High numerical aperture flow cytometer and method of using same

Details High numerical aperture flow cytometer and method of using same High numerical aperture flow cytometer and method of using same

2000-02-18

2001-11-20

Pham, Hoa Q. (Department: 2877)

Optics: measuring and testing

By particle light scattering

With photocell detection

C356S343000, C356S073000, C436S063000

Reexamination Certificate

active

06320656

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to particle discrimination by light scattering, and more particularly to a flow cytometer and method therefore that discriminates particles employing a high numerical aperture. Numerical aperture is defined as the refractive index of the medium through which light is collected multiplied by the sine value of one-half of the angle of light collection.
BACKGROUND OF THE INVENTION
The discrimination of particles is useful in numerous clinical assays including ascertaining the types and numerical quantity of cells in blood, ascertaining invasive particles in a fluid sample, such as bacteria and virus, and quantifying the density and volume of cells in a fluid sample.
One method of the above is disclosed in U.S. Pat. No. 5,017,497 issued to de Grooth et al. FIG. 1 of the present application illustrates the apparatus disclosed '497 Patent. Referring to FIG. 1, the '497 Patent discloses a flow cell 2 through which cells from, for example, blood or the like, flow substantially one by one therethrough. A laser input 4 emits a polarized beam of laser light that is oriented substantially orthogonally to the flow of blood cell through flow cell 2 such that the polarized laser light impinges upon the blood cells as they pass through flow cell 2. By “polarized” it is meant that the plane of the electric field oscillation of the laser light is uniform. An optical lens 6 has an aperture which limits the cone of scattered light from the blood cells that can be collected to 72° or less. The central axis of the cone of lens 6 is 90° to both the path of the polarized laser light and the flow of blood cells through flow cell 2. The scattered light emanating from lens 6 is collimated in a matter known in the art. The scattered light now has a mixed polarization that is characteristic of the cell type. The light next passes through a beam splitter 8 that divides the light into two separate beams. A first light beam, substantially concentric with the light beam that originally emanated from lens 6, passes through first polarization analyzer
10
. Polarization analyzer 10 is configured to pass therethrough only polarized light having a vector the same as the original laser light. The second beam emanating from beam splitter 8 is oriented substantially perpendicular to the orientation of the first beam emanating from beam splitter 8. This second beam enters second polarization analyzer 12. Second polarization analyzer 12 is configured to pass therethrough only light having a polarization vector substantially orthogonal to the polarization vector of the other beam from beam splitter 8 that passed through first polarization analyzer 10. The beams that pass through first polarization analyzer 10 and second polarization 12 enter polarized detector 14 and depolarized light detector 16, respectively. The ratio of the outputs of polarized light detector 14 and depolarized light detector 16, based on intensity, provide the depolarization ratio.
As shown in
FIG. 4
eosinophils, a subset of leukocytes (white blood cells), depolarize the right angle of scattered light quantified by the above configuration to a greater degree than other leukocytes.
FIG. 4
is a graphical representation having the output of polarized light detector
14
as one axis and the output of depolarized light detector
16
as the axis . While the above invention does provide some useful data regarding leukocytes, and more specifically eosinophils, as shown in
FIGS. 6B
,
7
B,
7
B and
9
B, the cluster points within the eosinophil cluster (the cluster points above the angled threshold line on the graphical representation having “DEPOL” as one axis and “ORTHAGONAL” as the other axis) are quite condensed. The dense nature of the points within the eosinophil cluster results in difficulty for the computer software programs that ascertain and identify clusters to accurately identify eosinophil clusters. Additionally, this prior art configuration requires expensive optical devices such as photo multiplier tubes, and lens
6
, first polarization amplifier
10
and second polarization amplifier
12
.
A need thus exists for a flow cytometer apparatus and related method in which the cell cluster points are less dense for ease of characterization of the different cell clusters. A need also exists for the above apparatus and method which has fewer and less expensive components.
SUMMARY OF THE INVENTION
The high numerical aperture flow cytometer of the present invention includes a flow cell and a laser input. The laser input emits a beam of light that is oriented substantially orthogonally to the flow of blood cells through the flow cell such that laser light impinges upon the blood cells as they pass through the flow cell. Unlike the prior art, the laser light emitted by the laser input need not be polarized for analysis of the cells according to the present invention. A portion of the beam from the laser input that impinges upon the blood cells in the flow cell is scattered at a substantially right angle to the beam of laser input (“right angle scatter light”). A second portion of the beam from the laser input that impinges upon the cells in the flow cell is scattered at a much lower angle than 90°. This scatter is termed “low angle forward scatter light” and has an angle of from about 2° to about 5° from the orientation of the original beam from laser input. A right angle scatter light detector is oriented to receive the previously mentioned right angle scatter light. The right angle scatter light detector is preferably located about 2 millimeters from the blood cells in the flow cell. An important aspect of the present invention is that, at the distance of about 2 millimeters from the blood cells, the right angle scatter light detector collects a cone of scattered light of at least 100° or greater, and preferably 130° or greater. It is this larger light cone value over the prior art light cone of about 72° that results in the greater cluster separation in the present invention due to the larger signal gathered. In contrast, the smaller 72° cone of the prior art results in missed signals and lesser cluster separation.
A low angle forward scatter light detector is oriented to capture the previously mentioned low angle forward scatter light oriented at about 2° to about 5° from the beam of the laser input.
In one embodiment of the present invention, both right angle scatter light detector and low angle forward scatter light detector are employed in order to produce a 2-dimensional cytrogram. However, it should be noted that in another embodiment of the present invention, only right angle scatter light detector is employed, low angle forward scatter light detector is not employed, and characterization of eosinophils is possible.


REFERENCES:
patent: 4565448 (1986-01-01), Abbott et al.
patent: 4606636 (1986-08-01), Monin et al.
patent: 4818103 (1989-04-01), Thomas et al.
patent: 4954715 (1990-09-01), Zöld
patent: 5017497 (1991-05-01), Grooth et al.
patent: 5179026 (1993-01-01), Matsuda et al.
patent: 5264369 (1993-11-01), Sakata et al.
patent: 5308772 (1994-05-01), Sakata et al.
patent: 5408307 (1995-04-01), Yamamoto et al.
patent: 5432601 (1995-07-01), Tanaka et al.
patent: 5467189 (1995-11-01), Kreikebaum et al.
patent: 5631165 (1997-05-01), Chupp et al.
patent: 5650847 (1997-07-01), Maltsev et al.
patent: 5747343 (1998-05-01), Tsuchiya et al.
patent: 5940177 (1999-08-01), Esser et al.

Affiliated with

Also associated with

Rating

High numerical aperture flow cytometer and method of using same does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with High numerical aperture flow cytometer and method of using same, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and High numerical aperture flow cytometer and method of using same will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2596279