- Email: [email protected]
[2.05] Optical monitoring of tissue coagulation during MRI-assisted interstitial laser thermotherapy (ILTT)
ARTICLE IN PRESS Congress Abstracts / Medical Laser Application 24 (2009) 120–142
120 fs) and a fiber-based system (Toptica, 90 MHz, 1090 nm, 20 fs) were used in combination with various fiber bundles (Sumitomo Electrics Industries). Two miniaturized objectives were examined, a GRIN-lens system (GRINTECH) and a custom made system (Storz), with a numerical aperture of 0.75 and 0.6, respectively. The fluorescent dyes, fluorescein and cresyl violet acetate, were used on sample materials and excised bladder wall tissue. Simulations of short-pulse propagation in the fiber materials were done with LAB2-A. Results: First measurements with our two-photon microendoscope showed that it is possible to detect fluorescence signals without compensating for linear and nonlinear pulse broadening. The outer diameter is already compatible with the working channel of a conventional cystoscope. Furthermore, the optical performance showed a good image quality at high axial resolution. Consequently, intracellular details could be resolved from bulky tissue with high-speed performance. For future optimizations, the simulations indicate an effective decrease of linear and non-linear pulse distortions by mainly quadratic pre-chirping, which may be implemented for example, using a simple grating compressor. Conclusion: The current microendoscope for two-photon imaging shows the potential to resolve intracellular details and can be integrated into a commercial endoscope. Two-photon endoscopy may be the possible next step to effectively overcome the limitations of confocal microendoscopy. In particular, the higher penetration depth may contribute to a broader acceptance for in-vivo tissue diagnostics. Acknowledgement: The work was carried out with the support of the ‘‘Bundesministerium fu¨r Bildung und Forschung (BMBF)’’ and the DFG Cluster of Excellence ‘‘Munich-Centre for Advanced Photonics’’. doi: 10.1016/j.mla.2009.02.010
[2.04] Multiphoton imaging of skin Karsten Ko¨niga,b a
Faculty of Mechatronics and Physics, Saarland University, Campus, 66123 Saarbruecken, Germany b JenLab GmbH, Schillerstraße 1, 07745 Jena, Germany Clinical multiphoton tomography and two-photon microendoscopy provide clinicians and researchers with high-resolution in-vivo optical biopsies based on twophoton autofluorescence, second harmonic generation, and fluorescence lifetime imaging. This review reflects state-of-the-art technology and reports on applications in the fields of early stage melanoma detection, skin
125
aging, nanoparticle imaging, tissue engineering, and in-situ screening of pharmaceutical and cosmetic products. So far, more than 500 patients and volunteers in Europe, Asia, and Australia have been investigated using these novel molecular imaging tools. The femtosecond multiphoton tomograph DermaInspects is the first multiphoton imaging system in clinical use. The novel tissue tomograph has a 0.5 m long flexible mirror arm in combination with piezo-driven focusing optics and multiple single-photon-counting PMT detectors. The photodetectors are particularly useful for obtaining information about the extracellular matrix as they simultaneous measure the two-photon autofluorescence of elastin as well as the second harmonic generation of collagen. A major application is the in-vivo determination of the age index of the skin. Multiphoton tomography has the potential to provide novel, non-invasive, high-resolution diagnostic tools on a cellular level under physiological conditions. doi: 10.1016/j.mla.2009.02.011
[2.05] Optical monitoring of tissue coagulation during MRI-assisted interstitial laser thermotherapy (ILTT) Michael Pellera, Maximilian F. Reisera, Ronald Srokab a
Josef Lissner Laboratory for Biomedical Imaging, Department of Clinical Radiology, University Hospital of Munich, Marchioninistr.15, 81377 Munich, Germany b Laser Research Laboratory, LIFE Center, Ludwig Maximilian University, Großhadern Medical Campus, Marchioninistr. 23, 81377 Munich, Germany Objective: Thermally altered tissue can be distinguished from native tissue by its optical parameters. Thus, a device, monitoring the evolution of diffuse backscattered test-light intensity, could detect this transition. The purpose of this study was to demonstrate technical feasibility of such a device, characterize its signal response during interstitial laser thermotherapy (ILTT) in comparison with MRI and to identify a switch-off parameter for ILTT. Material and methods: The backscattering light detector (BLD) is based on the interstitial measurement of a diffuse backscattered test light (HeNe laser: l=632 nm, P=5 mW) describing optical changes depending on the time at a certain position. The ILTT-induced spread of the coagulation zones, in muscle and liver tissue samples, were determined by macroscopic examination and MRI (1.5 T). MRI monitoring included temperature-sensitive parameter maps and postILTT T2-weighted images. The results were then correlated with the position and the time-dependent BLD signal.
