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Raman spectroscopy for neoplastic tissue differentiation: a pilot study
Raman Spectroscopy for Neoplastic Tissue Differentiation: A Pilot Study By Attila Lorincz, Daad Haddad, Ratna Naik, Vaman Naik, Alan Fung, Alex Cao, Prasad Manda, Abhilash Pandya, Greg Auner, Rajah Rabah, Scott E. Langenburg, and Michael D. Klein Detroit, Michigan and Dearborn, Michigan
Background: Several changes occur during the transformation of normal tissue to neoplastic tissue. Such changes in molecular composition can be detected by Raman spectroscopy. Raman spectroscopy is a nondestructive method of measuring these changes, which suggests the possibility of real-time diagnosis during medical procedures. Methods: This study seeks to evaluate the ability of Raman spectra to distinguish tissues. The Raman signatures of normal kidney, lung, and liver tissue samples from pigs and rats were characterized in vitro. Further, a human neuroblastoma and a hepatoblastoma, obtained at resection were also studied. Results: The Raman spectra of the animal samples of kidney, liver, and lung are distinctly different in the intensity distribution of the Raman peaks. Further, the spectra of a given
R
AMAN SPECTROSCOPY is a vibrational spectroscopic technique that originates from inelastic scattering of light by vibrating molecules. A Raman “spectrum” displays intensity as a function of frequency difference (the Raman shift) between the incident and scattered light. A Raman spectrum of a given molecule consists of a series of peaks or “bands” each shifted by one of the characteristic vibrational frequencies of that molecule. Each molecule has its own characteristic spectrum, and, thus, a Raman spectrum can provide a “fingerprint” of a substance from which molecular composition can be determined. Further, the intensity of a band is proportional to the concentration of the molecule from which the band arises. For example, proteins, nucleic acids, polysaccharides, and carotenoids have their own set of characteristic bands. It is important, however, to recognize that there is considerable overlap among the bands of different components of a tissue; hence, it is necessary to look at all/many bands of a given type of molecule. Thus, Raman spectroscopy provides detailed information about the biomolecular composition of tissues, which might be used to distinguish between normal, borderline, and malignant tissues. As a preliminary step, we chose an animal model to determine if Raman spectroscopy could provide unique tissue differentiation signatures. We sought to find the specific charac-
Journal of Pediatric Surgery, Vol 39, No 6 (June), 2004: pp 953-956
organ from pigs and rats, although similar, were different enough to distinguish between the 2 animals. In the patient tissues, the Raman spectra of normal liver, viable tumor, and fibrotic hepatoblastoma were very different. Fibrotic tissue showed a greater concentration of carotenoids, whereas viable tissue was rich in proteins and nucleic acids. The normal tissue showed both components. Similar differences were also seen in the neuroblastoma tissue. Conclusions: The results of this study show the potential use of Raman spectroscopy in clinical diagnosis. J Pediatr Surg 39:953-956. © 2004 Elsevier Inc. All rights reserved. INDEX WORDS: Raman spectroscopy, Raman spectra, peak intensity.
teristics of the spectra that represent the typical features of a given tissue. MATERIALS AND METHODS After receiving approval from the Animal Investigation Committee of Wayne State University (A-05-02-01) we harvested tissue from pigs that were undergoing surgery for other studies, just before their being killed. These pigs were well and had been used to evaluate minimally invasive surgery, so they had received no drugs other than anesthetic agents and antibiotics, and had undergone no procedures other than minimally invasive cholecystectomy, fundoplication, portoenterostomy, or intestinal resection and anastomosis. Tissue was similarly harvested from rats that were undergoing femoral artery microvascular
From the Department of Pediatric Surgery, Children’s Hospital of Michigan; the Departments of Physics and Astronomy, Biomedical Engineering, and Electrical and Computer Engineering, Wayne State University, the Department of Surgery, Wayne State University, School of Medicine, Detroit, MI; and Department of Natural Sciences, University of Michigan-Dearborn, Dearborn, MI. Presented at the 55th Annual Meeting of the Section on Surgery of the American Academy of Pediatrics, New Orleans, Louisiana, October 31-November 2, 2003. Supported in part by grants from the Maxine and Stuart Frankel Foundation, the Ensure Foundation, and the Festival of Trees. Address reprint requests to Michael D. Klein, MD, Department of Pediatric Surgery, Children’s Hospital of Michigan, 3901 Beaubien Blvd, Detroit, MI 48201. © 2004 Elsevier Inc. All rights reserved. 0022-3468/04/3906-0035$30.00/0 doi:10.1016/j.jpedsurg.2004.02.043
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Fig 1. Representative Raman spectra of kidney, liver, and lung samples. (A) Pig raw data for kidney, liver, and lung. (B) Rat raw data for kidney, liver, and lung. (C) Pig normalized data for kidney, liver, and lung. (D) Rat normalized data for kidney, liver, and lung.
