U.S. patent application number 10/688131 was filed with the patent office on 2006-09-07 for fluorescence detection instrument with reflective transfer legs for color decimation.
Invention is credited to Barry J. Blasenheim, Clifford A. JR. Oostman.
Application Number | 20060197032 10/688131 |
Document ID | / |
Family ID | 25476334 |
Filed Date | 2006-09-07 |
United States Patent
Application |
20060197032 |
Kind Code |
A9 |
Oostman; Clifford A. JR. ;
et al. |
September 7, 2006 |
FLUORESCENCE DETECTION INSTRUMENT WITH REFLECTIVE TRANSFER LEGS FOR
COLOR DECIMATION
Abstract
An optical instrument using a plurality of lasers of different
colors with parallel, closely spaced beams to stimulate scattering
and fluorescence from fluorescent biological particulate matter,
including cells and large molecules. A large numerical aperture
objective lens collects fluorescent light while maintaining spatial
separation of light stimulated by the different sources. The
collected light is imaged into a plurality of fibers, one fiber
associated with each optical source, which conducts light to a
plurality of arrays of detectors, with each array associated with
light from one of the fibers and one of the lasers. A detector
array has up to ten detectors arranged to separate and measure
colors within relatively narrow bands by decimation of light
arriving in a fiber. A large number of detectors is mounted in a
compact polygonal arrangement by using reflective transfer legs
from multiple beam splitters where the transfer legs arise from a
polygonal arrangement of beam splitters in a circumference within
the circumferential arrangement of detectors.
Inventors: |
Oostman; Clifford A. JR.;
(Hansville, WA) ; Blasenheim; Barry J.; (San Jose,
CA) |
Correspondence
Address: |
DAVID W. HIGHET, VP AND CHIEF IP COUNSEL;BECTON, DICKINSON AND COMPANY
1 BECTON DRIVE, MC 110
FRANKLIN LAKES
NJ
07417-1880
US
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20050104008 A1 |
May 19, 2005 |
|
|
Family ID: |
25476334 |
Appl. No.: |
10/688131 |
Filed: |
October 17, 2003 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09941357 |
Aug 28, 2001 |
6683314 |
|
|
10688131 |
Oct 17, 2003 |
|
|
|
Current U.S.
Class: |
250/458.1 |
Current CPC
Class: |
G01J 3/0205 20130101;
G01J 3/10 20130101; G01N 2021/6421 20130101; G01J 3/0237 20130101;
G01J 3/4406 20130101; G01J 3/02 20130101; G01J 3/0202 20130101;
G01J 3/0208 20130101; G01J 3/0291 20130101; G01N 21/645 20130101;
G01N 2021/6484 20130101; G01J 3/021 20130101; G01N 2021/6419
20130101; G01N 21/6428 20130101; G01J 3/36 20130101; G01N 2021/6463
20130101 |
Class at
Publication: |
250/458.1 |
International
Class: |
G01N 21/64 20060101
G01N021/64 |
Claims
1-34. (canceled)
35. A detector apparatus for analyzing light emitted from a
fluorescent material, wherein the light is collected by a light
collector and formed into an output beam for analysis, comprising a
means for collimating said output beam, the collimated output beam
having a projected optical axis, a plurality of dichroic mirrors
disposed along the projected optical axis in a manner separating
light at each mirror into a reflected beam and a transmitted beam,
wherein, at each mirror, one of the reflected and transmitted beams
is a transfer leg carrying the beam further to the next dichroic
mirror and the other is a leg carrying light to a detector, wherein
a majority of the dichroic mirrors receives light from a reflected
beam coming from another dichroic mirror.
36. The apparatus of claim 35 wherein all of the dichroic mirrors
except one receive light from a reflected beam coming from a
dichroic mirror.
37. The apparatus of claim 35 wherein the number of dichroic
mirrors is at least four.
38. The apparatus of claim 35, wherein a plurality of dichroic
mirrors are angled relative to an optical axis of the transfer leg
or the output beam at an angle of 20.degree. or less.
