CA2050761A1 - Flow imaging cytometer - Google Patents
Flow imaging cytometerInfo
- Publication number
- CA2050761A1 CA2050761A1 CA002050761A CA2050761A CA2050761A1 CA 2050761 A1 CA2050761 A1 CA 2050761A1 CA 002050761 A CA002050761 A CA 002050761A CA 2050761 A CA2050761 A CA 2050761A CA 2050761 A1 CA2050761 A1 CA 2050761A1
- Authority
- CA
- Canada
- Prior art keywords
- image capturing
- flow
- light
- light source
- specimen solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 238000003384 imaging method Methods 0.000 title claims abstract description 34
- 239000002245 particle Substances 0.000 claims abstract description 76
- 238000001514 detection method Methods 0.000 claims abstract description 16
- 239000007850 fluorescent dye Substances 0.000 claims abstract description 4
- 230000001678 irradiating effect Effects 0.000 claims description 12
- 239000000306 component Substances 0.000 claims 14
- 230000001934 delay Effects 0.000 claims 2
- 238000012544 monitoring process Methods 0.000 abstract description 11
- 206010056740 Genital discharge Diseases 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 47
- 238000010586 diagram Methods 0.000 description 27
- 239000000243 solution Substances 0.000 description 16
- 238000010276 construction Methods 0.000 description 8
- 210000000265 leukocyte Anatomy 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 239000004065 semiconductor Substances 0.000 description 2
- 230000001360 synchronised effect Effects 0.000 description 2
- 230000001960 triggered effect Effects 0.000 description 2
- 210000002700 urine Anatomy 0.000 description 2
- 241001520954 Cameraria <moth> Species 0.000 description 1
- 101710083262 Ectin Proteins 0.000 description 1
- 241001505295 Eros Species 0.000 description 1
- 208000003251 Pruritus Diseases 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 229910052736 halogen Inorganic materials 0.000 description 1
- 150000002367 halogens Chemical class 0.000 description 1
- 239000002674 ointment Substances 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000012192 staining solution Substances 0.000 description 1
Classifications
-
- G01N15/1433—
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Electro-optical investigation, e.g. flow cytometers
- G01N15/1425—Electro-optical investigation, e.g. flow cytometers using an analyser being characterised by its control arrangement
- G01N15/1427—Electro-optical investigation, e.g. flow cytometers using an analyser being characterised by its control arrangement with the synchronisation of components, a time gate for operation of components, or suppression of particle coincidences
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Electro-optical investigation, e.g. flow cytometers
- G01N15/1456—Electro-optical investigation, e.g. flow cytometers without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
- G01N15/1459—Electro-optical investigation, e.g. flow cytometers without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals the analysis being performed on a sample stream
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Electro-optical investigation, e.g. flow cytometers
- G01N15/1434—Electro-optical investigation, e.g. flow cytometers using an analyser being characterised by its optical arrangement
- G01N2015/144—Imaging characterised by its optical setup
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N2021/1765—Method using an image detector and processing of image signal
- G01N2021/177—Detector of the video camera type
- G01N2021/1772—Array detector
- G01N2021/1774—Line array detector
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N2021/1789—Time resolved
- G01N2021/1791—Time resolved stroboscopic; pulse gated; time range gated
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
Abstract
FLOW IMAGING CYTOMETER
ABSTRACT OF THE DISCLOSURE:
A flow imaging cytometer, in which an image capturing area for cells traveling in the flow of a specimen solution is constantly monitored to perform cell photography in an efficient manner, is improved so that fluorescent images of cells can be acquired with greater efficiency. Cell particles to be sensed in the specimen are treated with a fluorescent stain, and the specimen solution is irradiated with light from a pulsed light source for exciting fluores-cence, and with infrared light constantly for monitoring the passage of cells through the cytometer. When the monitoring light is made to serve also as the light for exciting fluorescence, the specimen flow is continuously irradiated at all times at a low luminance for the purpose of monitoring. Then, when a cell particle of interest is detected, the cell particle is irradiated with a light pulse of high luminance to obtain a still picture of the fluorescence-emitting cell. At detection of the cell, a source of white light can be actuated to acquire a white-light image of the cell.
