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Nonlinear Optical Microscopy: Use of Second Harmonic Generation and Two-Photon Microscopy for Automated Quantitative Liver Fibrosis Studies

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Nonlinear Optical Microscopy: Use of Second Harmonic Generation and Two-Photon Microscopy for Automated Quantitative Liver Fibrosis Studies Nonlinear optical microscopy: use of second harmonic generation and two-photon microscopy for automated quantitative liver fibrosis studies The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Citation Sun, Wanxin et al. “Nonlinear Optical Microscopy: Use of Second Harmonic Generation and Two-photon Microscopy for Automated Quantitative Liver Fibrosis Studies.” Journal of Biomedical Optics 13.6 (2008): 064010. Web. 16 Feb. 2012. © 2008 SPIE - International Society for Optical Engineering As Published http://dx.doi.org/10.1117/1.3041159 Publisher SPIE - International Society for Optical Engineering Version Final published version Citable link http://hdl.handle.net/1721.1/69125 Terms of Use Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use. Journal of Biomedical Optics 13͑6͒, 064010 ͑November/December 2008͒ Nonlinear optical microscopy: use of second harmonic generation and two-photon microscopy for automated quantitative liver fibrosis studies Wanxin Sun* National University of Singapore Institute of Bioengineering and Nanotechnology Department of Physiology and Graduate Programme in The Nanos #4-01 Bioengineering 31 Biopolis Way Graduate School for Integrative Science and Engineering Singapore, Singapore 138669 and Tissue-Engineering Programme DSO Labs * Singapore, Singapore 117597 Shi Chang and Central-South University Singapore-MIT Alliance Xiangya Hospital E4-04-10, 4 Engineering Drive 3 Department of General Surgery Singapore, Singapore 117576 Changsha, Hunan 410008, China and and Massachusetts Institute of Technology National University of Singapore Department of Mechanical Engineering Department of Physiology Cambridge, Massachusetts 02139 MD9, 2 Medical Drive Singapore, Singapore 117597 Abstract. Liver fibrosis is associated with an abnormal in- crease in an extracellular matrix in chronic liver diseases. Dean C. S. Tai Institute of Bioengineering and Nanotechnology Quantitative characterization of fibrillar collagen in intact The Nanos #4-01 tissue is essential for both fibrosis studies and clinical ap- 31 Biopolis Way plications. Commonly used methods, histological staining Singapore, Singapore 138669 followed by either semiquantitative or computerized im- age analysis, have limited sensitivity, accuracy, and Nancy Tan operator-dependent variations. The fibrillar collagen in si- National University of Singapore nusoids of normal livers could be observed through Department of Physiology ͑ ͒ MD9, 2 Medical Drive second-harmonic generation SHG microscopy. The two- Singapore, Singapore 117597 photon excited fluorescence ͑TPEF͒ images, recorded si- and multaneously with SHG, clearly revealed the hepatocyte K. K. Women’s and Children’s Hospital Department of Paediatrics morphology. We have systematically optimized the pa- 100 Bukit Timah Road rameters for the quantitative SHG/TPEF imaging of liver Singapore, Singapore 229899 tissue and developed fully automated image analysis algo- rithms to extract the information of collagen changes and Guangfa Xiao cell necrosis. Subtle changes in the distribution and Central-South University amount of collagen and cell morphology are quantita- Xiangya Hospital Department of General Surgery tively characterized in SHG/TPEF images. By comparing Changsha, Hunan 410008, China to traditional staining, such as Masson’s trichrome and and Sirius red, SHG/TPEF is a sensitive quantitative tool for National University of Singapore automated collagen characterization in liver tissue. Our Department of Physiology system allows for enhanced detection and quantification MD9, 2 Medical Drive Singapore, Singapore 117597 of sinusoidal collagen fibers in fibrosis research and clini- cal diagnostics. © 2008 Society of Photo-Optical Instrumentation Engi- Huihuan Tang neers. ͓DOI: 10.1117/1.3041159͔ Central-South University Xiangya Hospital Keywords: second harmonic generation; two-photon excited Department of General Surgery fluorescence; liver fibrosis; fibrillar collagen; quantification. Changsha, Hunan 410008, China Paper 08095R received Mar. 21, 2008; revised manuscript received Jul. 18, 2008; accepted for publication Aug. 28, 2008; published on- Hanry Yu line Dec. 15, 2008. Institute of Bioengineering and Nanotechnology The Nanos #4-01 31 Biopolis Way Singapore, Singapore 138669 1 Introduction and Liver fibrosis is associated with the excessive deposition of extracellular matrix ͑ECM͒ proteins, such as collagen, as a recurrent wound healing responses to most chronic liver *Contributed equally as first authors. diseases.1 The abnormal increase in collagen fibers causes the Address all correspondence to: Dean C. S. Tai, Institute of Bioengineering and Nanotechnology, The Nanos #4-01, 31 Biopolis Way, Singapore 138669; Tel: ͑65͒68247191; Fax: ͑65͒64789080; E-mail: [email protected]. 