We evaluate the diagnostic feasibility of the integrated polarized near-infrared (NIR) autofluorescence (AF) and NIR diffuse reflectance (DR) imaging technique developed for colonic cancer detection. A total of 48 paired colonic tissue specimens (normal vs. cancer) were measured using the integrated NIR DR (850-1100 nm) and NIR AF imaging at the 785 nm laser excitation. The results showed that NIR AF intensities of cancer tissues are significantly lower than those of normal tissues (p<0.001, paired 2-sided Student’s t-test, n = 48). NIR AF imaging under polarization conditions gives a higher diagnostic accuracy (of ~92-94%) compared to non-polarized NIR AF imaging or NIR DR imaging. Further, the ratio imaging of NIR DR to NIR AF with polarization provides the best diagnostic accuracy (of ~96%) among the NIR AF and NIR DR imaging techniques. This work suggests that the integrated NIR AF/DR imaging under polarization condition has the potential to improve the early diagnosis and detection of malignant lesions in the colon.
© 2010 OSA
Gastrointestinal malignancies are the second leading cause of cancer-related death, and also the third most common cancer in the world . In Singapore, colonic cancer has become the most common malignancy for males while the second most common for females . In general, the diagnosis of colonic cancer is based on conventional white-light colonoscopic inspections followed by the histopathological examinations of biopsied tissues. However, the flat and depressed neoplastic lesions are difficult to identify due to the lack of obvious morphological changes under white light colonoscopy which heavily relies on the observation of gross morphological changes of tissues associated with neoplastic transformation . The accurate diagnosis and localization of early neoplastic tissue represents an important measure for planning effective treatment to improve the survival rates of patients with colonic malignancies . Hence, it is highly desirable to develop advanced diagnostic techniques that complement white-light endoscopy for improving the noninvasive, in vivo diagnosis and screening of early cancer in the GI tract. In the past two decades, autofluorescence (AF) spectroscopy and imaging techniques [5–10], which are capable of probing the changes of tissue morphological structures and concentrations of intrinsic fluorophores such as collagen, elastin, nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD), and porphyrins in tissue associated with neoplastic transformation, have been comprehensively investigated for improving the diagnostic sensitivity of malignant lesions in various organs, including the colon [7–15]. But to date, most of tissue AF studies are centered on the use of ultraviolet (UV) or short visible (VIS) wavelengths of excitation light that has a limited penetration depth and cannot detect lesions in deeper areas. Unlike UV excitation light, the near-infrared (NIR) light is non-carcinogenic, and it is safe for tissue diagnosis [16–19]. Both the excitation light used and the resulting tissue AF are at NIR wavelengths that can penetrate deeper into the tissue up to 1 mm [17,18], and thus NIR AF could potentially be useful for the noninvasive in vivo detection of lesions located deeper inside the tissue. In this work, we extend the previous UV/visible AF work [12–15,20] to the NIR domain for colonic tissue diagnosis and characterization. We examine if colonic tissue exhibits observable NIR autofluorescence under the 785 nm laser excitation, and evaluate if the integrated NIR AF imaging and NIR diffuse reflectance (DR) imaging with polarization technique can be used for improving colonic cancer diagnosis and detection.