39. The apparatus of claim 35, wherein a plurality of dichroic
mirrors are angled relative to an optical axis of the transfer leg
or the output beam at an angle between 5.degree. and
20.degree..
40. The apparatus of claim 35, wherein a plurality of dichroic
mirrors are angled relative to an optical axis of the transfer leg
or the output beam at an angle of about 11.25.degree..
41. The apparatus of claim 35 wherein said detectors are arranged
in a polygonal pattern having a first circumference and said
dichroic mirrors are arranged in a polygonal pattern having a
second circumference smaller than said first circumference.
42. The apparatus of claim 41, further comprising a plurality of
filters arranged in a polygonal pattern having a third
circumference, wherein a filter is associated with each detector
and said third circumference is greater than said second
circumference but less than said first circumference.
43. The apparatus of claim 35 wherein said dichroic mirrors are
arranged such that said transfer legs carrying said beam from one
dichroic mirror further to the next dichroic mirror follow a zigzag
pattern.
44. The apparatus of claim 35 wherein said means for collimating
said output beam receives light by means of an optical fiber.
45. The apparatus of claim 35, wherein said detectors are
photomultiplier tubes or semiconductors.
Description
TECHNICAL FIELD
[0001] 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
[0002] 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.
[0003] 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.degree.
relative to the excitation access of the light beam 12. Axis 24
represents the 90.degree. 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.
[0004] 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.
[0005] 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.
[0006] 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.
[0007] 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.
[0008] 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
[0009] 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. Each array has a series of beam splitters in a
series or cascade arrangement receiving light from an associated
fiber and relaying part of the light to a downstream beam splitter,
spectrally filtering the light on each relay within the cascade
arrangement by means of coatings associated with the splitters.
Within each array, light reflected from a beam splitter is
forwarded to a downstream splitter, while light transmitted through
a beam splitter is sent to a detector. This means that the
reflected component is a broadband wavelength component and the
transmitted component is filtered to be a narrowband wavelength
component. For the last beam splitter, light from the reflective
leg may be sent to a detector, as well as light from the
transmitted leg. Since, for most optical coatings on a beam
splitter, the fraction of light reflected from a beam splitter
exceeds the transmitted fraction, the downstream beam splitters
receive more light from the reflective transfer legs than the prior
art arrangement where downstream beam splitters receive light from
the transmitted transfer leg. Each array of detectors is arranged
in a polygonal compact cluster. The detector configuration of the
present invention is modular because light from each laser is
spatially separated from other lasers and each detector cluster has
at least 6 detectors. The clusters may be physically separated
since optical fibers can feed light to clusters in remote locations
or in stacks or racks. In this instrument, collected light is
transmitted to a plurality of beam splitters. Note that the beam
splitters, split light into a transfer leg and a transmitted
detector leg, as in the prior art. However, unlike the prior art,
the transfer leg is reflected from beam splitters and forwarded to
another beam splitter and the transmitted detector leg is directed
to a detector. This is true for a majority of beam splitters, but
not for the last one receiving a maximally attenuated transfer leg
where the transfer leg is either sent to a detector or terminated.
So the last dichroic mirror may be associated with two detectors,
one for the reflected leg and one for the transmitted leg. By using
reflective transfer legs for most detectors, the detectors may be
clustered in a polygonal arrangement of between five and ten light
detectors in a common plane. Here, the term "most detectors" refers
to all transfer legs except the last one, but is not limited to the
last leg.