ABSTRACT OF THE DISCLOSURE:
A flow imaging cytometer, in which an image capturing area for cells traveling in the flow of a specimen solution is constantly monitored to perform cell photography in an efficient manner, is improved so that fluorescent images of cells can be acquired with greater efficiency. Cell particles to be sensed in the specimen are treated with a fluorescent stain, and the specimen solution is irradiated with light from a pulsed light source for exciting fluores-cence, and with infrared light constantly for monitoring the passage of cells through the cytometer. When the monitoring light is made to serve also as the light for exciting fluorescence, the specimen flow is continuously irradiated at all times at a low luminance for the purpose of monitoring. Then, when a cell particle of interest is detected, the cell particle is irradiated with a light pulse of high luminance to obtain a still picture of the fluorescence-emitting cell. At detection of the cell, a source of white light can be actuated to acquire a white-light image of the cell.
Description
-1- 2~76~
FLOW Ii~lAGING CYTOMETER
BAC~CGROUND QF THE INVENTION:
1. Field of the Invention This invention relates to an apparatus for forming 5 a particle-containing specimen soLution such as blood or urine into a flat sheathed flow, irradiating the flat flow of the specimen solution with pulsed light to obtain a still picture, and applying imaging processing to perform ?
analysis such as classi-~ication and enumeration of the 10 particle components contained in the specimen solution.
More particularly, the invention relates to a ~low imaging cytometer, which is a particle image analy~er, adapted so as to constantly monitor an image cap~uring area of the flat specimen flo~, irradiate the particles with light 15 when they reach the image capturing area, and acquire a ~luorescent image and/or image by white light of the particle components in an ef~icient manner.
FLOW Ii~lAGING CYTOMETER
BAC~CGROUND QF THE INVENTION:
1. Field of the Invention This invention relates to an apparatus for forming 5 a particle-containing specimen soLution such as blood or urine into a flat sheathed flow, irradiating the flat flow of the specimen solution with pulsed light to obtain a still picture, and applying imaging processing to perform ?
analysis such as classi-~ication and enumeration of the 10 particle components contained in the specimen solution.
More particularly, the invention relates to a ~low imaging cytometer, which is a particle image analy~er, adapted so as to constantly monitor an image cap~uring area of the flat specimen flo~, irradiate the particles with light 15 when they reach the image capturing area, and acquire a ~luorescent image and/or image by white light of the particle components in an ef~icient manner.
2. Description of the Prior Art A technique known in the art is to subJect particles 20 such as stained cells to exciting light and perform analysis such as classi*ication of the particles by detecting fluorescence emitted by the particles. Specific e~amples of apparatus which employ this technique are ~low cytometers and microscopes.
A ~low cytometer is capable of detectin~ the amount of ~luorescence emitted by individual particles.
A detailed ~lùorescent image can be observed by using a microscope. In addition, an àrrangement is availahle in which the fluorescent image obtained is subjected to image pro~essing. Furthermore, the specification of Japanese Patent Application Laid-Open (KOKAI) No. 63-269875 discloses an image capturing apparatus wherehy images can be acquired using three types of light, namely ultraviolet light, visible li~ht and infrared light.
An apparatus for acquiring the images of particles ~lowing as a ~l~t stream and analyzin~ the particles by image processing is disclosed in -the specifications of .
2~76.~
Japanese Patent Application Laid-Open INo. 57-~00995 and USP 4,338,024.
Further, the present applicant has previously filed an application for an apparatus adapted so as to constantly monitor an ima~e capturing area, detect particles in the flow when they arrive at this area, and acquire images o~
the particles in e-fficient ~ashion.
SU~M~RY OF THE INVENTION:
Though the conventional ~low cytometer exhibits a high processing capability per unit time, the fluorescence from particles can be obtained only as a gross value, and it is not possible to acquire detailed information as to which portions o~ a particle are emitting ~luorescence and the degree of this ~luorescence. Though a large quantity of in-formation can be obtained using a microscope, pre-treatment is laborious and a high processing speed cannot be obtained.
Accordingly, an obJect of the present invention is to provide a ~low imaging cytometer which overcomes these di~ficulties encountered in the prior art.
