Infrared laser–mediated gene induction in targeted single cells in vivo Yasuhiro Kamei 1,2,8 , Motoshi Suzuki 3,8 , Kenjiro Watanabe 4,8 , Kazuhiro Fujimori 2 , Takashi Kawasaki 2 , Tomonori Deguchi 2 , Yoshihiro Yoneda 5 , Takeshi Todo 1,6 , Shin Takagi 3 , Takashi Funatsu 4,7 & Shunsuke Yuba 2 We developed infrared laser–evoked gene operator (IR-LEGO), a microscope system optimized for heating cells without photochemical damage. Infrared irradiation causes reproducible temperature shifts of the in vitro microenvironment in a power-dependent manner. When applied to living Caenorhabditis elegans, IR-LEGO induced heat shock–mediated expression of transgenes in targeted single cells in a more efficient and less deleterious manner than a 440-nm dye laser and elicited physiologically relevant phenotypic responses. Heat stress induces transcription of genes encoding heat shock proteins 1–3 . In transgenic organisms this response is exploited to manipulate gene expression in vivo, using heat as a trigger to induce expression of a gene cloned downstream of a heat shock promoter. Moreover, by irradiating living specimens with a laser beam under a microscope, it is possible to induce genes that are under the control of a heat shock promoter in individual targeted cells. This method has advantages over conventional techniques for ectopic gene expression 4–9 . First, it allows induction of gene expression in a specific cell without a tissue-specific promoter. Second, for induc- tion of gene expression in different cell types, only a single strain carrying a heat shock promoter–driven transgene is required. Most importantly, it enables the induction of gene expression in single targeted cells at a defined time. A cell-ablation microscope system with a pulsed 440-nm dye laser has previously been used for inducing gene expression mediated by the heat shock response 4–7 . Although gene expression can be induced with a 440-nm laser 4–7 or other visible lasers 8,9 , leading to relevant phenotypic responses in irradiated cells 5,7 , relatively long irradiation times are required for gene induction, and irradiation often has detrimental effects on cells 5,9 . Here we report an application of an infrared (IR) laser for heat shock response–mediated gene expression using a newly developed microscope system called IR-LEGO (Fig. 1a). The wavelength of IR (1,480 nm; Fig. 1a) matches the combination of symmetric and antisymmetric OH stretching modes of water and can heat water with B10 5 -fold higher efficiency than the 440-nm laser. To evaluate the utility of IR-LEGO, we first examined its in vitro heating profiles: time course of temperature changes, controllability of heating and spatial distribution of heat around the laser focus. To measure microenvironmental temperature, we exploited the temperature dependence of the fluorescence intensity of fluorescent proteins. We found that fluorescence of GFP and Laser diode (473 nm) a c d b Dichroic mirror CCD camera Confocal unit Actuator 60 15 56–59 53–56 50–53 47–50 44–47 41–44 38–41 35–38 32–35 29–32 (°C) 10 5 0 0 10 15 20 25 r ( m) z ( m) 5 –5 –10 –15 40 30 20 0 5 10 Laser on 30 mW 10 mW 15 Time (s) Temperature (°C) Temperature (°C) 50 Mirror Lens 60 50 40 30 20 0 10 20 30 40 Power (mW) Focus Objective IR laser (1,480 nm) Figure 1 | Schematic illustration of microscopic system for IR-LEGO, and thermal profiles of IR-laser irradiation in vitro.(a) The optical path of the IR-LEGO system. The basic system used for the in vivo study has an IR-laser inlet path (right). For the in vitro study, a confocal imaging system with a fixed microscope objective (left) was additionally installed. (b) Time course of estimated temperature at the focus of the IR laser at 30 mW (red) and 10 mW (blue). Ambient temperature was 25 1C. Laser powers represent the value at the specimen plane. (c) Relationship between induced temperature and power of the IR laser. Induced temperature was the value 5 s after the start of irradiation. Ambient temperature was 25 1C. (d) Three-dimensional thermal map during IR-laser irradiation at a power of 33 mW. r , distance from the z axis; z, depth along the z axis. The position of (r , z ¼ 0, 0) is the focus of the IR laser. The tissue model was irradiated from below with a laser beam of conical shape, along the z axis. The lower coverslip of the tissue model is located at z ¼ –18 mm. RECEIVED 21 JULY; ACCEPTED 29 OCTOBER; PUBLISHED ONLINE 14 DECEMBER 2008; DOI:10.1038/NMETH.1278 1 Radiation Biology Center, Kyoto University, Yoshida Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan. 2 National Institute of Advanced and Industrial Science and Technology (AIST), 1-8-31 Midorigaoka, Ikeda, Osaka 563-8577, Japan. 3 Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan. 4 Major in Bioscience and Biomedical Engineering, Waseda University, 3-4-1 Okubo, Shinjuku, Tokyo 169-8855, Japan. 5 Department of Cell Biology and Neuroscience, Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan. 6 Department of Radiation Biology and Medical Genetics, Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan. 7 Laboratory of Bio-Analytical Chemistry, Graduate School of Pharmaceutical Sciences, The University ofTokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033, Japan. 8 These authors contributed equally to this work. Correspondence should be addressed to Y.K. (ykamei@radbio.med.osaka-u.ac.jp) or S.Y. (s-yuba@aist.go.jp). NATURE METHODS | VOL.6 NO.1 | JANUARY 2009 | 79 BRIEF COMMUNICATIONS © 2009 Nature America, Inc. All rights reserved.