U.S. patent number 3,785,735 [Application Number 05/219,187] was granted by the patent office on 1974-01-15 for photoanalysis method.
This patent grant is currently assigned to Bio/Physics Systems, Inc.. Invention is credited to Mitchell Friedman, Louis A. Kamentsky, Isaac Klinger.
United States Patent |
3,785,735 |
Friedman , et al. |
January 15, 1974 |
**Please see images for:
( Certificate of Correction ) ** |
PHOTOANALYSIS METHOD
Abstract
A narrow beam of light is directed through an optical chamber to
intersect a thin stream of small particles to be optically
analyzed. At least two photoresponsive pick-up elements are
positioned at different angular positions with respect to the beam
to simultaneously detect different optical reactions of each
particle to the beam illumination.
Inventors: |
Friedman; Mitchell (Yorktown
Hts., NY), Kamentsky; Louis A. (Briarcliff Manor, NY),
Klinger; Isaac (Yorktown Hts., NY) |
Assignee: |
Bio/Physics Systems, Inc.
(Kantonah, NY)
|
Family
ID: |
22818238 |
Appl.
No.: |
05/219,187 |
Filed: |
January 19, 1972 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
|
2750 |
Jan 14, 1970 |
3705771 |
Dec 12, 1972 |
|
|
Current U.S.
Class: |
356/39; 250/565;
356/339; 356/342; 356/343 |
Current CPC
Class: |
G01N
15/1434 (20130101); G01N 21/53 (20130101); G01N
15/1459 (20130101); G01N 2015/1413 (20130101); G01N
2021/4707 (20130101); G01N 2021/4711 (20130101); G01N
2021/4709 (20130101) |
Current International
Class: |
G01N
21/53 (20060101); G01N 21/47 (20060101); G01N
15/14 (20060101); G01r 033/16 () |
Field of
Search: |
;356/39,40,41,42,102,103,104,181,184 ;250/218X |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
"Viability Assay System" L. A. Kamentsky, IBM Tech. Disclosure,
Vol. 12 No. 3 8/69..
|
Primary Examiner: Corbin; John K.
Assistant Examiner: Clark; Conrad
Attorney, Agent or Firm: Ailes; Curtis
Parent Case Text
This is a division of application Ser. No. 2,750 filed Jan. 14,
1970, now U.S. Pat. No. 3,705,771 issued Dec. 12, 1972.
Claims
We claim:
1. A method for rapidly detecting biological cells having different
properties including the steps of exposing a liquid solution
containing the cells to be analyzed to a dye which is taken up by
cells having one characteristic and not taken up to a significant
extent by cells having other characteristics,
moving the liquid solution in a thin stream to convey the cells one
by one through a housing comprised of a material which transmits
light and defining an optical chamber while directing light from a
light source into one side of the housing to intersect with the
thin stream of cells in a narrow incident beam,
the narrow incident beam converging at the stream of cells and
intercepting the entire width of the particle stream,
and detecting optical signals by means of photoresponsive pick-up
elements positioned outside of the housing at different angular
positions with respect to the direction of the incident beam when
measured from the intersection of the incident beam with the stream
of cells,
at least one of the photoresponsive pick-up elements being
effective to detect optical absorption of said incident beam to
indicate cells which took up the dye,
and at least one other photoresponsive pick-up element being
effective to detect scatter of said incident beam by the cells to
thereby detect the cells which did not take up the dye.
2. A method as claimed in claim 1 wherein
said photoresponsive pick-up element which is effective to detect
absorption is positioned at an angular position out of alignment
with the direction of the incident beam so as to measure absorption
in terms of a scatter signal.
3. A method as claimed in claim 1 wherein
the photoresponsive pick-up element effective to detect absorption
is positioned at essentially a zero angular position with respect
to the direction of the incident beam so as to be in line with the
incident beam for the direct measurement of optical absorption by
the cells.
4. A method as claimed in claim 1 wherein
the liquid solution containing the cells is moved in a thin stream
to convey the cells one by one through the housing by causing the
stream of cells to be narrowed down at the entrance of the housing
as the solution is moved through the housing.
5. A method as claimed, in claim 4 wherein
the liquid solution is narrowed down to a thin stream by conveying
a second compatible liquid through the housing as an annular sheath
of liquid surrounding the liquid solution and causing the liquid
solution to be confined to a thin stream at the center of the
sheath of surrounding compatible liquid as both are moved through
the housing.
6. A method as claimed in claim 5 wherein
the liquid solution is narrowed down to a thin stream having a
maximum dimension of the same order of magnitude as the maximum
dimension of the individual cells.