1083-3668/2008/13͑6͒/064010/7/$25.00 © 2008 SPIE Journal of Biomedical Optics 064010-1 November/December 2008 b Vol. 13͑6͒ Downloaded from SPIE Digital Library on 07 Dec 2011 to 18.51.3.76. Terms of Use: http://spiedl.org/terms Sun et al.: Nonlinear optical microscopy: use of second harmonic generation… derangement of liver architecture, hampers intrahepatic blood Intraperitoneal injection of a 1:1 mixture of carbon tetra- ͑ ͒ flow, and subsequently causes persistent and progressive he- chloride CCl4 with vegetable oil was administered three patic dysfunction.2 The degree of liver fibrosis is a key indi- times a week at a dose of 0.1 mL/100 g, for up to three cator for staging and grading chronic liver diseases and for weeks, according to an established protocol for inducing liver therapeutic efficacy evaluation.3–6 Therefore, assessing col- fibrosis.29,30 Tissues were harvested on days 3, 14, and 21 lagen content in livers accurately and objectively is extremely ͑n=3 per group͒ under sodium pentobarbital. Cardiac perfu- important in both research and clinical context.3,6–8 sion with 4% paraformaldehyde was performed to flush out Grading of liver fibrosis is performed with descriptive or blood cells and fix the liver tissue before harvesting. Liver semiquantitative scoring systems using stained liver specimens were taken for both cryopreservation and conven- tissues.3–8 These methods focus on the qualitative rather than tional paraffin sections. Cryosections were processed at a quantitative properties of fibrosis development, and results are thickness of 50 ␮m while paraffin blocks were cut to a thick- 9–13 highly subjected to interpretations by the observers. It is ness of 4 ␮m. Human biopsy samples ͑three patients͒ were difficult to provide a highly reproducible and standardized fixed in buffered formalin, embedded in paraffin, and sec- 2 scoring system for diagnostic purposes. The intra- and inter- tioned at 4 ␮m in thickness. observer discrepancy is as high as 35% in the assessment of 9,14–17 liver fibrosis despite efforts to improve diagnostic accu- 2.2 Imaging System racy for qualitative scoring systems.4,7 These scoring systems 4 The nonlinear optical microscope was developed based on a remain rough classifications of the severity of fibrosis. confocal imaging system ͑LSM 510, Carl Zeiss͒ using an ex- Digital morphometric image analysis has been attempted ͑ 2,5,11,12,18,19 ternal tunable mode-locked Ti:sapphire laser Mai-Tai broad- to quantify liver fibrosis. A decrease of collagen band, Spectra-Physics͒. The laser was routed to an acousto- after treatment was observed in cases where conventional de- optic modulator ͑AOM͒ for power attenuation, then through a scriptive histology scoring failed to show any significant ͑ ͒ 20 dichroic mirror 490 nm and an objective lens to the tissue changes. Operator-input parameters are still required for fea- sample ͓Fig. 1͑a͔͒. TPEF emission generated in tissue was tures such as thresholding and segmentation, giving rise to collected by the same objective lens and recorded by a pho- interassay differences that bias the reproducibility and objec- tomultiplier tube ͑PMT͒, after passing through the dichroic tivity of quantifications.12 Here, we aim to develop a highly mirror and a 700-nm short-pass filter. SHG signal was col- objective and reproducible automated image-based liver fibro- lected using a high numerical aperture ͑NA͒ condenser and a sis quantification system.12,19,21,22 field diaphragm, and was filtered by a 450-nm bandpass filter Recent developments in both mode-locked lasers and ͑10-nm bandwidth͒ before entering PMT ͑Hamamatsu highly sensitive optical sensors have made nonlinear optical R6357͒ for detection. Note that, with the intrinsic optical sec- microscopy, such as the multiphoton excited fluorescence23,24 tioning characteristics for nonlinear optical process, the pin- and multiharmonic generation,25,26 an affordable option for hole function of the confocal microscope was not used. tissue imaging. Second-harmonic generation ͑SHG͒ micros- copy is used to measure highly ordered structures without 2.3 Imaging Acquisition central symmetry in tissue, such as type 1 collagen.27,28 Being a nonabsorption process that causes no photochemical dam- Samples were initially imaged using SHG/TPEF microscopy, age to specimens, SHG is ideal for tissue imaging. where tissue staining was not required. Two to three images We combined two-photon excited fluorescence ͑TPEF͒ and ͑4096ϫ4096 pixels with dimensions 3.68ϫ3.68 mm͒ were SHG microscopes and systematically optimized the param- scanned for each tissue slice, depending on sample size. eters
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