surgery and were planned to be killed. Tissues were placed in saline and taken directly to the Raman laboratory for immediate analysis. The same samples were examined and identified by histopathologist. Kidney, liver, and lung specimens were taken from 8 pigs and 4 rats. Three or 4 samples of tissue from each type of organ were used. About 20 spectra were recorded for a given type of organ for pigs and about 10 spectra for rats. In addition to the animal model, after receiving permission from the Institutional Review Board of Wayne State University (Protocol 018201MP4F), we obtained Raman spectra for a human neuroblastoma and a human hepatoblastoma. All Raman spectra were recorded using a single monochromator (Instruments SA-Triax 550, Edison, NJ), equipped with conventional optics, holographic supernotch filters, and a charge coupled device detector. A 514.5-nm excitation line was used to record spectra in a nearly back-scattering geometry. A laser power of approximately 15 mW was focused at the sample. The resolution of the instrument was approximately 4 cm⫺1, and the calibration of the spectrometer was periodically checked with Si phonon and Hg lines. The wavenumber accuracy of the peaks is estimated to be ⫾1 cm⫺1. Once the raw data were obtained, several steps were taken to normalize the raw data. The data were first denoised to remove the noise and boost the signal-to-noise ratio. The signal also was filtered of fluorescence backgrounds and then normalized using the standard normal variate method.
RESULTS
Typical Raman spectra for each organ are displayed in Fig 1 A-D from both pigs and rats. Also shown is the raw versus normalized data to illustrate the difference in the signal before and after processing. There were no differences in spectra of different samples from the same organ from the same animal species. For example, the Raman spectra of a set of kidney samples taken from 1 pig are identical to a second set of
samples taken from a different pig. Figure 1A shows typical Raman spectra for kidney, liver, and lung tissues of pigs. For a comparison, Fig 1 B shows similar spectra for tissues of rats. Although the spectral features are similar, there are subtle changes in the frequencies and the intensity distribution that distinguish the tissue from different organs of a given species, as well as from the same organ from different species. The Raman spectra of tissue components such as lipids, proteins, and nucleotides are distinct. Hence, the Raman spectra are tissue specific with respect to peak positions and their intensities. There is an overlap of Raman bands owing to several components. However, for a given type of tissue, one can distinguish the composition of the tissue in terms of its principal components (ie, lipids, proteins, nucleotides) by the position and intensities of the characteristic Raman bands of the components. For example, typical tissue Raman bands are seen at approximately 1,655 cm⫺1 (protein amide I), approximately 1545 cm⫺1 (protein amide II), and approximately 1455 cm⫺1 (CH2 bending mode). For clarity, Figs 2A-C make a comparison of pig and rat tissues from similar organs. Again, although the spectra from a given organ are similar, there are small, yet discernible differences. The observed similarity in the Raman spectra of kidneys in pig and rat suggest that they have similar molecular composition. Lungs have a prominent peak around 1,634 cm⫺1, which is attributed to the amide I band, although this is less pronounced in kidneys and livers. In the spectra of
RAMAN SPECTROSCOPY
Fig 2.
