Radiant energy – Luminophor irradiation
Reexamination Certificate
2001-01-26
2004-07-06
Porta, David (Department: 2878)
Radiant energy
Luminophor irradiation
C356S213000
Reexamination Certificate
active
06759662
ABSTRACT:
FIELD OF THE INVENTION
The present invention is related to optical detection systems. In particular, the present invention is related to optical detection systems which can analyze multiple samples simultaneously.
BACKGROUND OF THE INVENTION
On many occasions, in chemistry and biology, large numbers of samples need to be analyzed. Particularly in molecular biology, in the Human Genome Project, high speed analyses with high throughput are necessary to achieve the goals of the project. Genetic mapping and DNA sequencing on slab gels are currently performed by using automated DNA sequencing with mono-colour or multi-color fluorescent dye labeling. Because capillary electrophoresis (CE) and particularly CE combined with laser induced fluorescence (CE-LIF) offers rapid charged species analyte separation and high detection sensitivity, it is particularly attractive as a separation technique in DNA sequencing applications. However, the number of capillaries that can be analyzed at one time limits the total throughput of the analysis. To increase the throughput a technique called capillary array electrophoresis (CAE) has been introduced. In this technique multiple capillaries are used in parallel with some advantages over slab gels with multiple lanes. There is a substantial reduction on Joule heating effect. Therefore, higher electric fields can be applied and faster analysis can be obtained. The cost of material is reduced in terms of gel usage due to the reduced diameter of the capillaries, as well as samples usage due to a smaller sample size. Another advantage is the possibility to increase the sample throughput by increasing the number of channels in theory up to thousands, while the slab gels impose physical size and sample loading difficulties.
Various methods for acquiring signals from multiple channels have been described; however, the simultaneous detection of the different channels in CAE still presents some problems. A multiple capillary electrophoresis laser induced fluorescence detector that utilizes a confocal fluorescence scanner is described in U.S. Pat. Nos. 5,091,652 and 5,274,240. The scanner or computer controlled stage translates the capillary array past the light path of a laser beam and the optical detection system. Since relatively heavy components are being moved problems with misalignments of the capillaries relative to the light source are likely to occur. To avoid problems derived from the movement of bulky components in U.S. Pat. No. 5,675,155, a detection system is described where an excitation laser beam is focused and scanned across the is capillary array by the movement of a mirror which is aligned as well to receive the electromagnetic radiation from the sample. The advantages of these approaches are the use of a small local illumination and detection volumes requiring only modest excitation power for optimal signal to noise ratio. Crosstalk between adjacent capillaries is eliminated since only a single capillary is illuminated at a time. On the other hand, because the data acquisition is sequential, i.e., the scan modes are from the first capillary to the last capillary in the array, the use for a very large number of capillaries is limited by the observation time needed per capillary. Loss of information could happen in case of a large number of capillaries.
Another multiplexed detector system for capillary electrophoresis is described in U.S. Pat. No. 5,498,324. The invention involves laser irradiation of the sample in a plurality of capillaries through individual optic fibers inserted into the outflow of each capillary. Quesada and Zhang (Electrophoresis 17, 1841-1851, 1996) improved this design by using fiber optics for illumination and collection of the fluorescent emission orthogonally. One of the advantages of the this approach is that no moving parts are involved. However, in both systems the excitation energy that reaches each capillary does not have a homogeneous distribution and degrades as the numbers of fibers included in the fused taper splitter increases. In addition, detection of the arrays is simultaneous through a CCD combined with microscope or camera lens. Therefore, in this case the limitation of the number of capillaries that can be detected at one time depends on the number of them that can be packed in the imaging field of the detector and the resolution of the detector. The most critical problem in this approach may be cross talk between capillaries because fluorescence from adjacent capillaries can be refracted to reach the detector. Although cross talk between capillaries can be avoided by the use of spacers, it is evident that the use of spacers will reduce the number of capillaries in the array.
A greater number of capillaries can be measured at the same time (U.S. Pat. No. 5,730,850) by arranging capillaries two-dimensionally in a capillary array sheet and using a simultaneous two-dimensional detector. Employing modified sheath-flow cuvette detection, sensitivity is enhanced by eliminating light interferences. However, the simultaneous illumination of all the capillaries requires a complicated system of mirrors to transmit the light beam through the buffer solution path between the capillary holder and the detection window, which in turn may result in differences in intensity.
Accordingly, it is desirable to provide an economical and high sensitive detection system for multiple sample analysis which is easy to set up and easy to handle where bulky moving parts and complicated alignments are minimized.
OBJECT OF THE INVENTION
It is therefore an object of the present invention to provide a system to overcome the shortcomings as stated above.
It is another object to provide an optical detection system which allows only collimated light to reach the sample to be detected.
It is a further object to provide an embodiment of an optical detection system which can perform simultaneous detection in a plurality of samples with reduced scattering and cross-talk.
SUMMARY OF THE INVENTION
The present invention is an optical detection system comprising an electromagnetic radiation source, a source radiation focusing and collimating means, a photodetector, an emitted radiation focusing means and a source radiation blocking panel. The radiation source is used to direct source radiation onto a sample which is disposed in a sample platform. The source radiation focusing and collimating means is disposed between the radiation source and the sample for focusing and collimating the source radiation onto the sample. The photodetector is adapted for receiving radiation emitted from the sample which has been focused by the emitted radiation focusing means. The source radiation blocking panel, disposed between the source radiation focusing and collimating means and the sample, is unique in that it is capable of reducing light scattering and interference, such that a clear signal from each individual sample can be obtained by the photodetector.
In the most preferred embodiment, the source radiation focusing and collimating means comprises at least one convergent cylindrical rectangular lens and the source radiation blocking panel comprises a light absorbing panel with at least one pinhole. The samples are contained in channels or tubes aligned in parallel. For simplicity, the samples contained in the various channels or tubes are referred to as sample volumes. In one embodiment, the emitted radiation focusing means is a convex lens, while in another embodiment, it is a convergent cylindrical lens together with an emitted radiation blocking panel having pinholes. This panel with pinholes will be referred to simply as pinholes in the following description. The pinholes may be connected to scanning or conveying means to allow movement. The system may be used for the detection of radiation absorbance or for fluorescence, including epi-fluorescence. Static pinholes for reducing interference and moving pinholes for sequentially and repetitively illuminating selected sample volumes from an array of samples. In the cases of no cross talk between sample
CE Resources Pte. Ltd.
Porta David
Sung Christine
LandOfFree
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