7. A method as claimed in claim 6 wherein
there is included the step of diluting the solution containing the
cells so that the maximum concentration of cells is in the order of
one million cells per milliliter of solution to thereby assure that
the cells pass the point of intersection with the light beam one by
one.
8. A method as claimed in claim 1 wherein
the cells to be analyzed are blood cells and the characteristic to
be determined is whether or not the cells are alive,
the dye being taken up by the dead cells and not significantly
taken up by the live cells.
9. A method as claimed in claim 8 wherein the dye is trypan blue.
Description
The present invention relates to photoanalysis apparatus, and more
particularly to photoresponsive apparatus for detecting various
characteristics of small particles such as blood cells.
There is a great need for accurate analysis of the characteristics
of groups of small particles such as in the analysis of air
pollution and water pollution conditions. A particularly important
field for such analysis is in medical research and diagnosis. For
this purpose, blood cells and other biological cells must be
analyzed.
In the present invention the analysis of small particles is
accomplished optically by entraining the particles in a very thin
stream of liquid so that the particles pass one by one in the
stream through an optical scanning station. A photo-optical
detecting device is arranged to detect the optical reaction of each
particle to illumination from a beam of light. This method has been
shown to impart very important information about the particles
which have been scanned. The information derived is particularly
valuable when special procedures are followed for preliminary
preparation of the particles, such as the prior application of dyes
which are taken up by the particles in different ways related to
the differences in particles which are to be detected. The
particles having these differences may be differentially
counted.
In accordance with one aspect of the present invention, it has been
determined that in an apparatus of the above description, the value
of the information which can be derived photo-optically is
tremendously increased if at least two different optical reactions
of the particles can be detected simultaneously by means of optical
pick-up elements arranged at different angular positions with
respect to the direction of the beam of light directed at the
particle.
One of the most useful optical reactions to the illumination is a
measurement of the optical absorption of the particles. The amount
of optical absorption of the particles may be directly related to
the amount of dye which has been taken into each particle in a
preliminary conditioning step. Since the dye uptake by the
particles may be different based upon different characteristics,
the optical absorption may thus be used to distinguish these
characteristics. The combined detection of absorption and another
optical reaction, such as scatter, therefore provides a very useful
combination of information about the particles under observation.
For instance, in the analysis of biological cell samples, such as
blood cells, it is recognized that dead cells absorb the dye Trypan
blue while live cells do not. Accordingly, it is possible to
analyze a sample of such cells by first exposing the sample to such
a dye and then optically measuring both absorption and scatter. The
dead cells which take up the stain are then optically detected by
the absorption signal, and the live cells are detected by the
optical scatter signal, and the apparatus may be used to count and
record the numbers of dead and live cells in the sample.
Accordingly, it is an object of the present invention to provide a
method and an apparatus in which at least two different optical
reactions of particles can be detected, and more specifically in
which different conditions of individual cells, such as the
differentiation of live and dead cells may be accomplished reliably
and quickly.
In photoanalysis apparatus of the above description, it has been
recognized that the light scattering effect produced by particles
under observation varies according to different characteristics of
the particles, including such factors as particle size, refractive
index, and the presence of refractive and absorbent substances in
the particles. Accordingly, the detection of scattered light from
the particles provides an extremely valuable method for determining
the characteristics of the particles. Furthermore, the magnitude of
the scatter radiation as a function of the angles or the ranges of
angles over which the scattering of light occurs provide
distinctive information. In apparatus heretofore available, it has
generally been possible to detect light scattered by particles
under observation only for a very small fixed range of scatter
angles. This has been accomplished by projecting illumination to
the particle by means of a lens having a mask over the central
portion thereof to create a "cone of darkness" beyond the particle
positioned at the focal point of the lens. The scatter illumination
is detected within this cone of darkness.
In accordance with another aspect of the present invention, it is
another object of the present invention to provide a photoanalysis
apparatus for optical analysis of particles in which very accurate
measurements of scatter illumination can be made at any desired
angle, or over any desired range of angles, without specific
limitation to a particular cone of darkness.
In carrying out the above object, a structure is employed in which
there is produced an extremely narrow beam of illumination directed
at the particles, and photosensing devices are arranged at
positions displaced from the beam to receive illumination scattered
at predetermined angles from the beam by the particles.
Another problem in photoanalysis apparatus of the above description
has always been the economical production of a suitable optical
chamber through which the particles to be examined are passed and
subjected to illumination. It has been previously thought to be
absolutely essential to provide flat sides on this optical chamber
in order to avoid distortion of the light beam directed through the
chamber by the material of the side walls of the chamber.