955
Comparison of pig and rat normalized data for kidney, liver, and lung. (A) Kidney, (B) liver, (C) lung.
rats, the peak of approximately 1510 cm⫺1 (possibly owing to CH in-plane bending vibration from the adenine nucleotide or amino acid tyrosine) is well resolved compared with the spectra of the pigs. The same was found to be true for comparison of the liver and lung (Figs 2B & C). The relative intensities of such Raman bands will be key to the diagnosis of normal versus neoplastic tissue. The spectra of the 2 human tumors and the normal adjacent tissue are shown in Figs 3A & B. The Raman spectra of normal liver, viable tumor, and fibrotic hepatoblastoma tissues of the human samples are very different (Fig 3A). Fibrotic tissue shows a greater concentration of carotenoids, whereas viable tissue is rich in proteins and nucleic acids. However, the normal tissue shows both the components. If such assertions can be confirmed with biochemical analyses of these tissues for their relative content of carotenoids, lipids, proteins, and nucleic acids, then the Raman spectra can be used to distinguish the viable and normal hepatoblastoma tissues. Also shown is the Raman spectrum of the saline solution in which the sample was kept before testing. It’s clear that the saline solution does not contribute to any of the main Raman bands. Figure 3 B shows the Raman spectra from various parts of a neuroblastoma. Normal tissue was not avail-
able from this patient. We can, however, infer from the Raman data that the white nodule is rich in carotenoids, whereas red nodule is rich in proteins/nucleic acids. These observations are similar to the data presented in Fig 3 A for the fibrotic and viable tissues of hepatoblastoma. By a comparison of Raman spectra of neuroblastoma with hepatoblastoma tissues, one can infer that the red nodules in neuroblastoma correspond to malignant regions. DISCUSSION
Raman Spectroscopy is a nondestructive technique that uses specific excitation laser wavelength and laser power so that tissue sample damage is avoided. It can provide quantitative chemical composition, identify tissue constituents, and distinguish pathological changes. By measuring intensity of Raman marker bands of proteins, lipids, or nucleotides, the relative ratios and absolute concentration of each component can be determined and related to pathologic changes.1-8 Wolthuis et al9 studied the possibility of tissue characterization using near-infrared (780 nm while we used 514 nm) Raman spectroscopy and have presented a brief overview of the results of previously reported Raman studies for the detection of cancer. Most of these studies have reported differences in the intensities of the Raman bands ob-
Fig 3. Human tumor samples. (A) Hepatoblastoma from a 6-month old female. Arrows represent carotenoid bands, and asterisks represent proteins and nucleic acids. (B) Adrenal neuroblastoma of a 7-year-old female.
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served in normal breast compared with breast cancer. Raman spectra of normal and cancerous tissues of the gynecologic tract, the colorectal carcinomas, and bladder cancers also show spectral differences, which were attributed to a probable lower total lipid content and a higher protein content in cancerous tissues. Wolthuis et al9 used a multivariate statistical model to analyze the Raman spectra of the esophagus of 3 patients with adenocarcinoma. Their results showed that the multivariate statistical model was able to separate the normal epithelium from Barrett’s epithelium and adenocarcinoma. Gniadecka et al10,11 used Raman spectroscopy and
the artificial neural network to analyze the data and were able to distinguish normal skin from basal cell carcinoma for a set of 32 samples. We believe that a Raman probe can be constructed to allow real-time in vivo differentiation of neoplastic from normal tissue and perhaps real-time identification of the specific tumor. Using a commercially available Raman spectrometer, we were able to distinguish a variety of normal tissues from different animals. In addition, noticeable differences in the Raman spectra of normal and cancerous regions of human pediatric tumors has been shown.
