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Potential of near infrared fluorescence optical imaging in diagnostic radiology
European Journal of Radiology 81S1 (2012) S61–S62
Contents lists available at SciVerse ScienceDirect
European Journal of Radiology journal homepage: www.elsevier.com/locate/ejrad
Potential of near infrared fluorescence optical imaging in diagnostic radiology Ingrid Hilger*, Werner A. Kaiser Institute of Diagnostic and Interventional Radiology I, Department of Experimental Radiology, University Hospital Jena, Friedrich-Schiller Universit¨ at Jena, Erlanger Allee 101, 07747 Jena, Germany
1. Introduction Fluorescence optical imaging of molecular and cellular structures has been pursued with great interest over the last years. The methodology, which was originally introduced in the microscopy field, is now avery important tool in several preclinical research fields which use living laboratory animals. Today, dedicated fluorescence imaging systems are also being tested for first introduction into the clinical situation. These investigations are driven by the advantage that the methodology is a non-invasive one, that images can be obtained in real-time and with high-resolution, and that molecular informationis available at comparatively low costs compared to PET and SPECT. Nevertheless, the limited penetration of light into tissues is one of the main challenges. Consequently, the implementation of fluorescence optical imaging into the clinical practice will prospectively be confined to dedicated parts of the body using specialized light excitation, detection systems. Moreover, special attention must also be devoted to the design of corresponding fluorooptical probes. The signaling moiety of optical probes is normally much larger compared to optical tracer with corresponding impact on pharmacokinetics and dynamics. In this article we will discuss the potential and current challenges in optical imaging in diagnostic radiology. 2. Probe design Optical probes for fluorescence optical imaging should be able to fluoresce in the near infrared region, i.e. between 650 and 850 nm, which is important from the fact that tissue autofluorescence and absorption of light by intrinsic tissue components is comparatively low in this spectral range. This procedure allows us to minimize background fluorescence and to improve penetration of light into the tissue [1]. The most adequate NIRF probes exhibit a large stokes shift to reduceinterferences between absorption and fluorescence emission. Moreover, near infrared fluorescence (NIRF) optical probes should have adequate molar absorption coefficient and quantum yields which ultimately determine imaging sensitivity. Further features are very important in terms of accessibility to the target region such as chemical and photostability, solubility under biological conditions in order to avoid aggregation which could modify target affinity. The presence of a chemical functionality allows us bioconjugation to biological structures such as peptides, aptamers, oligonucleotides (e.g. siRNA) antibodies or antibody
fragments. Compounds which meet these criteria are cyanine dyes and its derivatives. They are constructed of two aromatic nitrogen-containing heterocycles which are connected with a polymethine chain. A typical example isindocyanine green (ICG) which is already used in the clinical situation since almost 50 years [2]. Nevertheless, the photostability and quantum yield of ICG are comparatively low, the plasma binding rate high, and high hydrophilicity aggregation may further impair fluorescence. This is the reason why great emphasis was placed in the development of further dye formulations for in vivo imaging. Beyond Cy5, Cy5.5, Cy7, also hemicyanine dyes, by which only one of the two heterocycles contains an aromatic amine, have been introduced. In this context, conjugation with sulfonic acids which increases hydrophilicity and reduces of binding with serum proteins and aggregation is favorable [3]. There are several classes of optical probes developed so far: (a) unspecific formulations to image vasculature; they are constructed of the optically active probe per se (b) targeted probes able to detect specific molecular structures; they contain an optically active probe and a biomolecule directed to a specific molecular marker of disease, (c) activatable probes, which can detect the activity of a target after specific activation, (d) transporter gene probes, addressinggene expression activity, (e) cell tracking probes they are useful in monitoring migration of cells after labeling them with optically active tags or after having introduced defined fluorescing or luminescing marker genes. In all cases the nature of the optical probe component and its ability to fluoresce in the NIR spectral range is of particular significance. Whereas nonspecific probes such as indocyanine green to image the vasculature have been introduced into clinical practice (see above), all other mentioned approaches need still to be approved for application in humans. Targeted probes against specific molecular structures in cancer, cardiovascular diseases and inflammation have been designed so far (e.g. [4,5]) etc. Beyond the detection of single molecules as described above, there are several attempts for the detection of protein-protein interactions via in vivo FRET imaging. In this context, most of the approaches have still been restricted to intravital microscopy. In just a few cases, whole body imaging was employed via the injection of specific contrast agents able to detect FRET in vivo [6]. 3. Current available NIRF optical imaging systems
* Ingrid Hilger, PhD, Institute of Diagnostic and Interventional Radiology I, University Hospital Jena, Erlanger Allee 101, 07747 Jena, Germany. Tel.: +49 3641 9325921; fax: +49 3641 9325922. E-mail address: [email protected] (I. Hilger). 0720-048X/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved.