[0010] By maintaining spatial separation for the input beams,
spatial separation can be preserved in the output transfer beams,
with each transfer beam directed into an optical fiber for delivery
to a detector cluster. This allows detector clusters to be stacked
or placed in racks, with optical fibers carrying transfer beams to
the location of an input port of each cluster. Once inside of a
cluster, the transfer beam is decimated by the dichroic beam
splitters, each beam splitter inclined to a transfer leg at a
preferred angle centered on 11.25 degrees. Other angles will work
but not as efficiently. Each beam splitter achieves color
separation in the usual way, i.e. by transmitting light of a
particular wavelength. This transmitted light is directed to a
photomultiplier tube, or the like, which is positioned, to the
extent possible, to detect light in the transmitted detector legs
associated with the split beam. A focusing lens and the detector
photomultiplier tubes are positionally relatively adjustable so
that an optimum detector position can be found by motion of a
detector element relative to a lens focusing incoming light. In
this manner, the fluorescence associated with each of several laser
beams is simultaneously decimated into bands characteristic of the
target material within the detector array of each cluster. A group
of clusters provides color decimation much greater than heretofore
available. Moreover, the apparatus is modular because a greater
number of fibers can feed a greater number of clusters. One of the
advantages of using a reflective transfer leg to relay the optical
signal for decimation, rather than the transmitted leg, is that the
reflective transfer leg is a stronger optical signal. After
encounters with several beam splitters, the signal attenuation in a
relayed reflective transfer leg signal is substantially greater
than for an optical signal in which the relay was transmitted
through an equal number of beam splitters, as in the prior art.
[0011] In one embodiment, the light collection and detection optics
are included in a system having a plurality of lasers producing
input beams of different wavelength profiles to simultaneously
illuminate a fluorescent target, usually fluorescent particulate
matter which could be discrete small particles, including cells, or
large biological molecules. The term "color decimation" refers to
the simultaneous spectral breakdown of polychromatic light beams
from a target substance into narrow bands of light arriving at
detectors. Scattered light is measured by other detectors not
relevant to this invention or this application. Scatter detectors
are not described herein. Collection of fluorescent light is by a
lens similar to a microscope immersion lens of large numerical
aperture, the lens forming output transfer beams directed to a
plurality of dichroic mirrors. After collection, the light is
imaged into fibers, then distributed to "n" clusters of "m"
detectors, yielding an "n" times "m" number of detectors resolving
the fluorescent light stimulated by the input beams. Each cluster
isolates light within the corresponding array of detectors.
Clusters may be mounted on rails, racks or stacks in a modular
arrangement.
DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 is a perspective plan view of a multi-color flow
cytometer with single wavelength excitation in accordance with the
prior art.
[0013] FIG. 2 is a perspective view of a light collector of the
prior art for use with a flow cytometer of the kind illustrated in
FIG. 1.
[0014] FIG. 3 is a perspective view of a multi-color flow cytometer
with plural wavelength excitation and a polygonal arrangement of
detectors in accordance with the prior art.
[0015] FIG. 4 is a plan view of a multi-color optical instrument of
the present invention.
[0016] FIG. 5 is a top plan of a planar polygonal detector
arrangement showing decimation of an incoming beam with reflective
transfer leg beam splitters in accordance with the present
invention, the incoming beam received from a fiber bundle
illustrated in FIG. 4.
[0017] FIG. 6 is a top plan of an alternate planar polygonal
detector arrangement showing decimation of an incoming beam with
reflective transfer leg beam splitters, as in FIG. 5 but with the
beam having a folded path for compact placement of the
detectors.
[0018] FIG. 7 is a perspective assembly view of the apparatus of
FIG. 6.
[0019] FIG. 8 is a perspective plan view of three detector arrays
of the kind shown in FIG. 7 mounted on a rack in accordance with
the present invention.
[0020] FIG. 9 is a perspective plan view of a detail of a mirror
holder used in the apparatus of FIG. 7.
[0021] FIG. 10 is a front elevation of the mirror holder
illustrated in FIG. 9.
[0022] FIG. 11 is a side sectional view of the mirror holder of
FIG. 10, taken along lines 10-10.
[0023] FIG. 12 is a side plan view of a detector photomultiplier
tube and optics used in the detector arrangements shown in FIGS. 5
and 6.
[0024] FIG. 13 is a side plan view of a motion to optimize the
sensitivity of the detector shown in FIG. 12.
[0025] FIG. 14 is a top plan view of a motion to optimize the
sensitivity of the detector shown in FIG. 13.
[0026] FIG. 15 is a plan view of a light collector lens for use in
the instrument input end arrangement illustrated in FIG. 4.