ARTICLE IN PRESS 126
Congress Abstracts / Medical Laser Application 24 (2009) 120–142
Results: The backscatter intensity showed a reproducible
sigmoidal time course during the appearance of tissue whitening. A mean final signal increase of 3679% and a maximum temperature of 6579 1C in muscle could be determined. The increased backscatter-signal level of the sigmoidal curve was shown to be a possible switch-off criterion for ILTT. This was confirmed with T2weighted MRI and by dissection. Conclusion: The BLD based on a single fiber for transmitting a test light and detecting diffuse backscattered light provides additional online information on local changes in the tissue state and may serve as a guard, defining a local online switch-off criterion for ILTT. Combining locally confined optical information with multidimensional MRI parameters may synergistically improve thermotherapy monitoring. doi: 10.1016/j.mla.2009.02.012
[2.06] Quantitative fluorescence spectroscopy for the detection of cancer-specific enzymatic activity Tobias Becka, Herbert Steppa, Rainer Wittigb, Peter Schubertc, Markus Bo¨hlc, Markus Sauerd, Sigrun Henkenjohannd, Axel Du¨rkope, Christian Stephan Betza, Ann Johanssona a
Laser Research Laboratory, LIFE Center, Ludwig Maximilian University, Großhadern Medical Campus, Marchioninistr. 23, 81377 Munich, Germany b Institute for Laser Technologies in Medicine and Metrology (ILM) at the University of Ulm, Helmholtzstr. 12, 89081 Ulm, Germany c R-Biopharm AG, Landwehrstr. 54, 64293 Darmstadt, Germany d Applied Laser Physics and Laser Spectroscopy, University of Bielefeld, PO Box 100131, 33501 Bielefeld, Germany e Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, Universita¨tsstr. 31, 93040 Regensburg, Germany Objective: The visualization of cancer-specific enzymatic activities holds potential as a useful tool for early cancer detection and identification of aggressive tumors. DNaseX, which is typically found in early lesions, and TKTL1, which identifies aggressive carcinomas with a high metastatic potential, have been proposed as such cancer-specific enzymes. The enzymatic activity of DNaseX can be detected via molecular beacons i.e. the reversed fluorescence quenching produced when DNase cleavage of the DNA hairpin occurs, containing fluorochrome and quencher. Furthermore, an increase in rhodamine B hydrazide (RBH) fluorescence can be used to detect the accumulation of putatively specific
intermediates of TKTL1-related metabolism. However, for quantitative analysis of fluorophore concentration it is important to disentangle the effects of light absorption and scattering of the sample being investigated. Material and methods: A fiber-based system was developed for the quantitative detection of small concentrations of fluorescent dyes in scattering and absorbing media. Both the raw fluorescence and the intrinsic fluorescence signals were employed for fluorophore quantification. The intrinsic fluorescence was evaluated via a theoretical model for light distribution based on the diffusion approximation, and the measured reflectance and fluorescence spectra. The ability of the algorithm to assess the intrinsic fluorescence spectra was evaluated for different fluorophores, wavelength ranges and background optical properties. Results: The resulting intrinsic fluorescence spectra show less dependence on the optical properties of the sample than on the raw fluorescence signals. The ability to separate the effects of light absorption caused by the surrounding material was particularly good. Furthermore, the intensity of the intrinsic fluorescence correlated with the fluorophore concentration. Fluorophore concentrations and concentration variations could be detected in tens of picomolar. Conclusion: The fluorescence diagnostic system and evaluation algorithm showed good detection efficiency and quantification of the fluorescent probes investigated. The lower detection limit was in the range expected for fluorophore concentration within clinically obtained samples. This configuration thus presents a useful method for quantifying fluorophore concentration in clinically obtained tissue samples, which are related to the enzymatic activity of TKTL1 and DNaseX. Acknowledgements: The work was carried out with the support of the ‘‘Bundesministerium fu¨r Bildung und Forschung’’ (TumorVision; 13N9105). A. Johansson gratefully acknowledges the financial support from the Alexander von Humboldt Foundation. doi: 10.1016/j.mla.2009.02.013
