1. Field of the Invention
The present invention relates to a focus detection device and a fluorescent observation device using the focus detection device, and particularly, the present invention relates to a microscope and an optical device etc. for fluorescent-observing a live cell with a high contrast, which can detect a boundary face between a glass surface on which an observation sample as an observation object is mounted and an observation example with a high degree of accuracy.
2. Description of the Related Art
In a study field of biology, a function of the interior of the cell and the structure of the cell have been analyzed by using a fluorescent observation method. However, since an illumination light is irradiated on an overall observation object in normal fluorescent observation, fluorescent information of the position other than the place to be observed (the position that is out of focus) is also acquired simultaneously. Therefore, a contrast is lowered, and observation of a minute unit such as, for example, one molecule, cannot be made.
In recent years, the technology for illuminating only a minute area such as several tens to several hundreds nm is established by using an evanescent light generated on the total reflection of light as an illumination light. Accordingly, the fluorescent observation of a minute unit at one molecule level becomes possible by preventing lowering of the contrast that is a problem of the normal fluorescent observation.
The evanescent light is a light generated by totally reflecting an illumination light when the illumination light is inputted on a boundary face having a different refractive index at an angle of a predetermined value or less. In addition, the evanescent light has a property that it does not propagate through a free space located in an area of which size is smaller than that of a wave length at the opposite side of the illumination light with respect to the above boundary face.
In a biological study, a method so-called time lapse is frequently employed in order to observe change over time within the live cell. An object of this method is to analyze a function of the interior of the cell by acquiring an observation image continuously or at predetermined intervals in a period of time, for example, one day or two days.
According to the method, the observation for a long time is required. However, a microscope that is an observation device is an assembly of a metal and a glass. For example, only if the internal temperature of a room where the experiment is conducted is slightly changed, a problem such that the microscope is transformed and a point of focus is deviated occurs. Particularly, this is a serious problem in the florescent observation using the evanescent light used to observe the minute area from several tens to several hundreds nm.
Jpn. Pat. Appln. KOKAI Publication No. 2003-270524 discloses means for detecting a position of a part of a cell located in an evanescent field with a high degree of accuracy by using an evanescent light dedicated for focus detection.