BEST MODE FOR CARRYING OUT THE INVENTION
[0027] With reference to FIG. 4, a first laser 85, a second laser
87, a third laser 89, and a fourth laser 91, all produce light with
unique wavelength profiles and all are connected to respective
power supplies and a cooling module 93. The lasers emit respective
beams 95, 97, 99 and 101 which are directed by means of
beam-turning mirrors toward flow stream 103 causing the beams to
intersect with the stream. Although the preferred embodiment
features a flow cytometer, this instrument is merely illustrative
of instruments which employ fluorescence detection and color
separation. Other instruments include microscopes, electrophoresis
instruments, spectrophotometers, and the like. The scope of the
present invention is therefore not limited to flow cytometers.
[0028] A fluidic system 105 feeds tagged target liquid substances
into a stream 103 in a controlled manner. Material which passes
through the beam illuminated zone is collected in collection cup
107. The illuminated zone is established by the four laser beams
that impinge upon fluid tagged target material, thereby causing
scattering and fluorescence. Each beam has a characteristic color
produced by different types of lasing material. For example,
characteristic laser illumination wavelength profiles may be
produced by CO.sub.2 lasers, argon ion lasers, copper vapor lasers,
and helium neon lasers. Other colors are available from different
types of lasers. The output power of each laser is typically
between 10 and 90 milliwatts. At such power levels, a sufficiently
strong optical signal is produced without damaging coatings on the
surfaces of mirrors, fibers or beam splitters. Coatings are
selected to achieve desired passband transmissions and may be
specified from coating manufacturers.
[0029] Light of different colors intersects the flow stream 103 and
interacts with fluid sample causing scattering and fluorescence
that is spatially separated along a line parallel to the flow
channel 109. Scattered light can be processed by well-known scatter
detectors. For simplicity, this description deals only with
fluorescent light. This light appears to be originating at four
spaced apart point sources or spots, each of which is imaged by a
lens light collector 111 to four respective optical fibers 123,
125, 127 and 129, all held in place by a movable holder 121 which
securely mounts the fibers and allows both rotational and axial
adjustment of the fibers relative to light collector 111. In other
words, the holder 121 may be moved so that the fibers optimize the
input light into the fibers from collector 111. The focal spots
produced by collector 111 enter the tip of each respective fiber
123, 125, 127 and 129, each of which is a multi-mode fiber.
[0030] Light collector 111 is a group of lens elements which is
described with reference to FIG. 15. The collector is placed very
close to the flow stream, within a few millimeters. The distances
shown in FIG. 4 are not to any scale and are out of proportion.
This collector gathers fluorescent light from the fluorescent
target material. The collector lens is a microscope objective lens
similar to the fluid emersion microscope objective lens shown in
U.S. Pat. No. 5,805,346, except that the lens of the present
invention is a positive meniscus lens, while the fluid emersion
microscope objective lens of the '346 patent is a negative meniscus
lens. Other differences exists, but the lenses are similar in the
number of optical elements and their arrangements. Various supports
may be used with the goal of reducing vibration and allowing proper
alignment of optical elements along the optical axes 133, 135, 137,
and 139 defined by collector 111. The optical axes are maintained
by each of the fibers, although each of the fibers may be bent to
remove light to the location of a detector array. Light in each of
the fibers 123, 125, 127 and 129 is transmitted to a respective
detector cluster 124, 126, 128 and 130 which houses an array of
detectors. Each array processes fluorescent light which has
maintained spatial separation, i.e. color independence, to a large
extent. In other words, the fluorescence stimulated by a particular
laser has been preserved and forwarded to an array of detectors
which operates independently from other arrays of detectors. Each
array of detectors differentially separates bands of light by
filtering, using coatings on beam splitters and lenses, in a known
manner. With each cluster having between 3-4 and 10 or more
detectors, each detector receiving a passband of between 10 and 75
nanometers, the instrument of the present invention has a wide
spectral response.
[0031] Details of a detector array or cluster are shown in FIG. 5.
