Label-free biochemical imaging of heart tissue with high-speed spontaneous Raman microscopy Mitsugu Ogawa a,b , Yoshinori Harada a , Yoshihisa Yamaoka a , Katsumasa Fujita c , Hitoshi Yaku b , Tetsuro Takamatsu a, * a Department of Pathology and Cell Regulation, Kyoto Prefectural University of Medicine, Graduate School of Medical Science, 465 Kajii-cho Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan b Department of Cardiovascular and Thoracic Surgery, Kyoto Prefectural University of Medicine, Graduate School of Medical Science, 465 Kajii-cho Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan c Department of Applied Physics, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan article info Article history: Received 25 February 2009 Available online 11 March 2009 Keywords: Label-free imaging Spontaneous Raman microscopy Resonance Raman effect Cytochrome Hemoglobin Collagen Biochemical change abstract Label-free imaging is desirable for elucidating morphological and biochemical changes of heart tissue in vivo. Spontaneous Raman microscopy (SRM) provides high chemical contrast without labeling, but presents disadvantage in acquiring images due to low sensitivity and consequent long imaging time. Here, we report a novel technique for label-free imaging of rat heart tissues with high-speed SRM com- bined with resonance Raman effect of heme proteins. We found that individual cardiomyocytes were identified with resonance Raman signal arising mainly from reduced b- and c-type cytochromes, and that cardiomyocytes and blood vessels were imaged by distinguishing cytochromes from oxy- and deoxy- hemoglobin in intact hearts, while cardiomyocytes and fibrotic tissue were imaged by distinguishing cytochromes from collagen type-I in infarct hearts with principal component analysis. These results sug- gest the potential of SRM as a label-free high-contrast imaging technique, providing a new approach for studying biochemical changes, based on the molecular composition, in the heart. Ó 2009 Elsevier Inc. All rights reserved. Introduction To elucidate morphological and biochemical changes of heart tissue in vivo, label-free imaging of tissue components is desirable. Any probes for particular substrates can potentially influence the environment of living tissues. Conventional histological examina- tions such as hematoxylin and eosin staining reveal distribution of tissue structural components, but cannot be applied in vivo. They require fixed and stained biopsy specimens, and thus are invasive and time-consuming procedures. Spontaneous Raman microscopy (SRM) provides specific vibra- tional signatures of chemical bonds as a spectrum [1], allowing identification of particular molecules without labeling [2]. The Raman spectra usually exhibit sharp spectral features based on specific molecular structures and conformations of tissues [3,4]. This makes SRM very useful for biomedical applications, such as quantitative histochemical analysis of atherosclerosis [5,6], disease diagnosis, especially for cancer [7,8], and physiological analysis of hemoglobin oxygen saturation [9,10]. However, these Raman spec- troscopic studies are not generally based on precise tissue imaging. Tissue imaging clarifies the distribution of cellular components in tissues, and is indispensable for morphometric analysis of a given tissue. As far as we know, there have been no reports on high-con- trast tissue imaging at the cellular level with Raman microscopy. The obstacles are low Raman scattering energy and thus require- ment of long measurement time. To overcome the drawbacks of low sensitivity and limited imag- ing speed of SRM, we have recently developed a Raman confocal microscope incorporating slit-scanning apparatus. The method of particular line illumination and parallel detection system short- ened the image acquisition time, from hours to minutes [11,12]. We then applied the well-established resonance Raman spectra of heme proteins for biomedical imaging, since the signal intensity, dependent upon the excitation wavelength, is 10 3 –10 5 times stron- ger than that of non-resonance signal, which markedly increases both the sensitivity and selectivity of Raman microscopy [13,14]. On the hypothesis that high-contrast imaging is possible by using our high-speed Raman microscope combined with resonance effect, we sought to visualize heart tissue components based on the molecular composition without staining. By using principal component analysis, we have performed for the first time label- free high-contrast imaging of cardiomyocytes, small artery and vein, and fibrosis based on Raman spectra of reduced b- and c-type 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.03.028 * Corresponding author. Fax: +81 75 251 5353. E-mail address: ttakam@koto.kpu-m.ac.jp (T. Takamatsu). Biochemical and Biophysical Research Communications 382 (2009) 370–374 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc