Chemical apparatus and process disinfecting – deodorizing – preser – Analyzer – structured indicator – or manipulative laboratory... – Means for analyzing liquid or solid sample
Reexamination Certificate
2002-03-27
2004-03-30
Wallenhorst, Maureen M. (Department: 1743)
Chemical apparatus and process disinfecting, deodorizing, preser
Analyzer, structured indicator, or manipulative laboratory...
Means for analyzing liquid or solid sample
C422S082050, C436S063000, C436S164000, C356S317000, C356S318000, C356S337000, C356S338000
Reexamination Certificate
active
06713019
ABSTRACT:
CROSS-REFERENCE TO RELATED APPLICATION
This application is related to Japanese Patent Applications Nos. 2001-094878 filed in Mar. 29, 2001 and 2001-182085 filed in Jun. 15, 2001, whose priorities are claimed under 35 USC §119, the disclosures of which are incorporated by reference in their entirety.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a flow cytometer for optically detecting and analyzing particles such as blood cells in blood, material components contained in urine or the like.
2. Description of the Related Art
Various particle analyzers have been developed for automatically detecting and analyzing particles contained in a specimen, for example, blood cells in blood such as red blood cells, white blood cells and blood platelets, or material components in urine such as bacteria, blood cells, white blood cells, epithelial cells or casts. A flow cytometer is well known as such a particle analyzer. The flow cytometer includes a detecting section with a flow cell, a laser and a photoelectric conversion element. The flow cell surrounds a sample liquid containing particles to be analyzed with a sheath fluid to converge the sample liquid into a thin flow so that the particles align and pass therethrough. The laser irradiates the passing particles with a laser beam to obtain light generated from each particle i.e., optical information. The optical information includes scattered light such as forward scattered light, sideward scattered light, backward scattered light, fluorescence or the like. The optical information is suitably selected depending upon an analyzed target. The photoelectric conversion element detects the optical information to generate a pulsed electrical signal.
A waveform of the electrical signal obtained as mentioned above is processed to calculate a parameter representing the characteristics of each particle and the particles to be analyzed are classified and counted based on the parameter.
In the optical information, an intensity of the light is recognized as a height (peak level) of the signal waveform, and at the same time, a light emitting period (pulse width) is clocked. That is, the above parameter includes the peak level and the pulse width. For example, the peak level of the signal of the forward scattered light (hereinafter referred to as a “particle signal”) represents a size of a particle, while the pulse width represents a length of a particle. In case where a fluorescent staining is performed in advance to particles, for example, nucleated cells, a fluorescent signal can be obtained from each particle. The peak level of the signal represents a staining degree of the nuclear or the like, while the pulse width represents a length of the fluorescent staining portion. A histogram, formed based upon the parameter representing the characteristics of each particle or a scattergram showing a distribution of the particles, is formed by combining a plurality of parameters, whereby the type or number of the particles contained in the sample is statistically analyzed.
In recent years, the above-mentioned flow cytometer generally utilizes a laser diode from which the laser beam is irradiated to the flow cell.
FIG. 1
is a schematic view showing one example of an optical system in a conventional flow cytometer using a laser diode
1
for a light source. A sample liquid T containing particles to be analyzed is supplied from a nozzle
11
into the flow cell
6
in the direction shown by an arrow A. A sheath liquid S is supplied to the flow cell
6
for surrounding the supplied sample liquid T, whereby the sample liquid T is converged into a thin sample flow by a hydrodynamic effect. As a result, the sample flow is passed through the flow cell
6
with the particles aligned. A radiant laser beam LB emitted from the laser diode
1
is collimated by a collimator lens
3
, and then, passes through a cylindrical lens
4
and a condenser lens
5
to form a beam spot at a position R of the flow of the sample liquid T in the flow cell
6
.
As shown in
FIG. 2
, the laser diode
1
has inherent features that the laser beam LB is diffusible and has an elliptic cross section. Thus, the laser beam LB emitted from the laser diode
1
along an optical axis in the direction of Z has radiation angles defined by a large angle &thgr;1 in the direction of a major diameter of the ellipse, i.e., in the direction of X, and a small angle &thgr;2 in the direction of a minor diameter of the ellipse, i.e., in the direction of Y.
In
FIG. 1
, increasing the radiation angle &thgr; of the laser beam with respect to the flowing direction of the sample liquid T, that is, increasing a collimated beam width d in the same direction brings the following merits:
1. The amount of light of the beam spot at the position R can be increased.
2. The diameter of the beam spot in the flowing direction of the sample liquid T can be decreased in view of a diffraction limit of the beam, since the laser beam is such a Gaussian beam that the Gaussian beam of a larger diameter is less divergent than that of a smaller diameter.
These merits bring the effects of enhancing detecting sensitivity and preventing simultaneous illumination to a plurality of particles. Therefore, in the conventional flow cytometer, the laser diode
1
is mounted so that the major diameter of the elliptic section of the laser beam LB is arranged so as to be parallel to the flowing direction of the sample liquid T in the flow cell
6
.
However, the above-mentioned arrangement causes a significant demerit.
FIG. 3
shows a relationship between such as an arrangement and an intensity of the detected forward scattered light signal. In
FIG. 3
, the laser beam LB has such a large radiation angle &thgr; in the direction parallel to the flowing direction of the sample liquid T, that the laser beam LB is partially kicked at upper and lower edges
3
a
and
3
b
of the collimator lens
3
to generate stray beams SB
1
and SB
2
. Therefore, the stray beams SB
1
and SB
2
are focused on focal points BS
1
and BS
2
above and below a beam spot BS
0
focused by a main beam MB in the flow of the sample liquid T. As a result, signals S
1
and S
2
(hereinafter referred to as “stray beam signal”) attributed to the stray beams SB
1
and SB
2
are detected in addition to a particle signal S
0
due to the main beam MB.
The stray signals S
1
and S
2
are mistakenly detected as small particle signals, which have a bad influence on the counting and classifying result of the particles to be analyzed. This demerit also applies to each detection of a sideward scattered light signal, backward scattered light signal and fluorescent signal, besides the detection of the forward scattered light signal.
To overcome the above-mentioned problems, the following attempt in a signal processing system is taken. In the system, a threshold value Vth is set to a value so as to detect the particle signal S
0
but the stray beam signals S
1
and S
2
as shown in FIG.
4
. That is, the “threshold value Vth” here is used for selecting the particle signal S
0
in a series of signals S
1
, S
0
, S
2
. The signal intensity (peak level) VP and the light emitting period (pulse width) PW are calculated based on a part of the signal which exceeds the threshold value Vth.
In case where there is a great difference in pulse size between the particle signal S
0
and the stray beam signals S
1
and S
2
(each of the stray beam signals S
1
and S
2
has a pulse smaller than that of the particle signal S
0
). For example, in case a relatively large-sized particle such as a blood cell is a particle to be detected, higher setting of this threshold value Vth enables to detect only the particle signal S
0
and not to detect the stray beam signals S
1
and S
2
. Accordingly, the laser diode
1
is provided in the conventional general flow cytometer such that the major diameter of the elliptic section of the emitted laser beam LB is arranged so as to be parallel to the flowing direction of the sample liquid T in the flow cell
6
, whereby the above-menti
Kosako Tatsuya
Ozasa Masatsugu
Birch & Stewart Kolasch & Birch, LLP
Sysmex Corporation
Wallenhorst Maureen M.
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