Accordingly, it is another object of the present invention to
provide a very satisfactory and easily produced optical chamber in
a photoanalysis apparatus which is extremely economical to produce
because of the manner in which provision is made for satisfactory
optical properties of the side walls.
In carrying out the above object of the invention, a photoanalysis
apparatus is provided having an optical chamber in the form of a
cylindrical tube.
Further objects and advantages of the invention will be apparent
from the following description and the accompanying drawings.
In carrying out the invention in one preferred form thereof, there
is provided an apparatus for simultaneous optical measurement of
several characteristics of each particle of a group of small
particles such as blood cells while the particles are suspended in
a liquid, including a source of light, a housing comprised of a
material which transmits light from the source and defining an
optical chamber. A means is provided for moving the particle
suspending liquid through the housing in a thin stream to convey
the particles in sequence through the stream one by one. Another
means is provided for directing light from the light source into
one side of the housing to intersect with the thin stream of
particles in a narrow beam, and at least two photoresponsive
pick-up elements are positioned outside of said housing at
different angular positions with respect to the direction of the
beam when measured from the intersection of the beam with the
stream of particles, the photoresponsive pick-up elements being
effective to simultaneously detect different optical reactions of
each particle to illumination from the beam.
In the accompanying drawings:
FIG. 1 is a schematic top view, partly, in section, of the most
essential elements of a photoanalysis apparatus in accordance with
the present invention.
FIG. 2 is a sectional side view of a portion of the apparatus shown
in FIG. 1 and illustrating additional photo-optical pick-up
elements for detecting scatter at additional angles.
FIG. 2A is a sectional side view corresponding to FIG. 2 and
illustrating, on a reduced scale, an alternative arrangement of the
apparatus including means for picking up and detecting fluorescence
of the particles.
FIG. 3 is an enlarged sectional front view taken through a section
at the center of the optical chamber of the apparatus of FIG. 1,
and illustrating the intersection of the beam of light with the
particles being measured.
FIG. 4 is a partial front view of a photoanalysis apparatus in
accordance with the invention and having modified photoelectric
pick-up elements for the detection of scattered illumination.
FIG. 5 is a top view, corresponding to FIG. 1, but illustrating a
modification of the photoresponsive pick-up elements in which
separate signals are derived to indicate the intensity of scattered
radiation and the angle of the scattered radiation.
And FIG. 6 is a top view of two optical chamber tube members for a
photoanalysis apparatus in the course of production, in accordance
with a preferred method of production.
Referring more particularly to FIG. 1, there is shown an optical
chamber formed by a glass tube member 10 clamped between metal
members 12, 14, which respectively include liquid tight annular
seals 16 and 18. The liquid 19 containing the particles to be
observed enters the apparatus through a tube 20 centrally disposed
within member 12. Another liquid 23, which forms a sheath for the
liquid 19 containing the particles enters the member 12 through an
entrance opening 22. The liquids come together in the cone or
funnel-shaped entrance portion 24 of the central bore 26 of the
cylindrical member 10.
The velocity and volume of flow of the particle-bearing liquid 19
entering through tube 20 and the other liquid 23 entering through
entrance 22 are such as to cause the stream of particle-bearing
liquid to be narrowed down at the end of the tube 20, as shown at
28, into a very narrow stream 29 having a maximum dimension of the
same order of magnitude as the maximum dimension of the particles
being carried by the stream. For instance, this dimension may be in
the order of a 25 micron stream diameter. The particles of greatest
interest are often somewhat smaller than this, being in the range
from 1 to 10 microns in diameter. The liquid 23 may be referred to
hereinafter as the "sheath flow" liquid since it forms a liquid
sheath about the narrowed stream 29. In order to provide a smooth
and non-turbulent flow of the sheath liquid 23, two or more radial
inlet openings 22 may be provided to the central bore of the member
12. The funnel-shaped entrance portion 24 of the cylindrical member
10 is preferably provided with a special exponential function
shape, as described more fully below, particularly in connection
with FIG. 6, in order to provide for smooth non-turbulent flow of
the liquids at the critical position 28 where the particle-carrying
liquid is narrowed down. Typically, the particle-carrying liquid
may be an aqueous solution and the sheath liquid 23 may be
water.
The stream 29 of particles is illuminated by a beam of light
emitted by a light source 30 which preferably consists of a laser.
One satisfactory laser, for instance, is a helium-neon laser. The
beam of light from the laser is reduced in diameter by a
combination of spherical lenses 32 and 34. The resultant reduced
diameter beam is collimated. This concentrated beam is narrowed by
a lens 36 to provide a very narrow beam at the point 38 where the
beam intersects with the stream 29 of particles under observation.