The optical fiber 123 has a fiber terminal 143, which allow
emergence of light and formation of a beam by means of a
collimating lens 145. The output beam 147 is directed toward a
beam-turning mirror 149 directing light at a first beam splitting
element 151 which is a dichroic mirror transmitting light in beam
155 in a transmitted detector leg toward a filter 156, a
beam-turning element 157, and a focusing element 158 which directs
the beam onto a light sensitive element of a photomultiplier tube
159. The tube is adjusted so that its most sensitive area is
exposed to the incoming beam. This beam is known as the transmitted
detector leg because it is transmitted through the beam splitting
element 151. The beam splitting element has a coating which allows
transmission of one band of optical signals, while reflecting light
in another band in the form of beam 153 which forms a reflective
transfer leg for the light which was originally in beam 147. The
reflective transfer leg 153 is seen to fall upon another beam
splitting element 161 which is a dichroic mirror having different
optical characteristics from the beam splitter 151. The colors
removed by beam splitter 161, as well as the other beam splitters,
are different, each beam splitter removing a selected band of
colored light in the same manner, but different wavelengths, as in
beam splitters of the prior art described with reference to FIG.
1.
[0032] Beam splitter 161 has a reflective transfer leg 163
reflected from the surface of the splitter, as well as a
transmitted detector leg 165 transmitted through the beam splitter
to the filtering element 166, the beam-turning element 167 and the
focusing element 168. The transmitted beam impinges upon a
sensitive portion of detector 160 where the amount of light
associated with the band transmitted by beam splitter 161 is
measured. In the same manner, beam splitters 171, 181, 191 and 201
split incoming beams which are light beams reflected from upstream
beam splitters, with beam splitter 191 being upstream of beam
splitter 201, beam splitter 181 being upstream from beam splitter
191, and beam splitter 171 being upstream from beam splitter 181,
etc. Each beam splitter, except for the last one, beam splitter
201, separates light into a reflective transfer beam, with the
transfer beam 173 being reflected from beam splitting dichroic
mirror 171 and the transmitted beam through dichroic mirror 171
being beam 165 impinging upon detector 169. On the other hand, the
reflected beam 173 is transmitted to beam splitter 181, with the
transmitted leg being directed to detector 170. Beam 175, passing
through the beam splitter 191, is directed to a detector 179 while
the reflected leg 193 goes to the last beam splitter, namely
dichroic mirror 201. This element, unlike the other beam splitters,
has two detectors associated with it. One detector 180, receives
light transmitted through the beam splitter 201 toward detector 180
while light reflected from beam splitter 201 forms a reflected leg
183 which impinges upon detector 189. In this manner, all of the
detectors illustrated in FIG. 5 form an array which decimates light
from a single fiber 123. As mentioned previously, the fiber 123 is
associated with scattered and reflected light collected from one of
the lasers mounted on the optical bench. Thus, for each laser there
is an array of detectors in a cluster. In FIG. 5, the transfer leg
forwarded upstream from one beam splitter to the next follows a
zigzag pattern. In FIG. 6, the transfer legs intersect in a
star-shaped pattern yielding a more compact polygonal arrangement
of detectors.
[0033] In FIG. 6, the optical fiber 123 is terminated in a terminal
143 directing an output beam 202 through a collimating lens 203 and
thence onto an array of beam splitters 211, thence to beam splitter
213, then to beam splitter 215, then to beam splitter 217, then to
beam splitter 219, and, lastly, to beam splitter 221. The
arrangement of beam splitters is in a polygonal pattern. In each
case, the transfer leg is reflective, with the beam splitter being
a dichroic mirror which is inclined at an angle of 11.25.degree. to
perpendicular, i.e. a small angle, say between 5.degree. and
20.degree. . It has been found that this angle optimizes balance
between reflection and transmission. On each bounce from a beam
splitter, part of the beam called the "detector leg" is transmitted
through the beam splitter toward one of the detectors. Detector 231
is associated with the transfer leg of beam splitter 221 while the
detector 233 is associated with the detector leg from the same beam
splitter. Detector 235 is associated with the detector leg coming
through beam splitter 211 while detector 237 is associated with the