Basically, there are several NIRF optical imaging systems available today. Whole body imaging systems are still confined to the preclinical analysis of defined mouse models. In contrast and due to
S62
I. Hilger, W.A. Kaiser / European Journal of Radiology 81S1 (2012) S61–S62
the limited penetration of light into tissue, clinical optical imaging is applied for the investigation of specific parts of the body. Examples are endoscopic, intraoperative and ophthalmologic imaging. From the biophysical point of view, optical imaging systems can be grouped into those measuring ballistic or diffuse photons [7]. The ballistic approach detects non-scattered photons. This approach allows for high-resolution but low penetration depth (several micrometers). The diffuse method measures scattered photons which enable the detection of deeper photons in the tissue (mm to cm) but the resolution is limited. In this view, photoacoustic tomography is a hybrid method. Hereto, diffuse photons are used to induce an acoustic signal, and this signal is detected in the ballistic mode. By doing so deep sensitation and high resolution is combined. These techniques are envisioned to be used in diverse diagnostic and interventional procedures in future radiology [8]. The currently available whole body imaging systems are based on the diffuse mode. In this context, planar systems are characterized by the illumination of the sample with light to excite the applied NIRF contrast agent and registration of the respective emerging photons from the tissue via a specific camera to give a planar 2D image [1]. Tomographic systems are fundamentally based on the so called light transport equation to gain 3D information. In this context, different techniques have been developed [9]. Whereas planar imaging systems have been intensively applied in studyingdisease models related to tissue alterations close to the surface of animals, tomographic systems detect abnormalities in deeper regions of the body, such as lung tumors. It is expected that a transfer to the clinical situation will be restricted to defined organs with good accessibility to light such as the breast, joints, etc. Examples for the transfer of fluorescence imaging to the clinical situation is represented by planar imaging of the hand after application of ICG as contrast agent for the detection of joint abnormalities [10] and intraoperative imaging [11]. Particularly, in relation to diffuse NIRF optical imaging approaches photon scattering is an important issue. Photon scattering occurs as a result reflection along intrinsic tissue boundary surfaces and interfaces (short “tissue inhomogeneities”), such as the presence of (lipid) cell membranes, cell organelles, fibrillar structures (e.g. collagen) etc. of the respective tissue sample to be analyzed. Another biophysical factor influencing detection is absorption of photons which are emitted after fluorescence excitation but do not reach the tissue surface. Accordingly, quantitative analysis of fluorescence intensity is a challenging issue. Albeit, numerous preclinical NIRF imaging approaches report on semi-quantitative analysis which can be interpreted as “trends” giving valuable information on imaging sensitivity and specificity. Beyond the diffuse fluorescence detection method, the ballistic approach is represented by optical coherence tomography [12] for
the detection of abnormalities in the eye or cardiovascular diseases using specific catheters. Attempts for the combination of NIR imaging with other modalities with high anatomical resolution such as MRI and CT are also being investigated [13]. 4. Conclusions The current status of knowledge indicates that fluorescence optical imaging is a promising technology for imaging of molecular structures in future diagnostic and interventional radiology. Competing interests: All authors indicate that they have no conflicting interests. Role of the funding source: The work was supported in parts by the DFG and BMBF. References 1. Ntziachristos V, Ripoll J, Wang LV, Weissleder R. Looking and listening to light: the evolution of whole-body photonic imaging. Nat Biotechnol 2005;23:313–20. 2. Scheider A, Nasemann JE, Lund OE. Fluorescein and indocyanine green angiographies of central serous choroidopathy by scanning laser ophthalmoscopy. Am J Ophthalmol 1993;115(1):50–6. 3. Hamann FM, Brehm R, Pauli J, et al. Controlled modulation of serum protein binding and biodistribution of asymmetric cyanine dyes by variation of the number of sulfonate groups. Mol Imaging 2011;10(4):258–69. 