For this purpose, the lens 36 is preferably a cylindrical lens
having its cylinder axis arranged in a plane perpendicular to the
axis of the chamber cylinder 10. Thus, the pattern of the
illumination of the beam at the point 38 where it strikes the
stream of particles is a very narrow ellipse which appears to be a
thin line of light transverse to the stream of particles. This will
be described more fully below in connection with FIG. 3.
Electrical photoresponsive pick-up elements are arranged around the
outside of cylindrical chamber member 10 to detect different
optical reactions of each particle to illumination from the beam
through lens 36. For instance, an electrical photo-responsive
pick-up element 40 is arranged in direct line with the beam to
measure the absorption of illumination by each particle. The
resultant electrical signals are connected to apparatus
schematically shown at 46 for amplification and recording or
display. In the absence of a particle at the intersection of the
beam, or in the absence of any substantial absorption, the beam
strikes the element 40 without any substantial diminution.
As illustrated in the drawing, the beam diverges to a certain
extent after having been converged at the center of the chamber at
38. The effective divergence in a practical embodiment has been
limited to approximately one degree on each side of the center line
of the beam as measured from the particle scanning point 38 at the
center of the chamber. Thus, photoresponsive pick-up elements 42
and 44 are arranged on opposite sides of the direct beam and can be
used to measure illumination scattered out of the direct beam by
the particles over a selected range of angles from one degree up to
a predetermined angular limit. For instance, this range of angles
may be from one to nine degrees. As shown in the drawing, the
photoresponsive pick-up elements 42 and 44 may be electrically
connected in parallel so that electrical signals resulting from
illumination scattered on either side of the beam will be detected
and may be recorded by the electrical apparatus schematically
illustrated at 46. Additional pairs of photoresponsive pick-up
elements for detecting scattered light at other ranges of angles
may be provided as shown at 47 and 48. For instance, this
additional pair of pick-up elements may detect scatter over the
scatter angle range from 90.degree. to 22.degree.. The wider angle
scatter sensors 47 and 48 may be employed for the purpose of
providing a measurement of optical absorption of the particles as
an alternative to the absorption measurement by pick-up element 40.
It is known that absorption of the incident beam will decrease the
amount of light scattered by the particles. This decrease in
scattered illumination is more pronounced for the wider angle
scatter, than for the near forward scatter detected by sensors 42
and 44. Accordingly, it has been found to be advantageous to use
the wider angle scatter measurements at 47 and 48 to detect
absorption because "noise" signals due to light source intensity
fluctuations and flow stream vibrations are much less for the
scatter sensors than for the direct measurement absorption sensor
40 which is in the direct path of the beam.
Scattering of illumination from the particles in the reverse
direction, called "back-scattering", can also be detected by
photoresponsive elements 50 and 52 arranged on the same side of the
chamber as the light source 30 and connected in parallel to an
electrical pick-up and recording apparatus 54. It will be
understood that the electrical apparatus 54 may be combined with
the apparatus 46, but it is shown separately here to simplify the
drawing.
The apparatus 46 and 54 may include amplifiers, logic circuitry,
digital counters, and electronic display devices. It is one of the
important features of the invention that different optical
reactions of each particle to illumination may be detected,
processed, and recorded substantially simultaneously. The
relationships between these different optical reactions may be
processed by analog and digital circuitry, displayed, recorded, and
plotted as a basis for making detailed determinations about the
particles, differentially classifying the particles, or determining
the frequency with which particular characteristics appear in
successive particles. Because of the unique features of this
invention, particle analysis and counting rates in the order of ten
thousand particles per second may be achieved. It will be
understood that this speed is well within the capacity of the
electronic and digital portions of the system.
The photoresponsive pick-up elements, such as elements 42 and 44
for the detection of scatter, are illustrated in FIG. 1 as though
they were fixed with relation to the cylindrical chamber member 10.
However, suitable means is provided for precisely changing the
position of those pick-up elements in relation to the beam from
source 30. This adjustment may be an adjustment from side to side
and it may also be an adjustment to place the elements in greater
or lesser proximity to the scanning position 38. By moving the
elements away from the scanning position, the inner margins of the
elements may be precisely positioned with respect to the outer
margins of the direct radiation beam directed to the absorption
pick-up element 40. Thus, the elements 42 and 44 are capable, when
so adjusted, of picking up scatter radiation over the narrowest
possible angle outside of the direct radiation beam path.