REFERENCES 1. Frank CJ, McCreery RL, Redd DC: Raman spectroscopy of normal and diseased human breast tissues. Anal Chem 67:777-783, 1995 2. Liu CH, Das BB, Sha Glassmann WL, et al: Raman, fluorescence, and time-resolved light scattering as optical diagnostic techniques to separate diseased and normal biomedical media. J Photochem Photobiol B 16:187-209, 1992 3. Mizuno A, Kitajima H, Kawauchi K, et al: Near infrared fourier transformed Raman spectroscopic study of human brain tissue and tumors. J Raman Spectrosc 25:25-29, 1994 4. Utzinger U, Heintzelman DL, Mahadevan-Jansen A, et al: Near infrared Raman spectroscopy for in vivo detection of cervical precancers. Applied Spectroscopy 55:955-959, 2001 5. Mahadevan-Jansen A, Richards-Kortum R: Raman spectroscopy for the detection of cancers and precancers. J Biomed Opt 1:31-70, 1996 6. Mahadevan-Jansen A, Mitchell MF, Ramanujam N, et al: Devel-
opment of a fiber optic probe to measure NIR Raman spectra of cervical tissue in vivo. Photochem Photobiol 68:427-431, 1998 7. Hanlon EB, Manoharan R, Koo TW, et al: Prospects for in vivo Raman spectroscopy. Phys Med Biol 45:R1-59, 2000 8. Alfano RR, Liu CH, Sha WL, et al: Human breast tissue studied by IR fourier transform Raman Spectroscopy. Laser Life Sci 4:23-28, 1991 9. Wolthuis R, Bakker Schut TC, Caspers PJ, et al: Raman spectroscopic methods for in vitro and in vivo tissue characterization, in Mason WT (ed): Fluorescent and Luminencent Probes for Biological Activity. Cambridge, UK, Academic Press, 1999, pp 433-455 10. Gniadecka M, Wulf HC, Nielson OF, et al: Distinctive molecular abnormalities in benign and malignant skin lesions: Studies by Raman spectroscopy. Photochem Photobiol 66:418-423, 1997 11. Gniadecka M, Wulf HC, Mortensen NN, et al: Diagnosis of basal cell carcinoma by Raman spectroscopy. Raman Spectros 28:125130, 1997
Discussion S.J. Shochat (Memphis, TN): My only comment would be that in many neuroblastomas that have been previously treated there’s a mixture of fibrosis with foci of viable tumor throughout the fibrosis. Do you really think that your technique is going to be able to sort that out? A. Lorincz (response): We are pioneering in this study, and we intend to buy a new Raman microscope, and we would like to do in vivo evaluation using the Raman microscope and obtain tissue from the OR directly. This
study was done first for pathologic evaluation and was proved by the pathologic examination. So we already know which is a fibrotic tissue and which are normal and abnormal tissues. In the future, we have to compare as much data as possible to see clearly the difference between normal and abnormal tissue. So, using the Raman, you can distinguish the normal and abnormal tissue, but you cannot tell exactly which grade is the tumor actually.
Background: Several changes occur during the transformation of normal tissue to neoplastic tissue. Such changes in molecular composition can be detected by Raman spectroscopy. Raman spectroscopy is a nondestructive method of measuring these changes, which suggests the possibility of real-time diagnosis during medical procedures. Methods: This study seeks to evaluate the ability of Raman spectra to distinguish tissues. The Raman signatures of normal kidney, lung, and liver tissue samples from pigs and rats were characterized in vitro. Further, a human neuroblastoma and a hepatoblastoma, obtained at resection were also studied. Results: The Raman spectra of the animal samples of kidney, liver, and lung are distinctly different in the intensity distribution of the Raman peaks. Further, the spectra of a given
R
AMAN SPECTROSCOPY is a vibrational spectroscopic technique that originates from inelastic scattering of light by vibrating molecules. A Raman “spectrum” displays intensity as a function of frequency difference (the Raman shift) between the incident and scattered light. A Raman spectrum of a given molecule consists of a series of peaks or “bands” each shifted by one of the characteristic vibrational frequencies of that molecule. Each molecule has its own characteristic spectrum, and, thus, a Raman spectrum can provide a “fingerprint” of a substance from which molecular composition can be determined. Further, the intensity of a band is proportional to the concentration of the molecule from which the band arises. For example, proteins, nucleic acids, polysaccharides, and carotenoids have their own set of characteristic bands. It is important, however, to recognize that there is considerable overlap among the bands of different components of a tissue; hence, it is necessary to look at all/many bands of a given type of molecule. Thus, Raman spectroscopy provides detailed information about the biomolecular composition of tissues, which might be used to distinguish between normal, borderline, and malignant tissues. As a preliminary step, we chose an animal model to determine if Raman spectroscopy could provide unique tissue differentiation signatures. We sought to find the specific charac-
Journal of Pediatric Surgery, Vol 39, No 6 (June), 2004: pp 953-956
organ from pigs and rats, although similar, were different enough to distinguish between the 2 animals. In the patient tissues, the Raman spectra of normal liver, viable tumor, and fibrotic hepatoblastoma were very different. Fibrotic tissue showed a greater concentration of carotenoids, whereas viable tissue was rich in proteins and nucleic acids. The normal tissue showed both components. Similar differences were also seen in the neuroblastoma tissue. Conclusions: The results of this study show the potential use of Raman spectroscopy in clinical diagnosis. J Pediatr Surg 39:953-956. © 2004 Elsevier Inc. All rights reserved. INDEX WORDS: Raman spectroscopy, Raman spectra, peak intensity.