4. Lisy MR, Goermar A, Thomas C, et al. In vivo near-infrared fluorescence imaging of carcinoembryonic antigen-expressing tumor cells in mice. Radiology 2008; 247(3):779–87. 5. Laabs E, Behe M, Kossatz S, Frank W, Kaiser WA, Hilger I. Optical imaging of CCK2/gastrin receptor-positive tumors with a minigastrin near-infrared probe. Invest Radiol 2011;46(3):196–201. 6. Busch C, Schroter ¨ T, Grabolle M, et al. An in vivo spectral multiplexing approach for the cooperative imaging of different disease-related biomarkers with nearinfrared fluorescent forster ¨ resonance energy transfer probes. J Nucl Med 2012; 53:1–9. 7. Palmer GM, Vishwanath K, Dewhirst MW. Application of optical imaging and spectroscopy to radiation biology. Radiat Res 2012;177(4):365–75. 8. Ntziachristos V. Clinical translation of optical and optoacoustic imaging. Philos Transact A Math Phys Eng Sci 2011;369(1955):4666–78. 9. Lasser T, Ntziachristos V. Optimization of 360 degrees projection fluorescence molecular tomography. Med Image Anal 2007;11(4):389–99. 10. Werner SG, Langer HE, Ohrndorf S, et al. Inflammation assessment in patients with arthritis using a novel in vivo fluorescence optical imaging technology. Ann Rheum Dis 2011 Oct 12 [Epub ahead of print]. 11. van Dam GM, Themelis G, Crane LMA, et al. Intraoperative tumor-specific fluorescence imaging in ovarian cancer by folate receptor-alpha targeting: first in-human results. Nat Med 2011;17(10):1315–9. 12. Huang D, Swanson EA, Lin CP, et al. Optical coherence tomography. Science 1991;254(5035):1178–81. 13. Ale A, Ermolayev V, Herzog E, Cohrs C, de Angelis MH, Ntziachristos V. FMTXCT: in vivo animal studies with hybrid fluorescence molecular tomography– X-ray computed tomography. Nat Meth 2012;9(6):615–20.
Contents lists available at SciVerse ScienceDirect
European Journal of Radiology journal homepage: www.elsevier.com/locate/ejrad
Potential of near infrared fluorescence optical imaging in diagnostic radiology Ingrid Hilger*, Werner A. Kaiser Institute of Diagnostic and Interventional Radiology I, Department of Experimental Radiology, University Hospital Jena, Friedrich-Schiller Universit¨ at Jena, Erlanger Allee 101, 07747 Jena, Germany
1. Introduction Fluorescence optical imaging of molecular and cellular structures has been pursued with great interest over the last years. The methodology, which was originally introduced in the microscopy field, is now avery important tool in several preclinical research fields which use living laboratory animals. Today, dedicated fluorescence imaging systems are also being tested for first introduction into the clinical situation. These investigations are driven by the advantage that the methodology is a non-invasive one, that images can be obtained in real-time and with high-resolution, and that molecular informationis available at comparatively low costs compared to PET and SPECT. Nevertheless, the limited penetration of light into tissues is one of the main challenges. Consequently, the implementation of fluorescence optical imaging into the clinical practice will prospectively be confined to dedicated parts of the body using specialized light excitation, detection systems. Moreover, special attention must also be devoted to the design of corresponding fluorooptical probes. The signaling moiety of optical probes is normally much larger compared to optical tracer with corresponding impact on pharmacokinetics and dynamics. In this article we will discuss the potential and current challenges in optical imaging in diagnostic radiology. 2. Probe design Optical probes for fluorescence optical imaging should be able to fluoresce in the near infrared region, i.e. between 650 and 850 nm, which is important from the fact that tissue autofluorescence and absorption of light by intrinsic tissue components is comparatively low in this spectral range. This procedure allows us to minimize background fluorescence and to improve penetration of light into the tissue [1]. The most adequate NIRF probes exhibit a large stokes shift to reduceinterferences between absorption and fluorescence emission. Moreover, near infrared fluorescence (NIRF) optical probes should have adequate molar absorption coefficient and quantum yields which ultimately determine imaging sensitivity. Further features are very important in terms of accessibility to the target region such as chemical and photostability, solubility under biological conditions in order to avoid aggregation which could modify target affinity. The presence of a chemical functionality allows us bioconjugation to biological structures such as peptides, aptamers, oligonucleotides (e.g. siRNA) antibodies or antibody