FIG. 2 is a partial sectional side detail view of the apparatus of
FIG. 1 including the cylindrical chamber member 10, the cylindrical
lens 36, the absorption and scattering pick-up elements 40, 44, and
48, and the back-scattering pick-up element 52. As illustrated in
FIG. 2, the cylindrical shape of the chamber member 10 causes a
refractive effect upon the light beam supplied through the
cylindrical lens 36 which causes the light beam to converge towards
the center bore 26 of the chamber member 10. This effect is shown
in an exaggerated form in FIG. 2. The diameter of the beam as it
enters the cylindrical lens 36 is actually selected so as to be
approximately equal to the diameter of the center bore 26. This
diameter is of the order of 250 microns. However, the convergence
of the beam in the plane of FIG. 2 (perpendicular to the axis of
the chamber member 10) is not a disadvantage since it serves to
concentrate the beam in the central portion of the center bore 26
where the particle carrying stream is located. Since the particle
carrying stream has a diameter of only about 25 microns, a
considerable convergence of the beam is desirable. This provides
relatively uniform illumination over the diameter of the
particle-carrying stream, even though the original energy
distribution from the laser beam is non-uniform. Furthermore, if
the beam were not caused to converge upon the center bore 26, the
outer portions of the beam would strike the interface between glass
and liquid at the center bore 26 at an angle greater than the
critical angle of refraction so that those outer portions of the
beam would be reflected away from the center bore, and lost,
without passing through the liquid.
FIG. 1 illustrated how pairs of photoresponsive pick-up elements
such as 42 and 44, and 46 and 48, can be arranged in positions
spaced away from the primary beam in directions parallel to the
axis of the cylindrical chamber member 10 for detecting small
angles of scattered illumination. However, as shown in FIG. 2, when
larger angles of scatter are to be detected, the angular
displacement of pick-up elements can be around the circumference of
the cylindrical chamber member. Thus, as shown in FIG. 2, a pair of
pick-up elements 56 and 58 may be circumferentially arranged to
detect light scattered in a range at about 45 degrees from the
particle scanning point 38. Similarly, pick-up elements 60 and 62
may be provided to detect scatter in a range near 90 degrees. It
will be understood that these arrangements of pick-ups are by way
of illustration only. Particular analyses will require the
detection of scatter for particular ranges of angles. The important
principle illustrated by FIGS. 1 and 2 is that light scattered by
particles under analysis can be detected for any selected ranges of
scatter angles from essentially 1.degree. up to 179.degree. with
the analyzer configuration as illustrated.
All of the components illustrated in FIG. 2, with the exception of
the cylindrical lens 36, are preferably mounted upon and movable
with a support block schematically shown as a box 55 pivotally
supported on a fixed mounting at 57. The support block 55 may be
vertically adjusted by rotation about the pivot 57 by means of a
thumb screw 59 engaging the lower edge of the block 55. Thumb screw
59 is threadedly engaged within a fixed support 61. The purpose for
this vertical adjustment is to precisely position the chamber 10
with respect to the light supplied from the light source through
the cylindrical lens 36. If the light beam is not vertically
centered upon the center bore 26 of the chamber 10, so that the
beam accurately intersects with the stream 29 of particle-carrying
liquid, then the device may be inoperative. The accurate
positioning of the chamber with respect to the beam is very
important because an offset in the positioning of the chamber with
respect to the beam causes undue loss of beam energy through
excessive refraction of beam energy at the bore 26.
In the embodiment of the invention illustrated in FIGS. 1 and 2,
there is unavoidably a certain amount of radiation which is
reflected radially outwardly from the scanning point 38 in a narrow
ring which is confined to a longitudinal dimension along the
cylinder of the cylindrical chamber member 10 generally
corresponding to the width of the entering beam of illumination
from source 30. Thus, the scatter detectors are always
longitudinally displaced out of this ring of radiation and
preferably arranged in pairs on opposite sides of the direct
radiation position as shown by detectors 42 and 44 in FIG. 1.
Similarly, in FIG. 2, the scatter detectors 56, 58, 60, and 62 each
represent a pair of detectors preferably arranged on opposite sides
of the ring of radiation.
The preferred photoresponsive pick-up elements to be employed in
the present invention as thus far described may consist of silicon
barrier layer photo diodes. These devices are photo-voltaic devices
commonly referred to as silicon solar cells. Suitable devices of
this kind are available from many commercial sources.