teristics of the spectra that represent the typical features of a given tissue. MATERIALS AND METHODS After receiving approval from the Animal Investigation Committee of Wayne State University (A-05-02-01) we harvested tissue from pigs that were undergoing surgery for other studies, just before their being killed. These pigs were well and had been used to evaluate minimally invasive surgery, so they had received no drugs other than anesthetic agents and antibiotics, and had undergone no procedures other than minimally invasive cholecystectomy, fundoplication, portoenterostomy, or intestinal resection and anastomosis. Tissue was similarly harvested from rats that were undergoing femoral artery microvascular
From the Department of Pediatric Surgery, Children’s Hospital of Michigan; the Departments of Physics and Astronomy, Biomedical Engineering, and Electrical and Computer Engineering, Wayne State University, the Department of Surgery, Wayne State University, School of Medicine, Detroit, MI; and Department of Natural Sciences, University of Michigan-Dearborn, Dearborn, MI. Presented at the 55th Annual Meeting of the Section on Surgery of the American Academy of Pediatrics, New Orleans, Louisiana, October 31-November 2, 2003. Supported in part by grants from the Maxine and Stuart Frankel Foundation, the Ensure Foundation, and the Festival of Trees. Address reprint requests to Michael D. Klein, MD, Department of Pediatric Surgery, Children’s Hospital of Michigan, 3901 Beaubien Blvd, Detroit, MI 48201. © 2004 Elsevier Inc. All rights reserved. 0022-3468/04/3906-0035$30.00/0 doi:10.1016/j.jpedsurg.2004.02.043
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Fig 1. Representative Raman spectra of kidney, liver, and lung samples. (A) Pig raw data for kidney, liver, and lung. (B) Rat raw data for kidney, liver, and lung. (C) Pig normalized data for kidney, liver, and lung. (D) Rat normalized data for kidney, liver, and lung.
surgery and were planned to be killed. Tissues were placed in saline and taken directly to the Raman laboratory for immediate analysis. The same samples were examined and identified by histopathologist. Kidney, liver, and lung specimens were taken from 8 pigs and 4 rats. Three or 4 samples of tissue from each type of organ were used. About 20 spectra were recorded for a given type of organ for pigs and about 10 spectra for rats. In addition to the animal model, after receiving permission from the Institutional Review Board of Wayne State University (Protocol 018201MP4F), we obtained Raman spectra for a human neuroblastoma and a human hepatoblastoma. All Raman spectra were recorded using a single monochromator (Instruments SA-Triax 550, Edison, NJ), equipped with conventional optics, holographic supernotch filters, and a charge coupled device detector. A 514.5-nm excitation line was used to record spectra in a nearly back-scattering geometry. A laser power of approximately 15 mW was focused at the sample. The resolution of the instrument was approximately 4 cm⫺1, and the calibration of the spectrometer was periodically checked with Si phonon and Hg lines. The wavenumber accuracy of the peaks is estimated to be ⫾1 cm⫺1. Once the raw data were obtained, several steps were taken to normalize the raw data. The data were first denoised to remove the noise and boost the signal-to-noise ratio. The signal also was filtered of fluorescence backgrounds and then normalized using the standard normal variate method.
