title: Development of an automated fluorescence microscopy system for photomanipulation of genetically encoded photoactivatable proteins (optogenetics) in live cells.
abstract: Photomanipulation of genetically encoded light-sensitive protein activity, also known as optogenetics, is one of the most innovative recent microscopy techniques in the fields of cell biology and neurobiology. Although photomanipulation is usually performed by diverting the photobleaching mode of a confocal laser microscope, photobleaching by the laser scanning unit is not always suitable for photoactivation. We have developed a simple automated wide-field fluorescence microscopy system for the photomanipulation of genetically encoded photoactivatable proteins in live cells. An electrically automated fluorescence microscope can be controlled through MetaMorph imaging software, making it possible to acquire time-lapse, multiwavelength images of live cells. Using the journal (macro recording) function of MetaMorph, we wrote a macro program to change the excitation filter for photoactivation and illumination area during the intervals of image acquisition. When this program was run on the wide-field fluorescence microscope, cells expressing genetically encoded photoactivatable Rac1, which is activated under blue light, showed morphological changes such as lamellipodial extension and cell surface ruffling in the illuminated region. Using software-based development, we successfully constructed a fully automated photoactivation microscopy system for a mercury lamp-based fluorescence microscope.
authors: Department of Histology and Cell Biology naraki@med.kagawa-u.ac.jp.
authors: Nobukazu
authors: N
authors: Araki
authors: Department of Histology and Cell Biology.
authors: Yuka
authors: Y
authors: Ikeda
authors: Department of Histology and Cell Biology Department of Urology, School of Medicine, Kagawa University, Miki, Kagawa 761-0793, Japan.
authors: Takuma
authors: T
authors: Kato
authors: Department of Histology and Cell Biology.
authors: Katsuhisa
authors: K
authors: Kawai
authors: Department of Histology and Cell Biology.
authors: Youhei
authors: Y
authors: Egami
authors: Department of Histology and Cell Biology.
authors: Katsuya
authors: K
authors: Miyake
authors: Technical Sales Department, Research/Clinical Division, Leica Microsystems, Takadanobaba, Shinjuku-ku, Tokyo 169-0075, Japan.
authors: Nobuhide
authors: N
authors: Tsurumaki
authors: Molecular Device Japan, Bakuro-cho, Nihonbashi, Chuo-ku, Tokyo 103-0002, Japan.
authors: Mitsunari
authors: M
authors: Yamaguchi
mesh_terms: D000818:Animals; D057205:Automation, Laboratory / Q000379:methods; D002477:Cells / Q000648:ultrastructure*; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods*; D062308:Optogenetics / Q000379:methods*; D020830:rac1 GTP-Binding Protein / Q000235:genetics / Q000648:ultrastructure
chemical_list: D020830:rac1 GTP-Binding Protein
keywords: fluorescence microscope; imaging software; live cell imaging; macro program; optogenetics; photomanipulation