detector leg through beam splitter 213. Detector 239 is associated
with the detector leg through beam splitter 215 while detector 241
is associated with the transfer leg from beam splitter 215 and the
detector leg through beam splitter 217. Correspondingly, the
detector 243 is associated with the transfer leg from beam splitter
217 and the detector leg through beam splitter 219. Each of the
beam splitters is a dichroic mirror having different wavelength
characteristics for decimating the input beam 202 into different
colors which register at the different detectors. Coatings applied
to the dichroic mirrors account for reflection of some wavelengths
and transmission of other wavelengths. Laser light of a particular
frequency will stimulate fluorescent emission in generally known
wavelength bands. Light in these bands is collected and passed
through an optical fiber to a detector array, the detectors
arranged in a polygonal pattern of greater circumference than the
polygonal pattern of beam splitters. Within each cluster decimation
of the light occurs, with passbands of 10-75 nanometers registering
at each detector, depending on the sharpness of filtering of the
coatings applied to the beam splitters. Additional selectivity of
the signals reaching the detector may be gained by a series of
filters 251, 253, 255, 257, 259, 261 and 263, the filters arranged
in a polygonal pattern with a polygonal circumference greater than
the circumference of the beam splitters but less than the
circumference of the detectors. Each of these filters is placed in
front of a corresponding detector. The filter has a bandpass over a
range of wavelengths which is of particular interest in the
corresponding detector. Associated with each detector is a focusing
lens 265 for focusing light in a detector leg on a sensitive spot
of the detector. Each lens 265 is movable for adjusting the focal
spot during calibration of the instrument.
[0034] In FIG. 7, a cluster with an array of detectors and an array
of beam splitters is seen to be held in place by a frame 200 which
generally supports detectors 237, 241, 233, 235, 239, 243 and 231
in a polygonal array where the polygon is drawn connecting the
axial centers of each of the cylindrical detectors, the detectors
being photomultiplier tubes. Each tube is seated in a tube mount
245. A tube connector base 247 makes contact with pins of each
tube. An electrical feed-through 249 allows power to come to
connector base 247 while signals exit the tube through another
feed-through 251. Similar connector bases and feed-throughs exists
for each tube. Within the center of the frame 200 is a coverplate
250. About a first close distance from the coverplate is a
polygonal array of dichroic mirror holders for the dichroic mirrors
213, 217, 221, 211, 215 and 219. A slightly further distance are
the filter holders 263, 261, 259, 257, 255, 253 and 251. The
dichroic mirror holders and the filter holders are mounted in
vertically removable housings so that dichroic mirrors and
associated filters may be interchanged or replaced.
[0035] In FIG. 8, a plurality of clusters 301, 303 and 305 is shown
to be vertically mounted on a rack 307 by means of standoff
supports 311, 313 and 315. The standoff supports are merely
illustrative of the manner in which three arrays may be mounted on
a rail or rack for easy replacement or modular supplementation.
Each cluster is of the type shown in FIG. 7.
[0036] In FIGS. 9-11, the construction of a removable beam splitter
holder is shown. The beam splitter mirror 321 is held in a mirror
holder frame 323 at a desired angle. Frame 323 is supported by a
block 325 having channels for finger contact in a non-slip manner.
The mirror holder frame 323 has a flat side 328 which presses
against the flat side 327 of a seating block 329 having a central
aperture 331 corresponding to the position of mirror 321. A facing
block 339 has an aperture 341 in alignment with aperture 331 and
with mirror 321. The flat side 327 is a reference surface for
positioning of the mirror 321. A wire spring 343 serves to push the
mirror holder frame 323 against the block 329.
[0037] FIGS. 12-14 show how the transmitted leg of a beam 361 may
impinge upon the focusing lens 265. With motion of the focusing
lens 265, as shown in FIG. 13 in the direction of the arrow C, the
focused beam 362 is moved to a more sensitive spot 364 on the
photomultiplier tube 231 in comparison to a less sensitive location
365 shown in FIG. 12. Additional sensitivity may be gained by
slightly rotating the photomultiplier tube 231 with its housing,
within the support frame to optimize the output signal for a
particular detector leg 361 focused on a detection element in the
photomultiplier tube. Motion is indicated by the arrows D.