2. Materials and methods
2.1 Integrated NIR autofluorescence and NIR diffuse reflectance imaging system
Figure 1 shows a schematic diagram of the integrated NIR AF and diffuse reflectance (DR) imaging system developed for tissue measurements. A 785 nm excitation light from a diode laser (maximum output: 300 mW, B&W Tec Inc, Newark, DE) is coupled into a 200 μm fiber and delivered into a collimator (F220SMA-B, Thorlabs, Newton, NJ) coupled with a narrow band-pass (BP) filter (LL01-785-12.5, Semrock Inc. Rochester, NY) for removing interference of fiber background fluorescence and laser noise. Then the filtered laser light is reflected by a dichroic mirror (Reflection: 450-800 nm, Transmission: 800-1400 nm; Semrock Inc., Rochester, NY) and shined onto the tissue specimen with a beam size of 10 mm. The induced AF emission from tissue passes through the dichroic mirror and a 850 nm long-pass filter (FEL0850, Thorlabs, Newton, NJ) and subsequently are collected by an NIR-optimized back-illuminated, deep-depletion charge-coupled device (CCD) detector (512x512 image pixels, 16 µm/pixel, Cascade II: 512, Photometrics, Tuscon, AZ). For the NIR DR imaging, a tungsten halogen light (HL-2000, Ocean Optics Inc., Dunedin, FL) is coupled into a 200 μm fiber and passes through a beam expander integrated with a polarizer to illuminate the tissue directly. The NIR diffuse reflectance photons from the tissue are collected by the CCD after passing through the dichroic mirror and the 850 nm long-pass filter. To acquire the AF and DR images under different polarization conditions, two linear polarizers (Model-10P109AR.16, Newport corporation, Irvine, CA) are placed along the AF and DR illumination light paths, and the parallel and perpendicular polarized AF/DR images can be acquired in tandem by rotating the analyzer positioned in front of the camera lens in the NIR AF/DR imaging system (Fig. 1). With the integrated NIR AF/DR imaging system developed, a set of six images can be acquired for colonic tissues in tandem, i.e., NIR AF image and the corresponding NIR DR image under three different excitation light polarization conditions (i.e., non-polarization; parallel and perpendicular polarization). The system acquires NIR AF images and DR images within the spectral bandwidth of 850-1100 nm, and each NIR AF image was acquired within 5 s with the 785 nm laser light irradiance of 0.15 W/cm2, while each NIR DR image was acquired within 0.01 s with the tungsten light incident power of 0.1 mW on the tissue surface.
2.2. Colonic tissue specimens
A total of 48 paired (i.e., normal vs cancer) colonic tissue specimens (average size of ~4 x 4 x 2 mm3) were collected from 48 patients (20 men and 28 women with a mean age of 62) who underwent partial colectomy or surgical resections with clinically suspicious lesions or histopathologically proven malignancies in the colon. All patients preoperatively signed an informed consent permitting the investigative use of the tissue, and this study was approved by the Institutional Review Board (IRB) of the National Healthcare Group (NHG) of Singapore. Immediately after surgical resections, the tissue specimens were immersed in physiological saline solution and sent to the Laboratory for NIR AF/DR imaging measurements. The paired tissue specimens from each patient were placed on a quartz glass slide (25 × 75 × 1.2 mm3) (cancer tissue was placed at the upper part of the slide while the normal tissue was placed at bottom part of the slide) for NIR imaging measurements (Fig. 1). After the NIR imaging acquisitions, the tissue specimens were fixed in 10% formalin solution and then submitted back to the hospital for histopathological examinations. The histopathogical examinations confirmed that 48 tissue specimens were normal, and 48 tissue specimens were cancer (moderately differentiated adenocarcinoma).
3. Results and discussion
Using the integrated NIR AF/DR imaging system developed, we have successfully acquired NIR AF/DR images of 48 paired colonic tissues under the three light excitation polarization conditions (i.e., non-polarization, parallel and perpendicular polarization). Figure 2(a) –2(c) shows the representative NIR AF images of the paired colonic specimens (normal (7.5 × 4 × 2 mm2 in size)) vs cancer (4 × 2.5 × 2.5 mm3 in size)) as confirmed by histological examinations): (a) NIR AF image without polarization; (b) NIR AF image with parallel polarization, and (c) NIR AF image with perpendicular polarization. Due to the heterogeneous nature of biological tissues, we observed the high intensity spots in both normal and tumor tissues. This could be attributed to the non-uniform distributions of related endogenous NIR fluorophores and surface morphologic effects in tissue. As such, the intensity line profiles [Fig. 2(d)] are drawn through the relatively homogenous regions of tissues located at around the center of the field of view (FOV) of NIR AF images [Fig. 2(a)–2(c)], illustrating that the cancer tissue shows a relatively lower NIR AF intensity than the normal tissue. The average intensities within the areas of 20x20 pixels in both normal and cancer tissue are calculated and compared. The first 20 pixels in the cancer tissue region, while the last 20 pixels in the normal tissue region in Fig. 2(d) are selected for intensity ratio calculations under the three different polarization conditions. The results show that NIR AF emission arising from cancer tissue reduces by 2.0-, 2.2-, and 2.4- fold, respectively, in intensity as compared to the normal tissue under the non-polarization, parallel and perpendicular polarization conditions. The differences of fluorescence intensity between normal and cancer tissue could be attributed to the changes of tissue optical properties of cancer tissue in the colon . For example, the proliferation of neoplastic cells caused the thickening of mucosal tissue in cancer tissue [20,21], which could significantly attenuate the excitation light penetration and also obscure the tissue AF emission from the tissue, resulting in an overall decrease of NIR AF intensity from cancer tissue as compared to normal colonic tissue. In addition, the changes in concentrations of endogenous fluorophores such as NADH, collagen, flavins, porphyrins, etc., in tissue associated with malignant transformation [20–22] may also attribute to the differences in the NIR AF emission between normal and cancer colonic tissue. But the origins of the specific endogenous fluorophores responsible for NIR AF emission in colonic tissue are still not well understood, which warrant further investigations. Nevertheless, the distinct differences in NIR AF emission between normal and cancer colonic tissue indicate that NIR AF imaging technique could be used to differentiate malignant lesions from normal tissue in the colon.
