RESUMO
In many phenomena of biological systems, not a majority, but a minority of cells act on the entire multicellular system causing drastic changes in the system properties. To understand the mechanisms underlying such phenomena, it is essential to observe the spatiotemporal dynamics of a huge population of cells at sub-cellular resolution, which is difficult with conventional tools such as microscopy and flow cytometry. Here, we describe an imaging system named AMATERAS that enables optical imaging with an over-one-centimeter field-of-view and a-few-micrometer spatial resolution. This trans-scale-scope has a simple configuration, composed of a low-power lens for machine vision and a hundred-megapixel image sensor. We demonstrated its high cell-throughput, capable of simultaneously observing more than one million cells. We applied it to dynamic imaging of calcium ions in HeLa cells and cyclic-adenosine-monophosphate in Dictyostelium discoideum, and successfully detected less than 0.01% of rare cells and observed multicellular events induced by these cells.
Assuntos
Células/citologia , Microscopia de Fluorescência/métodos , Animais , Encéfalo/citologia , Cálcio/análise , AMP Cíclico/análise , Dictyostelium/química , Dictyostelium/ultraestrutura , Cães , Entose , Células Epiteliais/ultraestrutura , Desenho de Equipamento , Proteínas de Fluorescência Verde , Células HeLa/química , Células HeLa/ultraestrutura , Humanos , Interneurônios/ultraestrutura , Proteínas Luminescentes , Células Madin Darby de Rim Canino , Camundongos , Microscopia de Fluorescência/instrumentação , Neurônios/ultraestrutura , Semicondutores , Proteína Vermelha FluorescenteRESUMO
Kinetic and physicochemical properties of hamster liver diacetyl reductase have been examined. The results of kinetic studies on the reduction of diacetyl and NADPH to acetoin and NADP+ suggest that the reaction follows an Ordered Bi Bi mechanism in which NADPH binds first before diacetyl. The enzyme is a tetrameric glycoprotein of single subunits of a molecular weight of 23,500 with a sedimentation coefficient of 6.0S. The enzyme does not contain Zn, Cu, or Fe. The amino acid composition revealed an unusually low proportion of proline residues (0.9%). p-Chloromercuriphenylsulfonate and phenylglyoxal inactivated the enzyme, but the presence of NADPH prevented the loss of activity due to thiol and arginine modification. The enzyme transferred the pro 4S hydrogen atom of NADPH to the substrate and the binding of the enzyme to NADPH resulted in a red shift of the ultraviolet absorption spectrum of the cofactor.