FIG. 2A corresponds generally to FIG. 2, but illustrates
alternative arrangements of the components, and also additionally
illustrates an arrangement for picking up and detecting
fluorescence of the particles. The light from the light source is
again supplied through the cylindrical lens 36 to the optical
chamber 10, and absorption is detected by photo detector 40. In
this embodiment of the invention, the scatter detector 44 includes
an optical reflector which may consist of a mirror 44B, and a photo
electric device 44C arranged to receive the scatter radiation
reflected by the optical reflector 44B. Since these two elements
accomplish the same combined function as the scatter pick-up
element 44 of FIG. 2, the combination of the two elements 44B and
44C may be referred to collectively as a photoresponsive pick-up
element. As explained above in connection with FIG. 1, the scatter
pick-up element 44 is preferably paired with another scatter
pick-up element 42, the combination of the two pick-up elements
being effective to detect scatter on the two sides of the light
beam passing through the scanning point. By means of the reflective
arrangement shown and described in connection with FIG. 2A, it is
possible to employ two separate reflectors on opposite sides of the
beam, both reflectors directing scatter radiation to a single
photoelectric device such as 44C. By this means, the scatter
radiation signals from the two sides of the beam need not be
electrically combined. They are, instead, optically combined by
being directed by separate reflectors to the single photoelectric
device.
The particular arrangement thus far described, with the reflector
44B, and a possible additional reflector, reflecting scatter
signals to a single photoelectric device 44C may be employed in the
embodiment previously shown and described in FIG. 2, and is not
necessarily limited to combination with other features of FIG. 2A
described immediately below.
One of the most useful categories of optical measurements available
in the photoanalysis of particles is the measurement of
fluorescence of the particles in response to the primary radiation
by the light beam directed through the cylindrical lens 36. The
particles may be stained with dyes so that when excited with light
they are caused to emit fluorescent radiation at one or more wave
lengths different from the wave length of the primary light beam.
The intensity of fluorescent radiation at various wave lengths is
an extremely useful indication of the properties and
characteristics of the particles. Since the fluorescent radiation
is emitted in virtually all directions from the particle, the
fluorescent radiation is gathered by the reflectors 63 and 65 and
generally directed, through a cylindrical lens 67, to a dichroic
mirror 69. As is well known, the dichroic mirror 69 is designed to
reflect radiation having a wave length shorter than a predetermined
limit such as 5,500 angstroms and to transmit radiation having a
longer wave length. The reflected radiation is directed through a
filter element 71 to a first photo multiplier tube 73. The
transmitted radiation is directed through a second optical filter
75 to a second photo multiplier tube 77. By means of the
combination of the dichroic mirror 69 and the optical filters 71
and 75, each of the photo multiplier tubes 73 and 77 receives only
that light at the wave length predetermined by the respective
filters. If desired, additional dichroic mirrors may be provided to
split up the fluorescent radiation into additional spectral
components in order to obtain additional optical analysis
information.
The reflective elements 63 and 65 generally extend axially along
the tubular chamber member 10. However, these reflective elements
are preferably split similarly to the scatter-sensing devices as
pictured in FIG. 1, so as to avoid reflecting and transmitting the
ring of radiation which is discussed immediately above. Avoiding
the reflection and transmission of the ring of radiation is
particularly important when dealing with radiation at the green end
of the visible spectrum because it is difficult to provide
efficient optical filters 71 and 75 at that end of the spectrum.
However, when dealing with fluorescence at the red end of the
visible spectrum, efficient filtering is available and it is then
preferable not to provide split reflective elements 63 and 65 and
to gather the ring of radiation, which includes fluorescence
signals at the desired wave length.
The reflector 63 is preferably arranged so that fluorescent
radiation emitted through the lower surface of the chamber member
10 is reflected up to the upper reflector 65. The upper reflector
65 is preferably arranged so that all of the radiation directed to
that reflector is ultimately reflected to and gathered by the left
portion of that reflector and directed, through lens 67, down to
the dichroic mirror 69.
When making fluorescence measurements, it has been found quite
effective to employ for the light source 30 an argon laser which
emits light in the blue part of the spectrum. Light of this wave
length has been found to provide a high degree of fluorescence
emission by biological particles which are often under observation.
Since the particles fluoresce in the "forward" direction, it has
been found to be quite desirable to provide a filter 79 which
permits the passage only of the argon blue direct radiation to the
scatter sensor 44B-44C, and to the absorption sensor 40.
It will be appreciated that the arrangement shown in FIG. 2A is
particularly useful because it permits simultaneous detection of a
least four different optical reactions of a particular particle
simultaneously. Thus, absorption is detected at 40, scatter is
detected at 44B-44C, and two different wave lengths of fluorescent
radiation are detected at photo multipliers 73 and 77.
Metachromatic, or combinations of fluorescent dyes are available
which, when used to stain cells, will fluoresce at two wave lengths
with each fluorescent intensity proportional to a given cellular
constituent.
