth. The fluorescence signal was detected and analyzed over a frequency range from 100 MHz to 700 MHz.
Results Optical parameters of phantom components Absorption of epoxy resin in the NIR is negligible. It exhibits some autofluorescence; however, the intensity of fluorescence emission is less than the equivalent autofluorescence of tissue (Fig. 2) [23]. Introduction of small concentrations of ICG dissolved (<10−4 Mol/L) in ethanol into the epoxy resin does not cause a problem. However, at a higher concentration of ethanol the curing process is prolonged and the material shrinks. The emission maximum of ICG fluorescence is above 800 nm, but shifts more to the infrared with increasing concentration. Comparative measurements show that the quantum yield of ICG in epoxy resin is about 70% that of phosphate buffered saline.
Figure 3 Absorption and scattering coefficient of tissue and phantom material for mammary tissue and skin, anisotropy factor g for phantom material and mammary tissue. EP stands for the phantom formulation for mammary tissue, HE for epidermal tissue, and HD for dermal tissue.
Fat and skin phantoms It was possible, using black epoxy color paste and TiO2 , to adjust the optical properties of the phantom to imitate those of mammary tissue (Fig. 3). The black epoxy paste used
exhibits an even absorption over a wide spectrum without distinct absorption bands. The anisotropy factor of TiO2 in epoxy resin is similar to that of mammary tissue and is almost constant over the wavelength region of interest, being at
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Figure 4 Relative error of absorption (mua = a ), scattering coefficient (red mus = s ) and anisotropy factor (g) from standard deviation when matrix and components are mixed together four times.
0.86 a little below the anisotropy factor for mammary tissue at 0.91. Other mixtures with the same formula gave slight variations of the optical parameters (Fig. 4). Of these the anisotropy factor showed the least variation; the reduced scattering coefficient showed a variation of about 5% and the absorption coefficient of about 20%. Phantoms were produced with drill holes of varying diameter for placing ICG rods at different levels in the sample (Fig. 5). The fluorescence emission of the ICG cores remained stable for the
Figure 6 Skin phantom with three layers: epidermis 200 m, dermis 2 mm, fatty tissue; containing lesions with ICG in different depth extensions (1, 2, 3, and 4 mm) and diameters (3, and 5 mm).
entire period of investigation (approx. 1 year). If the ICG cores were directly irradiated with the focused excitation laser radiation, the fluorescence was irreversibly bleached within a few minutes. However, when an ICG core was irradiated within the fatty tissue phantom, at less than a millimeter under the surface, no bleaching was observed. For the skin phantom, the individual layers were adjusted so that their optical characteristics corresponded to the known characteristics of epidermis and dermis [24,25] and those of fatty tissue were used to mimic subcutaneous fat. The optical characteristics remained stable for the entire investigation time (approx. 1 year). The phantom was made up of a 15 mm thick layer of subcutaneous fatty tissue, a 2 mm thick layer of dermis and 200 m thick layer of epidermis (Fig. 6).
Evaluation of the phantoms Figure 5 Cube phantom with tissue-like optical properties of mammary tissue and rods for simulation of lymph vessels or lymph nodes at different depths beneath the surface with different concentrations of ICG and different diameters. The dimension of the cube is 40 mm × 40 mm × 40 mm, the length of rods is 2 mm (not shown), 10 mm, and 30 mm, the diameters are 1.0, 1.5, 2, and 6 mm.
The remission measurement of the excitation light at different sites on a phantom shows that the value of the amplitude has a relative error of 6% compared to 0.3% for repeated measurements on the same site. Measurements made on different positions on four phantoms with the same formula resulted in a relative total error of 13% (Table 1). The
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Table 1 Error from measurement of remitted excitation light at different phantom positions.
Same position Different positions Different positions on multiple phantoms
Relative error amplitude (%)
Error phase (◦ )
0.3 6.0 13.0
0.6 0.6 0.8
phase was less susceptible to the position and the phantom variation. Considering the mammary tissue phantom (Fig. 7a), if an increasing depth of the fluorescence source was used, the modulation amplitude decreased exponentially and phase increased linearly (Fig. 7b). It was possible to detect amplitude and phase of fluorescence light from a depth of up to 6.5 mm below the surface at a modulation frequency of 625 MHz for rods with a diameter of 1.5 mm and an ICG content of 10−4 Mol/L. Rods with a lower ICG concentra-
Figure 7 (a) Amplitude and phase of fluorescence images (pseudo color) from a mammary tissue phantom with a fluorescence rod of 1.5 mm diameter at depth of 3 mm in horizontal plane at 625 MHz and signals derived from the acquired fluorescence images depending on the (b) depth of fluorescent inclusion and (c) the modulation frequency.