RESULTS
Typical Raman spectra for each organ are displayed in Fig 1 A-D from both pigs and rats. Also shown is the raw versus normalized data to illustrate the difference in the signal before and after processing. There were no differences in spectra of different samples from the same organ from the same animal species. For example, the Raman spectra of a set of kidney samples taken from 1 pig are identical to a second set of
samples taken from a different pig. Figure 1A shows typical Raman spectra for kidney, liver, and lung tissues of pigs. For a comparison, Fig 1 B shows similar spectra for tissues of rats. Although the spectral features are similar, there are subtle changes in the frequencies and the intensity distribution that distinguish the tissue from different organs of a given species, as well as from the same organ from different species. The Raman spectra of tissue components such as lipids, proteins, and nucleotides are distinct. Hence, the Raman spectra are tissue specific with respect to peak positions and their intensities. There is an overlap of Raman bands owing to several components. However, for a given type of tissue, one can distinguish the composition of the tissue in terms of its principal components (ie, lipids, proteins, nucleotides) by the position and intensities of the characteristic Raman bands of the components. For example, typical tissue Raman bands are seen at approximately 1,655 cm⫺1 (protein amide I), approximately 1545 cm⫺1 (protein amide II), and approximately 1455 cm⫺1 (CH2 bending mode). For clarity, Figs 2A-C make a comparison of pig and rat tissues from similar organs. Again, although the spectra from a given organ are similar, there are small, yet discernible differences. The observed similarity in the Raman spectra of kidneys in pig and rat suggest that they have similar molecular composition. Lungs have a prominent peak around 1,634 cm⫺1, which is attributed to the amide I band, although this is less pronounced in kidneys and livers. In the spectra of
RAMAN SPECTROSCOPY
Fig 2.
955
Comparison of pig and rat normalized data for kidney, liver, and lung. (A) Kidney, (B) liver, (C) lung.
rats, the peak of approximately 1510 cm⫺1 (possibly owing to CH in-plane bending vibration from the adenine nucleotide or amino acid tyrosine) is well resolved compared with the spectra of the pigs. The same was found to be true for comparison of the liver and lung (Figs 2B & C). The relative intensities of such Raman bands will be key to the diagnosis of normal versus neoplastic tissue. The spectra of the 2 human tumors and the normal adjacent tissue are shown in Figs 3A & B. The Raman spectra of normal liver, viable tumor, and fibrotic hepatoblastoma tissues of the human samples are very different (Fig 3A). Fibrotic tissue shows a greater concentration of carotenoids, whereas viable tissue is rich in proteins and nucleic acids. However, the normal tissue shows both the components. If such assertions can be confirmed with biochemical analyses of these tissues for their relative content of carotenoids, lipids, proteins, and nucleic acids, then the Raman spectra can be used to distinguish the viable and normal hepatoblastoma tissues. Also shown is the Raman spectrum of the saline solution in which the sample was kept before testing. It’s clear that the saline solution does not contribute to any of the main Raman bands. Figure 3 B shows the Raman spectra from various parts of a neuroblastoma. Normal tissue was not avail-
able from this patient. We can, however, infer from the Raman data that the white nodule is rich in carotenoids, whereas red nodule is rich in proteins/nucleic acids. These observations are similar to the data presented in Fig 3 A for the fibrotic and viable tissues of hepatoblastoma. By a comparison of Raman spectra of neuroblastoma with hepatoblastoma tissues, one can infer that the red nodules in neuroblastoma correspond to malignant regions. DISCUSSION
Raman Spectroscopy is a nondestructive technique that uses specific excitation laser wavelength and laser power so that tissue sample damage is avoided. It can provide quantitative chemical composition, identify tissue constituents, and distinguish pathological changes. By measuring intensity of Raman marker bands of proteins, lipids, or nucleotides, the relative ratios and absolute concentration of each component can be determined and related to pathologic changes.1-8 Wolthuis et al9 studied the possibility of tissue characterization using near-infrared (780 nm while we used 514 nm) Raman spectroscopy and have presented a brief overview of the results of previously reported Raman studies for the detection of cancer. Most of these studies have reported differences in the intensities of the Raman bands ob-
Fig 3. Human tumor samples. (A) Hepatoblastoma from a 6-month old female. Arrows represent carotenoid bands, and asterisks represent proteins and nucleic acids. (B) Adrenal neuroblastoma of a 7-year-old female.
