Radiant energy – Luminophor irradiation – With ultraviolet source
Reexamination Certificate
2001-08-28
2004-01-27
Epps, Georgia (Department: 2873)
Radiant energy
Luminophor irradiation
With ultraviolet source
C250S459100
Reexamination Certificate
active
06683314
ABSTRACT:
TECHNICAL FIELD
The invention relates to analytical instruments for flourescent light analysis from target specimens and, more particularly, to such an instrument employing increased color decomposition of fluorescent signals from target substances.
BACKGROUND ART
As an example of fluorescent light decomposition for bioanalytical studies, in high throughput screening, the ability to simultaneously detect a plurality of fluorescent dyes with good wavelength discrimination enables deeper multiplexing and higher throughput. In another example using fluorescent light analysis, simultaneous detection of multiple dyes associated with cells allows simultaneous assay of cell surface antigens, organelle states, nucleic acid assay, and intercellular protein content to be detected in a single assay. Multiple wavelength detection requires detectors which can separate many bands of colors. This has commonly been done using dichroic mirror beam splitters.
U.S. Pat. No. 5,317,162 to B. Pinsky and R. Hoffman, assigned to the assignee of the present invention, describes an instrument for phase resolved fluorescent analysis. The architecture of that instrument is similar to prior art instruments which rely upon color decomposition of a beam of fluorescent light derived from a laser impinging upon a fluorescent target. Such an apparatus is described in the book
Practical Flow Cytometry,
by H. M. Shapiro, Third Edition (1995), p. 9. The book describes an apparatus similar to what is shown in
FIG. 1. A
laser beam
12
from an air cooled argon ion laser
11
is used to generate a fluorescent signal which is subsequently decomposed or decimated. The beam
12
passes through focusing elements
13
,
14
and
15
to impinge upon a fluorescent substance in a flow cell
41
. Fluorescent target material, such as fluorescently tagged cells or particles within a liquid stream
16
flow through the flow cell
41
. Particles
43
having passed through flow cell
41
are collected in container
22
. Flow is adjusted by a fluidic system
18
which provides a hydrodynamically focused flow of cells within a sheath fluid. As the target substance passes through the flow cell, the focused light beam
12
intersects the liquid stream, causing fluorescent excitation, including the scattering of light. A photodiode detector
21
is positioned to receive forwardly scattered light. The fluorescent light is typically collected at an angle which is 90° relative to the excitation access of the light beam
12
. Axis
24
represents the 90° viewing axis for collection of fluorescent light. Objective lens
19
is placed across axis
24
to collect and collimate the fluorescent signal from the target substance. Fluorescent light collected by the lens
19
is formed into a beam
28
which impinges upon the dichroic mirror
25
. The dichroic mirror reflects light above 640 nm and transmits the remainder as the transmitted leg
30
. Reflected leg
31
is directed to the red light fluorescence detector photo multiplier tube (PMT) having a 660 nm longpass filter. Detector
32
thus registers the red light component of the collected fluorescent signal from the flow cell
41
. The transmitted leg
30
impinges upon the dichroic mirror
34
which reflects light above 600 nm. The reflected leg
35
is orange light which is detected by the orange fluorescence detector PMT 33 having a 620 nm bandpass filter. The transmitted leg
36
impinges upon the dichroic mirror
38
which reflects light above 550 nm and transmits the remainder in transmitted leg
42
. Reflected leg
39
is detected by the yellow fluorescence detector PMT 40 having a 575 nm bandpass filter.
The transmitted leg
42
impinges upon dichroic mirror
44
which reflects light above 500 nm. The reflected leg
46
impinges upon the green fluorescence detector PMT 47, while the transmitted leg
48
consists of essentially blue light which is directed into the orthogonal scatter detector PMT 50 with a 488 nm bandpass filter, registering blue light. In this manner, the fluorescent signal in beam
28
collected by collector lens
19
is decomposed into five colors with the amplitude of each detector being recorded simultaneously to form a spectral characteristic of the fluorescent material illuminated by the laser beam.
The flow cell
41
is typically a flat-sided quartz cuvet of square or rectangular cross-section with a flow path therethrough. Such a quartz cuvet of the prior art is described in international patent publication WO 01/27590 A2, owned by the assignee of the present invention, shown in FIG.
2
.
In that international patent application, the flow cell mentioned above is described with an aspheric reflective light collector, unlike the lens
19
shown in FIG.
1
. The apparatus of the international patent application mentioned above is shown in
FIG. 2
where a flow cell
17
is a quartz block having a flow channel
20
where a liquid stream containing fluorescent material is directed through the cell in a stream controlled by a nozzle. The flow cell of
FIG. 2
has a reflective aspheric light collector
51
collecting light scattered to a side of the flow cell opposite the side where lens
19
is situated. An aspheric reflective element
51
, placed on the side of flow cell
17
opposite collector
19
serves to augment the light directed toward lens
19
, or in some cases performs the function of lens
19
. The reflective collector
51
is coated with a broadband reflecting material for augmenting the amount of light collected from the flow cell. The aspheric shape may be parabolic or ellipsoidal, having focal properties to match light collector
19
of FIG.
1
.
The apparatus of
FIG. 3
is described in U.S. Pat. No. 4,727,020 to D. Recktenwald and assigned to the assignee of the present invention. This device shows a pair of lasers
52
and
54
directing light to a flow cell
78
so that two different illumination profiles may be used to illuminate a sample. Each laser is selected for stimulating the desired fluorescent emission from target substances. A set of detectors is associated with a different color band. For example, laser
52
generates a beam
53
impinging upon the flow stream
78
and producing a fluorescent signal collected by lens
56
, focused by lens
57
onto dichroic mirror
58
, a beam splitter, for analysis by detectors
60
and
62
. Similarly, laser
54
generates a beam
55
which impinges upon the flow which includes the particles under study in air and generates scattered fluorescent light, collected by light collector
56
and imaged by lens
57
onto dichroic mirror
64
where the beam is split between detectors
66
and
69
. In summary, it is known that groups of detectors can be associated with different lasers simultaneously illuminating the same target substance.
An object of the invention was to provide an improved system for detecting fluorescent light having multiple colors emitted from a target using a greater number of detectors than has been achieved in the prior art.
SUMMARY OF THE INVENTION
The above object has been achieved in an optical instrument having a detector arrangement featuring a larger number of spectrally diverse detectors than previously available. The detectors are fed by a plurality of lasers of different colors with parallel, spaced apart beams impinging upon fluorescent target material at different locations which may be in a channel, a plate, or the like. By using a plurality of lasers, a wide range of spectral responses may be stimulated from fluorescent target material. The target material may be fixed or flowing. Spatially separated fluorescence associated with each beam and emanating from the target material is collected by a large numerical aperture collector lens that preserves the spatial separation of the light originating from the plurality of sources, i.e. the fluorescent signatures of the laser beams on the target material is preserved. Fluorescent light stimulated by the different sources is imaged into a plurality of optical fibers that carry the light to separate detector arrays. Ea
Blasenheim Barry J.
Oostman, Jr. Clifford A.
Epps Georgia
Hanig Richard
Perry Douglas A.
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