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Acknowledgment
tion led to a linear decrease in the fluorescence signal. As a result, a concentration of 10−5 Mol/L was detectable to a depth of about 5.0 mm. At increasing modulation frequencies the amplitude was damped and the phase increased (Fig. 7c).
This work was co-financed by the Senate of Berlin and the European Union (European funds for regional development) (FKZ 10138596).
Discussion
Zusammenfassung
The relative errors caused by repeated mixes of the same phantom formula should be examined more closely. The anisotropy factor is a property of the material and is independent of the scatterer concentration over a wide range. Therefore the relative error of the anisotropy factor reflects the error of the measurement and calculation of the optical parameters without being affected by the weighing procedure. The scattering and absorption coefficient error depends on the accuracy of the weighing procedure, particularly when weighing out small amounts of the constituents. Regarding absorption, the larger error of about 20% is caused by the higher intrinsic absorption of the color paste. Only small amounts are weighed out, i.e. 15 mg color paste in 100 g epoxy resin which means that the accuracy of the weighing procedure has a significant influence on the error. Measurement of the reflected light at different excitation light positions using several phantoms with the same formula shows that the amplitude is more prone to error than the phase. The phase is the measure of the path length of light, whereas the amplitude is a measure of the attenuation of the modulation of light. Small differences in the phases indicate a high level of homogeneity and reproducibility. The more intensively fluctuating amplitude values are probably caused by inconsistencies in the surface of the phantom, resulting from the mechanical working of the phantom after demolding. It can lead to differences in the radiation profile properties of the sample and can be improved by additional polishing of the sample. The amplitude and phase of the fluorescence signal of the mammary tissue phantom exhibits behavior which is dependent on the depth of the fluorescence source and the modulation frequency. The stable fluorescence characteristics of the ICG core show that ICG in epoxy resin is photostable when used for measurements with physiologically compatible or slightly raised excitation intensities, and is therefore suitable for simulation of fluorescence sources in permanent and stabile phantoms.
Kalibrierstandards und Phantome für fluoreszenzoptische Messungen
Conclusion We were able to show that it is possible to build solid phantoms from epoxy resin with a well-defined shape and optical properties for the purpose of testing optical fluorescence measurement devices. A fluorescent dye was included in the epoxy matrix either permanently or in the form of exchangeable rods for simulating sources of fluorescence with different volumes and concentrations at different depths. The phantoms showed similar optical properties to the target tissue as well as a high degree of homogeneity and long-time stability.
Wir berichten in diesem Artikel über die Entwicklung und Herstellung von Phantomen mit gewebeähnlichen optischen Eigenschaften für die Detektion und Charakterisierung von oberflächennahen Fluoreszenzquellen. Die Phantome simulieren die optischen Parameter bezügliche Lichtstreuung und —absorption von weiblichem Brustgewebe bzw. Hautgewebe. Zur Simulation von pathologischen Gewebearealen, die mit einem spezifischen molekularen Marker belegt sind, an den ein Fluoreszenzfarbstoff gekoppelt ist, wurden in die Phantome Fluoreszenzquellen mit definierter Position und Ausdehnung integriert. Die Phantome bestehen aus Epoxidharz sowie Titandioxid und einer schwarzen Epoxid-Farbpaste zum Einstellen der gewebeähnlichen Streu- und Absorptionseigenschaften. Als Fluoreszenzfarbstoff wurde Indocyaningrün verwendet. Die Phantome wurden mit einem Experimentalaufbau für ein neues Verfahren einer In-vivo-Laserfluoreszenztomografie in der Frequency-Domain evaluiert. Schlüsselwörter: Phantome; Fluoreszenz; Indocyaningrün; Epoxidharz; Titandioxid; Optische Eigenschaften; Brust; Haut
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