Optics: measuring and testing – By dispersed light spectroscopy – With sample excitation
Reexamination Certificate
2000-09-22
2003-02-04
Evans, F. L. (Department: 2877)
Optics: measuring and testing
By dispersed light spectroscopy
With sample excitation
C356S318000, C356S417000, C250S458100
Reexamination Certificate
active
06515743
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to a fluorescence detection apparatus for detecting a fluorescence signal from a specific substance contained in a sample and quantifying the specific substance from the detected fluorescence signal amount and in particular to a fluorescence detection apparatus useful for monitoring a large number of samples in real time (tracing change of the fluorescence signal amount with time) in a clinical diagnosis field requiring incubation at a predetermined temperature, such as an enzyme reaction.
2. Description of the Related Art
To monitor producing a fluorescent reaction product by an enzyme reaction in real time, etc., it is necessary to detect fluorescence while incubating a sample (reaction liquid) at a predetermined temperature. Moreover, a large number of samples need also to be treated promptly at the same time in fields of clinical diagnosis, etc.
A first method used in the conventional clinical diagnosis field, etc., is a method of detecting fluorescence in order while transporting samples along a temperature-adjusted guide. For example, the temperature of a guide manufactured with a material having good thermal conductivity such as an aluminum alloy is adjusted by a heater, etc., samples placed in a holder are transported along the guide using a chain, a turn table, or the like one or more than one at a time, and a fluorescence signal is detected in order by a fluorescence detector placed along the guide.
In addition, a second method of detecting fluorescence at the same time about a large number of samples, for example, by placing a joint-type sample vessel, titer plate, etc., capable of storing a large number of samples on temperature adjustment means is also known. A fluorescence detection apparatus used for the purpose comprises (a) a plurality of photosensors or (b) a multichannel-type photosensor or has (c) mechanical move means for moving photosensor or light guide (means for guiding a fluorescence signal emitted from a sample vessel to photosensor, such as an optical fiber).
The apparatus (a) is a fluorescence detection apparatus for using as many photosensors as samples for detecting fluorescence at the same time to detect a fluorescence signal separately from each sample. In such an apparatus, it is common practice to use a light guide for dividing excitation light from a light source and guiding to each sample.
The apparatus (b) is a fluorescence detection apparatus for using an image sensor such as CCD or a photodiode array in place of a plurality of photosensors, thereby detecting fluorescence signals from aligned samples as an image in a state in which the positional relationship between light emission points is held. In such an apparatus, it is also common practice to guide excitation light from a light source to each sample by using a division-type light guide, such as an optical device or an optical fiber.
In the apparatus (c), the photosensor is moved mechanically on a large number of samples or samples are moved to the fluorescence detection position of the photosensor in order; most used is a configuration of moving the light guide mechanically. In this configuration, an excitation light guide and a fluorescence light guide are used and the ends of both guides placed on the sample side are made integral with each other, then both guides are moved at the same time, whereby fluorescence is detected while a large number of samples are excited in order.
To use the fluorescence detection apparatus in the conventional arts to monitor change of a fluorescence signal with time from a specific substance contained in a sample in real time while incubating the sample at a predetermined temperature, the following problems are involved:
The first method described above involves the risk of insufficient temperature adjustment accuracy, the limit of the number of treated samples, carry-over, etc., because samples are transported along the temperature-adjusted guide and fluorescence is detected in order. That is, it is difficult to adjust the whole sample transport guide at a uniform temperature and hold the thermal conductivity between the transport guide and each sample constant over the whole guide; consequently, temperature change of the sample may occur during transporting or the samples may differ in temperature. Since fluorescence is detected about the transported samples one at a time, the same sample must be transported repeatedly to monitor change of a fluorescence signal with time for a long time, thus the number of samples that can be treated is limited. Further, the risk of contamination (carry-over) between the samples caused by a sample splash cannot be excluded.
The second method described above can solve the problems of the first method, but may introduce the following new problems:
First, the conventional apparatus (a) comprises a plurality of photosensors, thus the manufacturing costs are increased and the space matching the number of photosensors also becomes necessary. If an attempt is made to miniaturize the apparatus, several photosensors can only be installed because of the limit of the space; after all, the number of samples that can be treated at the same time is only a few. Although use of small-sized photosensors such as photodiodes can also be considered, there is a problem of insufficient sensitivity to feeble fluorescence, and it becomes necessary to correct the sensitivity of each photodiode. Further, the strength of a fluorescence signal is proportional to the excitation light strength and thus if excitation light from the light source is divided, detection sensitivity is worsened; this is also a problem.
Next, the apparatus (b) has insufficient sensitivity to feeble fluorescence and thus is not adequate. To augment insufficient sensitivity, an element for amplifying the light quantity via electron amplification by a microchannel plate (so-called image intensifier) or the like may be used in combination, but is used only in special research application under the present circumstances because of an extremely rise in costs. Since fluorescence from a wide range is detected as an image, there are also problems of unevenness of light quantity detection caused by lens aberration and data processing load caused by an enormous data amount.
With the apparatus (c), the move range is limited because of the limit of the bendability of the light guide and moreover there is a possibility of breaking light guide. Since light communication efficiency is changed because the light guide is bent, it is difficult to make fluorescence detection good in reproducibility. On the other hand, a mechanical move of the photosensor also involves a move of attached cables, etc., thus the move range is limited and there is a possibility of breaking the cable, etc.
In addition, scanner-type fluorescence detection apparatus as disclosed in Japanese patent Unexamined Publication No. 2000-088752 (P2000-88752A) and an apparatus as shown in
FIG. 6
invented as means for solving problems of the above-described apparatus are also available. The scanner-type fluorescence detection apparatus described in Japanese patent Unexamined Publication No. 2000-088752 is as follows: As shown in
FIGS. 5A AND 5B
, sample vessels are arranged like a circular arc and a ring section of a ring-type light guide
21
is placed closely facing the sample vessels with putting a partition plate
23
therebetween and excitation light optical means
25
and fluorescence optical means
26
are fixed to the partition plate for rotation integrally, whereby separately gathered fluorescence signals are communicated through the ring-type light guide
21
to a photosensor
22
. In the scanner-type fluorescence detection apparatus as shown in
FIG. 6A and 6B
, sample vessels are arranged like a plurality of circular arcs and a ring-type light guide
31
is placed facing the sample vessels with a partition plate
33
between and excitation light optical means
35
and fluorescence optical means
36
containing at least one light g
Hayashi Toshinori
Ishiguro Takahiko
Kurihara Yoshifumi
Evans F. L.
Sughrue & Mion, PLLC
Tosoh Corporation
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