To compare the diagnostic performance of NIR AF imaging under different polarization conditions, NIR AF intensities are calculated from the relatively homogenous regions (~2 x 2 mm2) on the normal and cancer NIR AF images, respectively. We apply the threshold value of 1 as a decision line for the normal to tumor NIR AF intensity ratio algorithm for differentiation between normal and tumor tissues (i.e., NIR AF ratio of >1 classified as normal, whereas NIR AF ratio of 1 ≤ classified as tumor). Figure 3(a) –3(c) shows the pair-wise comparison of NIR AF intensities of all 48 paired (normal vs cancer) colonic tissues under the three polarization conditions (i.e., (a) non-polarization, (b) parallel and (c) perpendicular polarization). NIR AF intensities of cancer tissue are significantly lower than those of normal tissue with the p-values of 3.5E-4, 3.2E-8 and 5.8E-9, respectively, under the non-polarization, parallel and perpendicular polarization light excitation conditions (paired 2-sided Student’s t-test, n = 48). Based on the NIR AF intensity ratio of normal to cancer tissues (Inormal/Icancer), the diagnostic accuracies of 79.2% (38/48), 91.7% (44/48) and 93.8% (45/48), respectively, can be achieved by using the NIR AF imaging under the non-polarization, parallel and perpendicular polarization light excitation. Hence, the polarized NIR AF imaging was able to enhance the contrast between normal and cancer colonic tissue (Figs. 2 and 3) with a higher diagnostic accuracy (of ~92-94%) compared to the non-polarized AF imaging (accuracy of ~79%).
To explore the possible reason that the polarized NIR AF imaging performs better than the non-polarized NIR AF imaging technique for colonic cancer detection, we have also studied NIR AF polarization properties of normal and cancer colonic tissue by calculating the polarization ratio values (Ratio = (Ipar-Iper)/(Ipar + Iper)) in NIR AF images [Fig. 4(a) ]. It is observed that the polarization ratio values of cancer colonic tissue are in the range of 0.0001 to 0.01, while the polarization ratio values of normal tissue are much higher, ranging from 0.012 to 0.075 as shown in Fig. 4(b). Similar to the polarized reflectance imaging , the parallel-polarized NIR AF imaging contains the information mainly from the surface or shallower layer of the tissue, whereas the perpendicular-polarized NIR AF imaging reveals the information predominantly from deeper areas of the tissue [23,24]. A much reduced polarization ratio of cancer tissue reflects that much more multiple light scatterings may occur in deeper regions of tissue due to the disorganized structures of tissue in colonic adenocarcinoma, resulting in a larger contribution of the perpendicular polarized light component as compared to the normal tissue. As such, the polarized NIR AF imaging technique has the ability to selectively probe the AF light photons that arise from the subsurface or deep areas of tissue for improving cancer diagnosis and characterization.