120 fs) and a fiber-based system (Toptica, 90 MHz, 1090 nm, 20 fs) were used in combination with various fiber bundles (Sumitomo Electrics Industries). Two miniaturized objectives were examined, a GRIN-lens system (GRINTECH) and a custom made system (Storz), with a numerical aperture of 0.75 and 0.6, respectively. The fluorescent dyes, fluorescein and cresyl violet acetate, were used on sample materials and excised bladder wall tissue. Simulations of short-pulse propagation in the fiber materials were done with LAB2-A. Results: First measurements with our two-photon microendoscope showed that it is possible to detect fluorescence signals without compensating for linear and nonlinear pulse broadening. The outer diameter is already compatible with the working channel of a conventional cystoscope. Furthermore, the optical performance showed a good image quality at high axial resolution. Consequently, intracellular details could be resolved from bulky tissue with high-speed performance. For future optimizations, the simulations indicate an effective decrease of linear and non-linear pulse distortions by mainly quadratic pre-chirping, which may be implemented for example, using a simple grating compressor. Conclusion: The current microendoscope for two-photon imaging shows the potential to resolve intracellular details and can be integrated into a commercial endoscope. Two-photon endoscopy may be the possible next step to effectively overcome the limitations of confocal microendoscopy. In particular, the higher penetration depth may contribute to a broader acceptance for in-vivo tissue diagnostics. Acknowledgement: The work was carried out with the support of the ‘‘Bundesministerium fu¨r Bildung und Forschung (BMBF)’’ and the DFG Cluster of Excellence ‘‘Munich-Centre for Advanced Photonics’’. doi: 10.1016/j.mla.2009.02.010
[2.04] Multiphoton imaging of skin Karsten Ko¨niga,b a
Faculty of Mechatronics and Physics, Saarland University, Campus, 66123 Saarbruecken, Germany b JenLab GmbH, Schillerstraße 1, 07745 Jena, Germany Clinical multiphoton tomography and two-photon microendoscopy provide clinicians and researchers with high-resolution in-vivo optical biopsies based on twophoton autofluorescence, second harmonic generation, and fluorescence lifetime imaging. This review reflects state-of-the-art technology and reports on applications in the fields of early stage melanoma detection, skin
125
aging, nanoparticle imaging, tissue engineering, and in-situ screening of pharmaceutical and cosmetic products. So far, more than 500 patients and volunteers in Europe, Asia, and Australia have been investigated using these novel molecular imaging tools. The femtosecond multiphoton tomograph DermaInspects is the first multiphoton imaging system in clinical use. The novel tissue tomograph has a 0.5 m long flexible mirror arm in combination with piezo-driven focusing optics and multiple single-photon-counting PMT detectors. The photodetectors are particularly useful for obtaining information about the extracellular matrix as they simultaneous measure the two-photon autofluorescence of elastin as well as the second harmonic generation of collagen. A major application is the in-vivo determination of the age index of the skin. Multiphoton tomography has the potential to provide novel, non-invasive, high-resolution diagnostic tools on a cellular level under physiological conditions. doi: 10.1016/j.mla.2009.02.011
[2.05] Optical monitoring of tissue coagulation during MRI-assisted interstitial laser thermotherapy (ILTT) Michael Pellera, Maximilian F. Reisera, Ronald Srokab a
Josef Lissner Laboratory for Biomedical Imaging, Department of Clinical Radiology, University Hospital of Munich, Marchioninistr.15, 81377 Munich, Germany b Laser Research Laboratory, LIFE Center, Ludwig Maximilian University, Großhadern Medical Campus, Marchioninistr. 23, 81377 Munich, Germany Objective: Thermally altered tissue can be distinguished from native tissue by its optical parameters. Thus, a device, monitoring the evolution of diffuse backscattered test-light intensity, could detect this transition. The purpose of this study was to demonstrate technical feasibility of such a device, characterize its signal response during interstitial laser thermotherapy (ILTT) in comparison with MRI and to identify a switch-off parameter for ILTT. Material and methods: The backscattering light detector (BLD) is based on the interstitial measurement of a diffuse backscattered test light (HeNe laser: l=632 nm, P=5 mW) describing optical changes depending on the time at a certain position. The ILTT-induced spread of the coagulation zones, in muscle and liver tissue samples, were determined by macroscopic examination and MRI (1.5 T). MRI monitoring included temperature-sensitive parameter maps and postILTT T2-weighted images. The results were then correlated with the position and the time-dependent BLD signal.