FIG. 3 is an enlarged detail sectional front view of that portion
of the apparatus of FIG. 1 where the light beam actually strikes
the stream of particles at point 38 in the chamber 10. The stream
of particles 29 is shown to contain actual entrained particles 64
which are transported one at a time through the elliptically shaped
beam of light at 66. Because of the elliptical shape of the beam of
light, the stream 29 carrying the particles 64 may vary in its
position within the chamber without having the stream actually move
out of the path of the beam. Thus, the beam always intercepts the
entire stream of particles. On the other hand, the narrow
elliptical shape of the beam, in which the width of the beam is of
substantially the same order of magnitude as the diameter of the
particles, means that each particle causes an optimum optical
reaction with the beam. Furthermore, even if the particles are
closely spaced together, it is virtually impossible for more than
one particle to be acted upon by the beam at any particular
instant.
FIG. 4 is a sectional front view illustrating a modification of the
apparatus of FIG. 1 in which arcuate shaped pick-up elements 42A,
44A, and 46A, 48A are employed. This view is taken at a section
corresponding to the section line indicated at "4-4" in FIG. 2,
although it will be understood that the actual modified structure
of FIG. 4 is not illustrated in FIG. 2. The scatter pick-up
elements 42 and 44 and 46 and 48 of FIG. 1 are arranged to
intercept scatter over selected ranges of angles simply by virtue
of a displacement in position along the axis of the cylindrical
chamber member 10. As illustrated in FIG. 2, for larger scatter
angles, the measurements can be made by circumferential
displacement of the pick-up elements about the cylindrical chamber.
However, a combination of these techniques is possible in which a
high efficiency is achieved by means of the semicircular pick-ups
illustrated at 42A, 44A, and 46A, 48A in FIG. 4. Thus, the
illumination scattered over a particular range of angles may be
scattered anywhere within a possible cone of scatter radiation
terminating in a substantially circular radiation area such as the
area defined by the pick-up elements 46A and 48A. Thus, the
semicircular pick-up elements are particularly efficient in
intercepting substantially all of the scatter radiation over the
range of scatter angles for which they are designed.
FIG. 5 is a top view corresponding to FIG. 1 and illustrating a
modification of the apparatus of FIG. 1 employing special light
position-sensing photo-detectors 68 and 70 which are capable of
determining both the intensity and the angular position of scatter
radiation. These are silicon barrier photo diodes of the type which
are commercially available for instance from United Detector
Technology of Santa Monica, Calif., the name of the product being
designated as Light Position Sensing Photo-detectors. The photo
detectors 68 and 70 are direct current voltage source indicated at
72. The current from source 72 enters the detectors 68 and 70 at
the center detector terminals 74 and 76. The current passes through
either or both of the detectors and is emitted at the end terminals
78 and 80, or at the end terminals 82 and 84, the ratio of the
current through the different end terminals of each detector device
is determined by the position of the radiation striking the device.
For instance, if the radiation strikes the device 70, and if the
radiation position is closer to the terminal 84 than it is to the
terminal 80, there will be a greater current from the terminal 84
than there is from the terminal 80. The currents from terminals 82
and 84 are returned to the source 72 through a load resistor 86 to
ground and from ground through a load resistor 88. Similarly, the
currents from terminals 78 and 80 are returned through a load
resistor 90 to ground and thus through load resistor 88 to the
source 72. The resistance values of load resistors 86 and 90 are
preferably equal and the relative values of the load currents
through those resistors are measured in terms of voltage drops in a
differential amplifier 92 to provide a scatter angle signal fed to
a detection and recording circuit 46A.
Similarly, the intensity of the scatter illumination is detected in
terms of the voltage drop across the common load resistor 88 by an
amplifier 94, the output of which is also supplied to the circuit
46A. Thus, the arrangement of FIG. 5 does not simply record scatter
radiation in specified ranges of scatter angles, but rather it
records the intensity of the scatter radiation and indicates the
angle at which that radiation occurs. This provides a particularly
valuable analysis tool where the significant range of scatter angle
is not previously known.
In accordance with the present invention it has been determined
that a new method may be followed, employing the apparatus
described above, for rapidly analyzing samples of particles, and
particularly biological samples of cells such as blood cells. As
mentioned above, different conditions of biological cells may
control the amount of dye which individual cells will pick up when
the cell sample is exposed to a dye. For instance, when a mixture
of live cells and dead cells is exposed to Trypan blue dye, the
dead cells take up the dye to a significant extent, while the live
cells do not.