One notes that tissue NIR AF image acquired depends on not only the tissue status (e.g., tissue surface structures, physiology or histopathology status, etc.), but also the measurement conditions (e.g., light excitation-tissue-collection configurations with respect to the tissue surface, illumination light power variation, etc.) . To eliminate the geometrical effects on NIR AF measurements such as the variations of the light source-tissue distance, the varying angles for the incident light and tissue fluorescence collections, and the irregularities of the tissue surface which are naturally encountered in practical tissue fluorescence imaging, we have also measured the NIR DR images from normal and cancer tissue serving as background image to normalize the NIR AF image for correcting the artifacts of NIR AF image non-uniformity. Figure 5 shows the NIR DR images of normal and cancer colonic tissue acquired under the three polarization conditions, which give no significance differences in NIR DR intensities between normal and cancer tissue (p-values of 0.20, 0.28 and 0.17, respectively for the non-polarization, parallel and perpendicular polarization conditions, paired 2-sided Student’s t-test, n = 48).
However, when normalize the NIR DR images [Figs. 5(a)–5(c)] to the corresponding NIR AF images [Figs. 2(a)–2(c)], much enhanced differences in NIR ratio imaging between normal and cancer tissue can be observed clearly in Figs. 6(a) –6(c) [with the p-values of 5.0E-5, 2.5E-9 and 7.8E-10, respectively under the non-polarization, parallel and perpendicular polarization conditions (paired 2-sided Student’s t-test, n = 48)], and the NIR DR/NIR AF ratio values of cancer tissue can be ~2.8-fold larger than those of normal tissue [Fig. 6(d)]. The diagnostic accuracies of 83.3% (40/48), 93.8% (45/48) and 95.8% (46/48), respectively, can also be achieved by using the NIR DR/NIR AF ratio imaging under the non-polarization, parallel and perpendicular polarization conditions. Therefore, with the ability of correcting the geometrical effects on NIR AF measurements, the NIR DR/NIR AF ratio imaging technique can further improve the diagnostic accuracy (of ~94 to 96%) for colonic cancer detection, and may also have potential to be used for assisting in delineating the margins of tumors for surgical operation. One notes that the NIR laser light and the white light are incident onto the tissue surface with different angles in the current NIR AF and NIR DR imaging system (Fig. 1), resulting in the intensity gradient changes across the tissue surfaces (Figs. 5 and 6). With further optimizations of the integrated NIR AF/NIR DR imaging system using a collinear-illumination configuration (i.e., the white light is coupled into the same optical path of the NIR laser light in the NIR imaging system), the NIR DR/NIR AF ratio imaging technique can totally remove the tissue surface geometric effects for better tissue diagnosis and characterization. On top of these, due to heterogeneous properties of biological tissues, there are certain regions are with common intensities in both normal and cancer tissues. Hence, further investigations on correlating the NIR AF/DR images with the exact histological mapping of tissues are required to fully evaluate the detection power of the integrated NIR AF/NIR DR imaging technique.
In summary, we found that under the 785 nm laser excitation, NIR AF emission from colonic tissue can be detected and imaged by the sensitive NIR imaging system. Significant differences in AF intensity are observed between normal and cancer colonic tissue, indicating the feasibility of NIR AF imaging technique for colonic cancer detection. The ratio imaging of NIR DR to NIR AF under polarization condition further improves the colonic cancer diagnosis and characterization. We anticipate that with further miniaturization of the current NIR excitation and imaging system coupled with an endoscope, the integrated NIR AF and NIR DR imaging with polarization excitation technique developed in this work may have the potential to be a clinically useful tool for in vivo diagnosis and detection of colonic cancer during colonoscopic examination.
This research was supported by the Biomedical Research Council, the National Medical Research Council, and the Faculty Research Fund from the National University of Singapore.