ARTICLE IN PRESS 126
Congress Abstracts / Medical Laser Application 24 (2009) 120–142
Results: The backscatter intensity showed a reproducible
sigmoidal time course during the appearance of tissue whitening. A mean final signal increase of 3679% and a maximum temperature of 6579 1C in muscle could be determined. The increased backscatter-signal level of the sigmoidal curve was shown to be a possible switch-off criterion for ILTT. This was confirmed with T2weighted MRI and by dissection. Conclusion: The BLD based on a single fiber for transmitting a test light and detecting diffuse backscattered light provides additional online information on local changes in the tissue state and may serve as a guard, defining a local online switch-off criterion for ILTT. Combining locally confined optical information with multidimensional MRI parameters may synergistically improve thermotherapy monitoring. doi: 10.1016/j.mla.2009.02.012
[2.06] Quantitative fluorescence spectroscopy for the detection of cancer-specific enzymatic activity Tobias Becka, Herbert Steppa, Rainer Wittigb, Peter Schubertc, Markus Bo¨hlc, Markus Sauerd, Sigrun Henkenjohannd, Axel Du¨rkope, Christian Stephan Betza, Ann Johanssona a
Laser Research Laboratory, LIFE Center, Ludwig Maximilian University, Großhadern Medical Campus, Marchioninistr. 23, 81377 Munich, Germany b Institute for Laser Technologies in Medicine and Metrology (ILM) at the University of Ulm, Helmholtzstr. 12, 89081 Ulm, Germany c R-Biopharm AG, Landwehrstr. 54, 64293 Darmstadt, Germany d Applied Laser Physics and Laser Spectroscopy, University of Bielefeld, PO Box 100131, 33501 Bielefeld, Germany e Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, Universita¨tsstr. 31, 93040 Regensburg, Germany Objective: The visualization of cancer-specific enzymatic activities holds potential as a useful tool for early cancer detection and identification of aggressive tumors. DNaseX, which is typically found in early lesions, and TKTL1, which identifies aggressive carcinomas with a high metastatic potential, have been proposed as such cancer-specific enzymes. The enzymatic activity of DNaseX can be detected via molecular beacons i.e. the reversed fluorescence quenching produced when DNase cleavage of the DNA hairpin occurs, containing fluorochrome and quencher. Furthermore, an increase in rhodamine B hydrazide (RBH) fluorescence can be used to detect the accumulation of putatively specific
intermediates of TKTL1-related metabolism. However, for quantitative analysis of fluorophore concentration it is important to disentangle the effects of light absorption and scattering of the sample being investigated. Material and methods: A fiber-based system was developed for the quantitative detection of small concentrations of fluorescent dyes in scattering and absorbing media. Both the raw fluorescence and the intrinsic fluorescence signals were employed for fluorophore quantification. The intrinsic fluorescence was evaluated via a theoretical model for light distribution based on the diffusion approximation, and the measured reflectance and fluorescence spectra. The ability of the algorithm to assess the intrinsic fluorescence spectra was evaluated for different fluorophores, wavelength ranges and background optical properties. Results: The resulting intrinsic fluorescence spectra show less dependence on the optical properties of the sample than on the raw fluorescence signals. The ability to separate the effects of light absorption caused by the surrounding material was particularly good. Furthermore, the intensity of the intrinsic fluorescence correlated with the fluorophore concentration. Fluorophore concentrations and concentration variations could be detected in tens of picomolar. Conclusion: The fluorescence diagnostic system and evaluation algorithm showed good detection efficiency and quantification of the fluorescent probes investigated. The lower detection limit was in the range expected for fluorophore concentration within clinically obtained samples. This configuration thus presents a useful method for quantifying fluorophore concentration in clinically obtained tissue samples, which are related to the enzymatic activity of TKTL1 and DNaseX. Acknowledgements: The work was carried out with the support of the ‘‘Bundesministerium fu¨r Bildung und Forschung’’ (TumorVision; 13N9105). A. Johansson gratefully acknowledges the financial support from the Alexander von Humboldt Foundation. doi: 10.1016/j.mla.2009.02.013