In a specific example of the practice of this method, the method
may be employed to determine the feasibility of an organ transplant
from one individual to another by checking for compatability of
blood cells. For such a compatability test, lymphocytes may be
extracted from the blood of a donor to be matched with the sera of
potential recipients. To effect this match the lymphocytes are
diluted in 0.85 percent sodium chloride solution or an appropriate
nutrient medium to a concentration of 5,000,000 cells per
milliliter and added to the diluted serum of the potential donor.
The mixture is then incubated at a temperature in the range from
35.degree. to 40.degree. Centigrade for approximately 45 minutes.
The incubation is then stopped by placing the samples on ice.
Immediately before examination a freshly prepared Trypan blue
aqueous solution containing 0.24 percent Trypan blue and 0.85
percent sodium chloride is added to the sample in a volume equal to
50 percent of the sample volume so as to increase the sample volume
by about 50 percent. The final suspension should have a cell
concentration in the range of 1,000,000 cells, or less, per
milliliter of solution to provide reasonable assurance that the
cells will pass through the apparatus one at a time. The sample is
then caused to pass through the apparatus as described above in
connection with FIG. 1, and the optical absorption of the cells is
detected concurrently with the optical scatter over a range from
about 1.degree. to about 30.degree. away from the axis of the beam.
The stained cells, which provide a large absorption signal, are
indicated as dead, and the unstained cells, which provide a high
scatter signal, and a low absorption signal are indicated as live.
A high count of dead cells in this compatability analysis indicates
a lack of compatibility between donor and recipient for the purpose
of transplants.
For this analysis, a helium neon laser emitting illumination at
6300 angstroms may be employed. Other dyes may be alternatively
used for this live-dead cell analysis method. For instance,
nigrosine dyes are useful for this purpose.
FIG. 6 illustrates an essential step in the preferred method of the
production of the optical chamber member 10 of the apparatus of the
present invention. In practicing this method, a piece of glass
tubing having inside and outside diameters of the desired size for
the finished optical measurement portion of the tubular optical
chamber is cut to a length which is somewhat greater than twice the
desired length of the finished optical chamber. The tubing must be
substantially free of any cracks, scratches or blemishes. The glass
tubing is then heated uniformly around the central portion of the
axial length thereof to the softening temperature of the glass.
This may be simply accomplished by rotating the tubing while the
central portion of the tubing is held in a gas flame.
Next, the central bore of the tubing is placed under pressure. This
causes enlargement, or blowing out, of the central bore of the
tubing at the heated central portion thereof, as shown at 96 in
FIG. 6.
When the proper degree of enlargement has been achieved, the
pressure is released and the tubing is allowed to cool and harden.
As illustrated in FIGS. 1, 4, 5, and 6, a workable enlargement has
been found to be an enlarged inside diameter which is in the order
of five to six times the original inside diameter. It is then cut
apart through the centerline as indicated at 98. The two resultant
separate pieces 10A and 10B can each be used as optical chambers in
an apparatus such as previously described in connection with FIG. 1
after the next step. As the final step, in order to provide a
liquid-tight seal at the ends of each chamber, both ends of each of
the glass parts 10A and 10B are preferably ground flat and mutually
parallel to eliminate the slight inaccuracies of the glass cutting
procedure.
There are a number of useful variations in the process described
just above. For instance, the central bore of the tubing may be
placed under pressure while the tubing is being heated so that the
enlargement of the tubing occurs at the earliest possible moment
when the heating has progressed sufficiently to soften the glass.
Also, it has been found to be quite practical and desirable to
perform the heating and pressurizing steps at spaced axial
positions along an extended length of glass tubing. All of the tube
cutting operations are then done after the enlargements have been
formed, cutting at each enlargement and midway between adjacent
enlargements to thereby obtain a yield of two optical chambers for
each tubing enlargement.
It has been found that the above method of production of the
optical chambers is very simple and inexpensive and also very
satisfactory. It has been determined that the changes in the
diameter in the resultant enlarged funnel-shaped end 24 of the
central bore 26 is substantially an exponential function. That is,
the change in the diameter of the central bore is substantially an
exponential function of the displacement along the axis of the tube
at which the diameter is measured. This provides a graded change in
the bore diameter which is very valuable and useful in promoting
smooth and non-turbulent liquid flow in the critical bore narrowing
region where the sheath liquid is reducing the diameter of the
particle carrying liquid stream.
While this invention has been shown and described in connection
with particular preferred embodiments, various alterations and
modifications will occur to those skilled in the art. Accordingly,
the following claims are intended to define the valid scope of this
invention over the prior art, and to cover all changes and
modifications falling within the true spirit and valid scope of
this invention.
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