References and links
2. W. Du, J. T. L. Mah, J. Lee, R. Sankila, R. Sankaranarayanan, and K. S. Chia, “Incidence and survival of mucinous adenocarcinoma of the colorectum: a population-based study from an Asian country,” Dis. Colon Rectum 47(1), 78–85 (2004). [CrossRef] [PubMed]
3. R. M. Soetikno, T. Kaltenbach, R. V. Rouse, W. Park, A. Maheshwari, T. Sato, S. Matsui, and S. Friedland, “Prevalence of nonpolypoid (flat and depressed) colorectal neoplasms in asymptomatic and symptomatic adults,” JAMA 299(9), 1027–1035 (2008). [CrossRef] [PubMed]
5. A. L. McCallum, J. T. Jenkins, D. Gillen, and R. G. Molloy, “Evaluation of autofluorescence colonoscopy for the detection and diagnosis of colonic polyps,” Gastrointest. Endosc. 68(2), 283–290 (2008). [CrossRef] [PubMed]
7. S. Anandasabapathy, “Endoscopic imaging: emerging optical techniques for the detection of colorectal neoplasia,” Curr. Opin. Gastroenterol. 24(1), 64–69 (2008). [CrossRef]
10. D. Roblyer, R. Richards-Kortum, K. Sokolov, A. K. El-Naggar, M. D. Williams, C. Kurachi, and A. M. Gillenwater, “Multispectral optical imaging device for in vivo detection of oral neoplasia,” J. Biomed. Opt. 13(2), 024019 (2008). [CrossRef] [PubMed]
11. D. Pantalone, F. Andreoli, F. Fusi, V. Basile, G. Romano, G. Giustozzi, L. Rigacci, R. Alterini, and M. Monici, “Multispectral imaging autofluorescence microscopy in colonic and gastric cancer metastatic lymph nodes,” Clin. Gastroenterol. Hepatol. 5(2), 230–236 (2007). [CrossRef] [PubMed]
13. R. Richards-Kortum, R. P. Rava, R. E. Petras, M. Fitzmaurice, M. Sivak, and M. S. Feld, “Spectroscopic diagnosis of colonic dysplasia,” Photochem. Photobiol. 53(6), 777–786 (1991). [PubMed]
14. K. T. Schomacker, J. K. Frisoli, C. C. Compton, T. J. Flotte, J. M. Richter, N. S. Nishioka, and T. F. Deutsch, “Ultraviolet laser-induced fluorescence of colonic tissue: basic biology and diagnostic potential,” Lasers Surg. Med. 12(1), 63–78 (1992). [CrossRef] [PubMed]
15. H. Aihara, K. Sumiyama, S. Saito, H. Tajiri, and M. Ikegami, “Numerical analysis of the autofluorescence intensity of neoplastic and non-neoplastic colorectal lesions by using a novel videoendoscopy system,” Gastrointest. Endosc. 69(3 Pt 2), 726–733 (2009). [CrossRef] [PubMed]
17. Z. Huang, H. Zeng, I. Hamzavi, A. Alajlan, E. Tan, D. I. McLean, and H. Lui, “Cutaneous melanin exhibiting fluorescence emission under near-infrared light excitation,” J. Biomed. Opt. 11(3), 34010 (2006). [CrossRef] [PubMed]
18. Z. Huang, H. Lui, D. I. McLean, M. Korbelik, and H. Zeng, “Raman spectroscopy in combination with background near-infrared autofluorescence enhances the in vivo assessment of malignant tissues,” Photochem. Photobiol. 81(5), 1219–1226 (2005). [CrossRef] [PubMed]
19. E. B. Hanlon, I. Itzkan, R. R. Dasari, M. S. Feld, R. J. Ferrante, A. C. McKee, D. Lathi, and N. W. Kowall, “Near-infrared fluorescence spectroscopy detects Alzheimer’s disease in vitro,” Photochem. Photobiol. 70(2), 236–242 (1999). [PubMed]
20. Z. Huang, W. Zheng, S. Xie, R. Chen, H. Zeng, D. I. McLean, and H. Lui, “Laser-induced autofluorescence microscopy of normal and tumor human colonic tissue,” Int. J. Oncol. 24(1), 59–63 (2004).
21. G. I. Zonios, R. M. Cothren, J. T. Arendt, J. Wu, J. Van Dam, J. M. Crawford, R. Manoharan, and M. S. Feld, “Morphological model of human colon tissue fluorescence,” IEEE Trans. Biomed. Eng. 43(2), 113–122 (1996). [CrossRef] [PubMed]
22. R. Drezek, K. Sokolov, U. Utzinger, I. Boiko, A. Malpica, M. Follen, and R. Richards-Kortum, “Understanding the contributions of NADH and collagen to cervical tissue fluorescence spectra: modeling, measurements, and implications,” J. Biomed. Opt. 6(4), 385–396 (2001). [CrossRef] [PubMed]
25. J. Y. Qu, J. Hua, and Z. Huang, “Correction of geometrical effects on fluorescence imaging of tissue,” Opt. Commun. 176, 319–326 (2000). [CrossRef]