Optics: measuring and testing – By dispersed light spectroscopy – With sample excitation
Reexamination Certificate
2000-01-27
2002-05-28
Evans, F. L. (Department: 2877)
Optics: measuring and testing
By dispersed light spectroscopy
With sample excitation
C356S417000, C250S458100
Reexamination Certificate
active
06396581
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a scanner type fluorescence detection apparatus for detecting fluorescence signals emitted from a specific substance in a sample and determining quantitatively the substance from the quantity of the detected signals. Particularly, the present invention relates to a scanner type fluorescence detection apparatus which is useful for real-time monitoring (monitoring of the change with time of the fluorescence signal quantity) of numerous samples in clinical diagnosis, like samples incubated at a prescribed temperature in an enzyme reaction or a like reaction.
2. Description of the Related Art
For real-time monitoring of the progress of formation of a fluorescent product in an enzymatic reaction, for example, the fluorescence from the sample is detected while the sample (liquid reaction mixture) is being incubated at a prescribed temperature. In clinical diagnosis, the detection should be conducted rapidly for a large number of samples simultaneously.
In a first method employed conventionally in clinical diagnosis, the samples are conveyed along a temperature-controlled guide and the fluorescence is detected successively. For example, a guide is made of a highly heat-conductive material like an aluminum alloy; the temperature of the guide is controlled by a heater or a like means; the samples are conveyed one by one or in plural at a time successively by a chain, a turn table, or the like along the guide; and the fluorescence signal is detected successively by a fluorescence detector arranged along the guide.
In a second method, for example, a connected sample vessel or a titer plate which is capable of holding numerous samples is placed on a temperature-controlling means, and the fluorescence of the numerous samples is detected simultaneously. Such a system is characterized by (1) plural photosensors, (2) a multi-channel type photosensor, or (3) a mechanical moving means for moving a photosensor or a light guide (a means for introducing the fluorescence signals emitted from the sample vessels) such as optical fibers.
The apparatus employing the plural photosensors (1) requires photosensors in number corresponding to the number of the samples to be detected simultaneously, and the fluorescence signals are detected separately for the respective samples. In such a system generally, the excitation light is split and the split rays are introduced through light guides to the respective samples.
The aforementioned multi-channel type photosensor (2) employs an image sensor such as a CCD and a photodiode array instead of the plural photosensors. The image sensor detects the fluorescence signals emitted by the arrayed samples are detected as an image with retention of the light-emitting positional relations. In such a system also, the excitation light is generally split and the split rays are introduced through light guides (optical instrument or optical fibers) to the respective samples.
The aforementioned mechanical moving means (3) moves a photosensor mechanically over the plural samples, or moves the respective samples successively to the fluorescence detection position where the fluorescence is detected by the photosensor. In such a system frequently, a light guide is moved mechanically. In this constitution, a light guide for the excitation light and another light guide for the fluorescence are employed and the sample sides of the both guides are combined and are moved together to excite the samples and detect the fluorescence therefrom successively.
For solving the problems mentioned later which are involved in the above systems, another scanner type fluorescence detection apparatus is disclosed in Japanese Patent Application No. 10-254913. In this apparatus, as shown in
FIG. 3
, sample vessels are deployed and arranged along a circle line, and a ring portion of a ring-shaped light guide is opposed close thereto with interposition of a partition plate. An optical means for excitation light and an optical means for fluorescence light are fixed to the partition plate, and are rotated together with the partition plate. Fluorescence signals collected from the respective samples are transmitted through the ring-shaped light guide to the photosensor.
Conventional fluorescence detection apparatuses have the problems mentioned below in the real-time monitoring of the change with time of fluorescence emitted from a specified substance contained in a sample incubated at a prescribed temperature.
The aforementioned first method, in which the samples are conveyed along a temperature-controlled guide and the fluorescence is successively detected, may cause insufficient temperature-control accuracy, limitation of the speed of the treatment of a number of samples, and carry-over (contamination of the samples by sample splashing), disadvantageously. In other words, it is difficult to keep the entire of the sample delivery guide at a uniform temperature and to make uniform the thermal conduction between the delivery guide and the samples throughout the guide. Consequently, the temperature of the samples may vary during the delivery, or may differ between the samples. Further, in this method, in monitoring the change of the fluorescence signals for a long time, the same samples are delivered repeatedly, the fluorescence of the delivered samples is detected one by one successively, thereby the number of the treated samples being limited. Moreover, the carry-over cannot be prevented completely.
The aforementioned second method may cause different problems although the problems caused in the first method are solved.
The method employing the plural photosensors (1) requires high cost owing to the plural photosensors, and an installation space corresponding thereto. For size reduction of the apparatus, the number of the photosensors should be reduced for the limited installation space, which limits the number of the sample treated at one time. A photosensor of a small size such as a photodiode has not sufficient sensitivity to the faint fluorescence. The plural photodiodes should be calibrated individually. Further, since the intensity of the fluorescence signal is proportional to the intensity of the excitation light, the splitting of the excitation light from the light source will lower the detection sensitivity.
The method employing the multi-channel type photosensor (2) is not suitable because of low sensitivity to faint fluorescent light. To raise the sensitivity, an element (so-called image intensifier or the like) can be employed which amplifies the light quantity through electronic amplification by a micro-channel plate. However, this is extremely costly and is used only in special researches. Furthermore, this system detects the fluorescence over a broad range as an image, which may give rise to disadvantages of nonuniformity of light quantity detection caused by lens aberration and the additional treatment of an enormous amount of data.
The method employing the mechanical movement means (3) is restricted in movement range of the light guide by the limitation by flexibility of the light guide and may cause disconnection. In this method, the light transmission efficiency of the light guide is varied by flection, which makes difficult the detection of fluorescence with high reproducibility. The mechanical movement of the photosensor is also limited in the movement range by the attached cables, and the cable may cause disconnection.
The scanner type fluorescence detection apparatus disclosed in aforementioned Japanese Patent Application No. 10-254913, which solves the above problems, becomes larger in size of the apparatus and higher in cost with the increase of the number of the sample vessels to be held, disadvantageously. Specifically, the ring-shaped input end is counterposed close to the sample vessels arranged along a circle line with interposition of a partition plate. Therefore, the diameter of the ring constituted of optical fibers should be made larger with the increase of the number of the held sample
Hayashi Toshinori
Ishiguro Takahiko
Evans F. L.
Tosoh Corporation
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