fragments. Compounds which meet these criteria are cyanine dyes and its derivatives. They are constructed of two aromatic nitrogen-containing heterocycles which are connected with a polymethine chain. A typical example isindocyanine green (ICG) which is already used in the clinical situation since almost 50 years [2]. Nevertheless, the photostability and quantum yield of ICG are comparatively low, the plasma binding rate high, and high hydrophilicity aggregation may further impair fluorescence. This is the reason why great emphasis was placed in the development of further dye formulations for in vivo imaging. Beyond Cy5, Cy5.5, Cy7, also hemicyanine dyes, by which only one of the two heterocycles contains an aromatic amine, have been introduced. In this context, conjugation with sulfonic acids which increases hydrophilicity and reduces of binding with serum proteins and aggregation is favorable [3]. There are several classes of optical probes developed so far: (a) unspecific formulations to image vasculature; they are constructed of the optically active probe per se (b) targeted probes able to detect specific molecular structures; they contain an optically active probe and a biomolecule directed to a specific molecular marker of disease, (c) activatable probes, which can detect the activity of a target after specific activation, (d) transporter gene probes, addressinggene expression activity, (e) cell tracking probes they are useful in monitoring migration of cells after labeling them with optically active tags or after having introduced defined fluorescing or luminescing marker genes. In all cases the nature of the optical probe component and its ability to fluoresce in the NIR spectral range is of particular significance. Whereas nonspecific probes such as indocyanine green to image the vasculature have been introduced into clinical practice (see above), all other mentioned approaches need still to be approved for application in humans. Targeted probes against specific molecular structures in cancer, cardiovascular diseases and inflammation have been designed so far (e.g. [4,5]) etc. Beyond the detection of single molecules as described above, there are several attempts for the detection of protein-protein interactions via in vivo FRET imaging. In this context, most of the approaches have still been restricted to intravital microscopy. In just a few cases, whole body imaging was employed via the injection of specific contrast agents able to detect FRET in vivo [6]. 3. Current available NIRF optical imaging systems
* Ingrid Hilger, PhD, Institute of Diagnostic and Interventional Radiology I, University Hospital Jena, Erlanger Allee 101, 07747 Jena, Germany. Tel.: +49 3641 9325921; fax: +49 3641 9325922. E-mail address: [email protected] (I. Hilger). 0720-048X/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved.
Basically, there are several NIRF optical imaging systems available today. Whole body imaging systems are still confined to the preclinical analysis of defined mouse models. In contrast and due to
S62
I. Hilger, W.A. Kaiser / European Journal of Radiology 81S1 (2012) S61–S62
the limited penetration of light into tissue, clinical optical imaging is applied for the investigation of specific parts of the body. Examples are endoscopic, intraoperative and ophthalmologic imaging. From the biophysical point of view, optical imaging systems can be grouped into those measuring ballistic or diffuse photons [7]. The ballistic approach detects non-scattered photons. This approach allows for high-resolution but low penetration depth (several micrometers). The diffuse method measures scattered photons which enable the detection of deeper photons in the tissue (mm to cm) but the resolution is limited. In this view, photoacoustic tomography is a hybrid method. Hereto, diffuse photons are used to induce an acoustic signal, and this signal is detected in the ballistic mode. By doing so deep sensitation and high resolution is combined. These techniques are envisioned to be used in diverse diagnostic and interventional procedures in future radiology [8]. The currently available whole body imaging systems are based on the diffuse mode. In this context, planar systems are characterized by the illumination of the sample with light to excite the applied NIRF contrast agent and registration of the respective emerging photons from the tissue via a specific camera to give a planar 2D image [1]. Tomographic systems are fundamentally based on the so called light transport equation to gain 3D information. In this