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served in normal breast compared with breast cancer. Raman spectra of normal and cancerous tissues of the gynecologic tract, the colorectal carcinomas, and bladder cancers also show spectral differences, which were attributed to a probable lower total lipid content and a higher protein content in cancerous tissues. Wolthuis et al9 used a multivariate statistical model to analyze the Raman spectra of the esophagus of 3 patients with adenocarcinoma. Their results showed that the multivariate statistical model was able to separate the normal epithelium from Barrett’s epithelium and adenocarcinoma. Gniadecka et al10,11 used Raman spectroscopy and
the artificial neural network to analyze the data and were able to distinguish normal skin from basal cell carcinoma for a set of 32 samples. We believe that a Raman probe can be constructed to allow real-time in vivo differentiation of neoplastic from normal tissue and perhaps real-time identification of the specific tumor. Using a commercially available Raman spectrometer, we were able to distinguish a variety of normal tissues from different animals. In addition, noticeable differences in the Raman spectra of normal and cancerous regions of human pediatric tumors has been shown.
REFERENCES 1. Frank CJ, McCreery RL, Redd DC: Raman spectroscopy of normal and diseased human breast tissues. Anal Chem 67:777-783, 1995 2. Liu CH, Das BB, Sha Glassmann WL, et al: Raman, fluorescence, and time-resolved light scattering as optical diagnostic techniques to separate diseased and normal biomedical media. J Photochem Photobiol B 16:187-209, 1992 3. Mizuno A, Kitajima H, Kawauchi K, et al: Near infrared fourier transformed Raman spectroscopic study of human brain tissue and tumors. J Raman Spectrosc 25:25-29, 1994 4. Utzinger U, Heintzelman DL, Mahadevan-Jansen A, et al: Near infrared Raman spectroscopy for in vivo detection of cervical precancers. Applied Spectroscopy 55:955-959, 2001 5. Mahadevan-Jansen A, Richards-Kortum R: Raman spectroscopy for the detection of cancers and precancers. J Biomed Opt 1:31-70, 1996 6. Mahadevan-Jansen A, Mitchell MF, Ramanujam N, et al: Devel-
opment of a fiber optic probe to measure NIR Raman spectra of cervical tissue in vivo. Photochem Photobiol 68:427-431, 1998 7. Hanlon EB, Manoharan R, Koo TW, et al: Prospects for in vivo Raman spectroscopy. Phys Med Biol 45:R1-59, 2000 8. Alfano RR, Liu CH, Sha WL, et al: Human breast tissue studied by IR fourier transform Raman Spectroscopy. Laser Life Sci 4:23-28, 1991 9. Wolthuis R, Bakker Schut TC, Caspers PJ, et al: Raman spectroscopic methods for in vitro and in vivo tissue characterization, in Mason WT (ed): Fluorescent and Luminencent Probes for Biological Activity. Cambridge, UK, Academic Press, 1999, pp 433-455 10. Gniadecka M, Wulf HC, Nielson OF, et al: Distinctive molecular abnormalities in benign and malignant skin lesions: Studies by Raman spectroscopy. Photochem Photobiol 66:418-423, 1997 11. Gniadecka M, Wulf HC, Mortensen NN, et al: Diagnosis of basal cell carcinoma by Raman spectroscopy. Raman Spectros 28:125130, 1997
Discussion S.J. Shochat (Memphis, TN): My only comment would be that in many neuroblastomas that have been previously treated there’s a mixture of fibrosis with foci of viable tumor throughout the fibrosis. Do you really think that your technique is going to be able to sort that out? A. Lorincz (response): We are pioneering in this study, and we intend to buy a new Raman microscope, and we would like to do in vivo evaluation using the Raman microscope and obtain tissue from the OR directly. This
study was done first for pathologic evaluation and was proved by the pathologic examination. So we already know which is a fibrotic tissue and which are normal and abnormal tissues. In the future, we have to compare as much data as possible to see clearly the difference between normal and abnormal tissue. So, using the Raman, you can distinguish the normal and abnormal tissue, but you cannot tell exactly which grade is the tumor actually.