Radiant energy – Luminophor irradiation
Reexamination Certificate
2001-11-28
2003-11-11
Porta, David (Department: 2878)
Radiant energy
Luminophor irradiation
C250S459100
Reexamination Certificate
active
06646271
ABSTRACT:
PRIORITY INFORMATION
This application claims priority to Japanese Application Serial No. 361237/2000, filed Nov. 28, 2000.
FIELD OF THE INVENTION
The present invention relates to a method and an apparatus for determining an amount of light for each of a plurality of microspots arranged on a plane. More particularly, the present invention relates to a method and an apparatus for reading fluorescence on a biochip where biological substances such as DNAs or proteins labeled with a time-resolved fluorescent substance are arranged as a high-density spot array.
BACKGROUND ART
Currently-practiced methods for analyzing chemical and physical properties of biological substances such as DNA and proteins often utilize fluorescence. These methods use a biochip on which biological substances such as DNAs or proteins that are labeled with a fluorescent substance marker are arranged as a high-density microspot array. In order to read these spots, a fluorescence reading apparatus is necessary which scans the spots with a laser beam to excite the fluorescent label existing in each spot on the biochip, thereby reading the excited fluorescence from each spot.
FIG. 7
is a schematic view of a conventional fluorescence reading apparatus. A biochip
101
is a rectangular glass plate whose surface is provided with DNAs labeled with fluorescent substance Cy3 (excitation wavelength: 552 nm, fluorescence wavelength: 565 nm, duration: 1.3 ns) which are aligned as microspots in a matrix along x- and y-directions. The biochip
101
is placed on a stage
121
which travels stepwisely in the y-direction by a y-direction driving motor
103
. A reading head
111
placed above the biochip
101
is continuously driven in the x-direction by a x-direction driving motor
114
. A laser beam
110
emitted from a laser light source
104
is radiated on the biochip
101
via the reading head
111
. The fluorescence generated on the biochip
101
is captured by a photomultiplier
116
via the reading head
111
. The laser light source
104
emits a laser beam at a wavelength of 552 nm, a wavelength appropriate to excite the marker Cy3.
Upon reading the biochip
101
, the reading head
111
is continuously transferred in the x-direction along an x-axis rail
122
by the x-direction driving motor
114
under the control of a control computer
108
. Similarly, the stage
121
holding the biochip
101
is transferred stepwisely in the y-direction along a y-axis rail
123
under the control of the control computer
108
.
FIGS. 8A
to
8
E are schematic diagrams showing the order for reading the respective spots on the biochip
101
according to a conventional reading. The spots are arranged in a two-dimensional matrix along x- and y-directions. A black dot represents a spot whose fluorescence has been read, while a white dot represents a spot whose fluorescence has not yet been read.
As shown in
FIGS. 8A
to
8
C, the spots
119
on the biochip
101
are irradiated with the laser beam at a constant rate. Irradiation by the laser beam as well as reading the fluorescence resulting from the laser beam irradiation take place simultaneously and continuously. As shown in
FIG. 8C
, once scanning for a single row in the x-direction is completed, the reading head
111
returns to the left end of the same row. Then, the stage
121
is transferred stepwisely to send the biochip
101
in the y-direction for one resolution distance. Then, as shown in
FIGS. 8D and 8E
, the next row is scanned continuously in the x-direction. By repeating this series of steps, the entire area of the biochip
101
is completely scanned.
When a fluorescent substance such as Cy3 is used to label a biological substance, fluorescence should be read while radiating excitation light since the duration of fluorescence is as short as a few ns. Although the fluorescence capturing member is provided with an optical filter or the like that only passes the wavelength of the fluorescence and blocks the excitation light, it is hard to detect only the fluorescence by completely blocking the excitation light since the fluorescent intensity of the fluorescent label is as extremely weak as about {fraction (1/1000000)}the intensity of the excitation laser beam.
Thus, a time-resolved fluorescent substance such as europium (duration: 400 &mgr;s) with a very long fluorescent duration as compared to, for example, Cy3 (duration: 1.3 ns) may be used. The time-resolved fluorescent substance excited by laser beam irradiation retains the excitation state even after the laser beam irradiation depending on the relative duration. By utilizing this property, the laser beam irradiation and the fluorescence reading may be performed asynchronously so that fluorescence reading takes place after the excitation light irradiation, thereby preventing deterioration of the S/N ratio caused by capturing the excitation laser beam during the fluorescence reading. However, even when such a time-resolved fluorescent substance is employed as a label, the S/N ratio of the fluorescence detection of the spots can be poor since the conventional fluorescence reading apparatus sequentially reads the time-resolved fluorescent substances on the biochip along the laser beam irradiation path, and thus residual fluorescence resulting from the longer duration may become a noise and interfere with the reading of the adjacent spot.
In view of the above-mentioned problems, the present invention has an objective of providing a method and an apparatus for reading fluorescence, with which deterioration of the S/N ratio caused by residual fluorescence from an adjacent spot resulting from the use of the time-resolved fluorescent label can be prevented, thereby realizing fluorescence reading at a high S/N ratio.
SUMMARY OF THE INVENTION
In order to achieve the above-mentioned objective, the method for reading fluorescence of the present invention reads fluorescence by radiating excitation light on a plurality of spots arranged at predetermined intervals each containing a substance labeled with a time-resolved fluorescent substance and detecting fluorescence generated from each of the plurality of spots, the method comprising: a first step of irradiating a first spot with excitation light for a predetermined time; a second step of detecting fluorescence generated from the first spot after the irradiation with the excitation light; a third step of irradiating a second spot with excitation light, which is distanced from the first spot by a distance that is two or more times the distance between adjacent spots; and a fourth step of detecting fluorescence generated from the second spot after the irradiation with the excitation light, wherein, the third and fourth steps are repeated to detect fluorescence from all of the spots.
The plurality of spots may be arranged in a two-dimensional matrix along x- and y-directions, in a concentric pattern or in a spiral pattern. A second spot, which is distanced from the first spot by a distance two or more times the distance between the spots, is typically two spots away from the first spot that has just underwent fluorescence detection.
The time-resolved fluorescent substance according to the present invention refers to a fluorescent substance that lasts for a duration of 10 ns or longer. Examples of the time-resolved fluorescent substance include europium (excitation wavelength: 326 nm, fluorescence wavelength: 612 nm, duration: 400 &mgr;s) and luciferin. In order to enhance the detection sensitivity, longer duration is desirable.
According to above-described method, undesirable capture of the excitation light as well as undesirable capture of the time-resolved fluorescence emitted from already-detected spots can be avoided, thereby greatly improving the SIN ratio of fluorescence detection.
A method according to the present invention reads fluorescence by radiating excitation light on a plurality of spots arranged in a line at predetermined intervals each containing a substance labeled with a time-resolved fluorescent substance and detecting fluorescence generated from each of the plur
Tachibana Mitsuhiro
Yokokawa Naoki
Hitachi Software Engineering CO, Ltd.
Porta David
Sung Christine
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