context, different techniques have been developed [9]. Whereas planar imaging systems have been intensively applied in studyingdisease models related to tissue alterations close to the surface of animals, tomographic systems detect abnormalities in deeper regions of the body, such as lung tumors. It is expected that a transfer to the clinical situation will be restricted to defined organs with good accessibility to light such as the breast, joints, etc. Examples for the transfer of fluorescence imaging to the clinical situation is represented by planar imaging of the hand after application of ICG as contrast agent for the detection of joint abnormalities [10] and intraoperative imaging [11]. Particularly, in relation to diffuse NIRF optical imaging approaches photon scattering is an important issue. Photon scattering occurs as a result reflection along intrinsic tissue boundary surfaces and interfaces (short “tissue inhomogeneities”), such as the presence of (lipid) cell membranes, cell organelles, fibrillar structures (e.g. collagen) etc. of the respective tissue sample to be analyzed. Another biophysical factor influencing detection is absorption of photons which are emitted after fluorescence excitation but do not reach the tissue surface. Accordingly, quantitative analysis of fluorescence intensity is a challenging issue. Albeit, numerous preclinical NIRF imaging approaches report on semi-quantitative analysis which can be interpreted as “trends” giving valuable information on imaging sensitivity and specificity. Beyond the diffuse fluorescence detection method, the ballistic approach is represented by optical coherence tomography [12] for
the detection of abnormalities in the eye or cardiovascular diseases using specific catheters. Attempts for the combination of NIR imaging with other modalities with high anatomical resolution such as MRI and CT are also being investigated [13]. 4. Conclusions The current status of knowledge indicates that fluorescence optical imaging is a promising technology for imaging of molecular structures in future diagnostic and interventional radiology. Competing interests: All authors indicate that they have no conflicting interests. Role of the funding source: The work was supported in parts by the DFG and BMBF. References 1. Ntziachristos V, Ripoll J, Wang LV, Weissleder R. Looking and listening to light: the evolution of whole-body photonic imaging. Nat Biotechnol 2005;23:313–20. 2. Scheider A, Nasemann JE, Lund OE. Fluorescein and indocyanine green angiographies of central serous choroidopathy by scanning laser ophthalmoscopy. Am J Ophthalmol 1993;115(1):50–6. 3. Hamann FM, Brehm R, Pauli J, et al. Controlled modulation of serum protein binding and biodistribution of asymmetric cyanine dyes by variation of the number of sulfonate groups. Mol Imaging 2011;10(4):258–69. 4. Lisy MR, Goermar A, Thomas C, et al. In vivo near-infrared fluorescence imaging of carcinoembryonic antigen-expressing tumor cells in mice. Radiology 2008; 247(3):779–87. 5. Laabs E, Behe M, Kossatz S, Frank W, Kaiser WA, Hilger I. Optical imaging of CCK2/gastrin receptor-positive tumors with a minigastrin near-infrared probe. Invest Radiol 2011;46(3):196–201. 6. Busch C, Schroter ¨ T, Grabolle M, et al. An in vivo spectral multiplexing approach for the cooperative imaging of different disease-related biomarkers with nearinfrared fluorescent forster ¨ resonance energy transfer probes. J Nucl Med 2012; 53:1–9. 7. Palmer GM, Vishwanath K, Dewhirst MW. Application of optical imaging and spectroscopy to radiation biology. Radiat Res 2012;177(4):365–75. 8. Ntziachristos V. Clinical translation of optical and optoacoustic imaging. Philos Transact A Math Phys Eng Sci 2011;369(1955):4666–78. 9. Lasser T, Ntziachristos V. Optimization of 360 degrees projection fluorescence molecular tomography. Med Image Anal 2007;11(4):389–99. 10. Werner SG, Langer HE, Ohrndorf S, et al. Inflammation assessment in patients with arthritis using a novel in vivo fluorescence optical imaging technology. Ann Rheum Dis 2011 Oct 12 [Epub ahead of print]. 11. van Dam GM, Themelis G, Crane LMA, et al. Intraoperative tumor-specific fluorescence imaging in ovarian cancer by folate receptor-alpha targeting: first in-human results. Nat Med 2011;17(10):1315–9. 12. Huang D, Swanson EA, Lin CP, et al. Optical coherence tomography. Science 1991;254(5035):1178–81. 13. Ale A, Ermolayev V, Herzog E, Cohrs C, de Angelis MH, Ntziachristos V. FMTXCT: in vivo animal studies with hybrid fluorescence molecular tomography– X-ray computed tomography. Nat Meth 2012;9(6):615–20.