[0038] FIG. 15 shows the construction of light collector 111 in
FIG. 2. A flow cell 103 is shown at the left of the drawing with
flow channel 109 and input fluid from fluidic system 105 passing
through channel 109. As has been previously noted, a flow system is
but one type of optical instrument where fluorescence can be
observed. Non-flow systems may also be employed with the detection
apparatus of the present invention. A large numerical aperture
("N.A.") lens system, i.e. N.A. greater than one, positioned as
shown in FIG. 4 is described according to the lens data contained
in the following table. The numbered optical surfaces in the figure
correspond to surface numbers in the leftmost column of the table.
All radii and thickness values are in millimeters. Surface
curvature tolerances for the lens data include 5 fringes for power
(deviation of actual curvature from nominal curvature) and 1 fringe
for irregularity (deviation from a perfect spherical surface). Tilt
tolerance is 0.05 degrees from normal in any direction. Material
tolerances are 0.0005 for refractive index and 0.8% for Abbe
number. TABLE-US-00001 Clear Sur- Radius of Thickness Aperture
Aperture Ma- face Curvature Thickness Tolerance Radius Radius
terial 109 4 0.0889 -- 0.2 Wa- ter 301 4 1.94 -- 4.6 Silica 302 4
0.1682 -- 4.6 Gel 303 4 0.8 .025 5.1 4.6 BK7 304 4 3.915 .025 4.6
4.6 BK7 305 -4.66 1.5 .025 4.6A 4.6 Air 306 -16.918 5 .05 8.5 7.3
BK7 307 -10.894 1 .025 10 8.8 Air 308 -26.836 5 .10 11.5 10.2 BK7
309 -15.008 1 .025 12.5 11.1 Air 310 -103.704 9 .10 13 11.9 BK7 311
-14.012 3 .10 13 12.2 SF8 312 -34.38 2 .05 15.5 14.1 Air 313
+123.446 3 .10 17 14.9 SF8 314 +34.38 12 .10 17 15.1 BK7 315
-36.554 126.731 .50 17 15.5 Air
[0039] The lens proper (surfaces 303 through 315) in this system is
adapted to magnify and view cellular material within a cytometry
flow cell or cuvette 103 (the flow cell inner and outer wall
surfaces being optical surfaces 301 and 302 above). As indicated in
the table, a flow cell has 0.007 inch (0.1778 mm) interior
dimensions (wall-to-wall) and the fluorescent targets 109 to be
detected and analyzed are immersed in saline water flowing through
the cell 103, nominally for lens design purposes through the center
of the cell a distance of 0.0889 mm from the cell's inner wall. The
1.94 mm thick, fused silica, planar cell wall has a refractive
index.sub.nD of 1.45857 and an Abbe number.sub.vD of 67.7. An
optical gel layer provides an interface between the cytometry flow
cell and the lens proper and improves lens mounting tolerances. The
gel material is preferably NyoGel OC-431A sold by William F. Nye,
Inc. of New Bedford, Mass., and has refractive indices at the 0.40
.mu.m, 0.55 .mu.m and 0.70 .mu.m principal lens design wavelengths,
respectively, of 1.487, 1.467, and 1.459. The gel should have a
thickness less than 0.5 mm, and is selected in the above design to
be 0.1682 mm thick. Other cytometry flow cells with different
interior and wall dimensions, and other optical gels or oils could
be used, with appropriate modifications in the lens specifications,
optimized using commercially available software. Although lens
positioning tolerances would be much tighter (0.025 mm or less),
the lens could also be integrated with or mounted to the flow cell
without using optical gel.
[0040] The lens glass types BK7 and SF8 (Schott glass designations)
have been selected because they are relatively inexpensive stock
materials that are easy to obtain in quantity, and because they are
easy to grind and polish and don't stain easily. Other glass types
could be used instead, including similar glass types from other
optical glass suppliers, with appropriate modifications in the lens
specifications. The optical glass designated BK7 [517642] has a
refractive index.sub.nD of 1.51680 and an Abbe number.sub.vD of
64.17, and the optical glass designated SF8 [689312] has a
refractive index.sub.nD of 1.68893 and an Abbe number.sub.vD of
31.18. All of the lenses in the preferred embodiment have spherical
surfaces because they are inexpensive, more readily available in
bulk, are more alignment tolerant, and are easier to assemble and
test than aspheric lenses. However, if desired, modified lens
specifications using one or more aspheric lenses have lower on-axis
aberrations and could be used, although from a commercial
standpoint the performance improvement likely would not be
sufficient to justify their significantly greater expense and
assembly difficulty.
[0041] The basic lens requirements include a numerical aperture of
at least 1.17. (An object N.A. of 1.20.+-.0.01 was used in
obtaining the preferred embodiment that is set forth in the table
above. A numerical aperture of 1.20 provides about 10 to 15%
greater light collection than one of 1.17) The field of view should
be at least 200 .mu.m diameter and, if possible, as much as 400
.mu.m or better. The present preferred embodiment has a field of
view of 400 .mu.m diameter. The working distance should be at least
1.75 mm, (2.2 mm is achieved in the preferred embodiment.) Most
importantly, a lens system of less optical aberrations and high
image quality is required for better resolution compared to
existing cytometry lenses. In particular, the RMS spot size (a
measure of resolution) in image space (for hypothetical point
objects) for all wavelengths and all field points should be at most
100 .mu.m. The present preferred embodiment achieves a calculated
geometrical spot size of 85.04 .mu.m at full field and of 71.86
.mu.m on-axis. This puts a minimum of 80% of the optical energy of
the image of an infinitely small point source within a circle of
less than 200 .mu.m diameter. This is a significant improvement
over one existing cytometry lens design's 442.6 .mu.m full field
and 365.2 .mu.m on-axis spot sizes and 800 .mu.m diameter circle
energy (at 80% energy).
[0042] Other design parameters for the lens optimization software
include a magnification of at least 10.times., and preferably
between 10.5.times. and 11.5.times., and a back focal length of
127.+-.2 mm (as seen for surface 15 in the table, a back focal
length of 126.731 mm is obtained for the present embodiment), and a
wavelength range at least from 400 mm to 700 mm (the entire visible
light range). The total length and lens barrel diameter should be
as small as possible, i.e. less than 57 mm and 41 mm respectively,
since space near the flow cell is in high demand in cytometry
instruments. A lens length of 47.2 mm (combined thickness for
surfaces 3 to 14) and a maximum aperture radius (for less surfaces
13 to 15) of 17 mm show that these size goals have been met.
[0043] The lens is seen to comprise (a) a nearly hemispheric
plano-convex crown glass lens (surface 308 through 305 in the above
table including the cemented plate of identical material added for
handling) with its planar side 303 closest to the cytometry flow
cell and its convex surface 305 having a radius of curvature in a
range from 3.5 to 5.5 mm (4.66 mm in the present preferred
embodiment); (b) a pair of positive meniscus lenses (surfaces 306
to 309) with their concave sides 306 and 308 closest to the flow
cell (i.e. on the object side of the lens system) and with the
surfaces 308 and 309 of the second meniscus lens being less sharply
curved than the corresponding surfaces 306 and 307 of the first
meniscus lens, which are in turn less sharply curved than the
convex surface 305 of the plano-convex lens; and (c) a pair of
positive doublet lens elements (surfaces 310-315) to compensate for
chromatic aberrations from the first three lens elements. The near
hemispheric shape of the plano-convex lens (total axial thickness
of the lens plus the attached plate of identical crown glass
material being 4.715 mm compared to the 4.66 mm radius of curvature
of the convex surface 305, of difference of less than 1.2%) gives
the lens system its large field of view. The convex radius of
curvature range provides for a long working distance of at least
1.75 mm (about 2.2 mm in the present embodiment). Use of two
meniscus lenses, and also the use of crown glass material
(refractive index less then 1.55) for both the meniscus lenses and
the plano-convex lens, reduce aberrations, which are generally
proportional to the square of the amount of light bending at each
refractive surface. The lower aberrations provide improved
resolution, as indicated above the image spot size and circle
energy. The doublets are not achromats themselves, but are over
compensated so that the chromatic aberrations are reduced for the
entire lens system.
* * * * *