Another object o~ the present invention is to provide a flow imaging cytometer having a high analyzing capability and adapted to obtain, efficiently and at high speed, a large quantity of particle in~ormation which includes detailed information relating to individual particles.
According to the present invention, the ~oregoing obJects are attained by providing a flow imaging cytometer comprising a ~low cell ~ormed to include a ~lat ~low path ~or causing a specimen solution containing particle 30 ~ components to be sensed to ~low as a ~lat stream a first light source arranged on a ~irst side o~ the flow cell for irradiating the specimen solution in the flo~ cell with pulsed light, first image capturing means arranged on a second side of the~low cell ~or capturing still pictures of the particle components in the specimen solution irradiated by the first light source,~a second light source arranged on the ~irst side o~ the flow cell ~or irradiating the specimen solution in the flow cell with light constantl~, second 7 ~ ~
A ~low cytometer is capable of detectin~ the amount of ~luorescence emitted by individual particles.
A detailed ~lùorescent image can be observed by using a microscope. In addition, an àrrangement is availahle in which the fluorescent image obtained is subjected to image pro~essing. Furthermore, the specification of Japanese Patent Application Laid-Open (KOKAI) No. 63-269875 discloses an image capturing apparatus wherehy images can be acquired using three types of light, namely ultraviolet light, visible li~ht and infrared light.
An apparatus for acquiring the images of particles ~lowing as a ~l~t stream and analyzin~ the particles by image processing is disclosed in -the specifications of .
2~76.~
Japanese Patent Application Laid-Open INo. 57-~00995 and USP 4,338,024.
Further, the present applicant has previously filed an application for an apparatus adapted so as to constantly monitor an ima~e capturing area, detect particles in the flow when they arrive at this area, and acquire images o~
the particles in e-fficient ~ashion.
SU~M~RY OF THE INVENTION:
Though the conventional ~low cytometer exhibits a high processing capability per unit time, the fluorescence from particles can be obtained only as a gross value, and it is not possible to acquire detailed information as to which portions o~ a particle are emitting ~luorescence and the degree of this ~luorescence. Though a large quantity of in-formation can be obtained using a microscope, pre-treatment is laborious and a high processing speed cannot be obtained.
Accordingly, an obJect of the present invention is to provide a ~low imaging cytometer which overcomes these di~ficulties encountered in the prior art.
Another object o~ the present invention is to provide a flow imaging cytometer having a high analyzing capability and adapted to obtain, efficiently and at high speed, a large quantity of particle in~ormation which includes detailed information relating to individual particles.
According to the present invention, the ~oregoing obJects are attained by providing a flow imaging cytometer comprising a ~low cell ~ormed to include a ~lat ~low path ~or causing a specimen solution containing particle 30 ~ components to be sensed to ~low as a ~lat stream a first light source arranged on a ~irst side o~ the flow cell for irradiating the specimen solution in the flo~ cell with pulsed light, first image capturing means arranged on a second side of the~low cell ~or capturing still pictures of the particle components in the specimen solution irradiated by the first light source,~a second light source arranged on the ~irst side o~ the flow cell ~or irradiating the specimen solution in the flow cell with light constantl~, second 7 ~ ~
image capturing means arranged on the second side of the flow cell for capturing an image of the particle componen~s i.rradiated by the second light source, processing means for executing prescribed analysis based upon image data from the first and second image capturing means, and control means ~or detecting the particle componen~s based upon the ima~e data from the second image capturing means, and on the basis of such detection, ~or actuating the first light source within an image capturîng period of the first image captur-ing means., wherein the particle components to be sensed aretreated with a fluorescent stain and the ~irst light source is for exciting fluorescence.
In another aspect of the present invention, the foregoing objects are attained by providing a flow imaging lS cytometer comprising a flow cell ~ormed to include a flat flow path for causing a specimen solution containlng particle components to be sensed to flow as a flat stream, a ~irst light source arranged on a first side o~ the flow cell ~or irradiating the specimen solution in the flow cell with light quantity of which is switched, first image capturing means arranged on a second side of the flow cell for capturing still pictures of particle components in the specimen solution irradiated with high-luminance pulsed light ~rom the first light source, second image capturing means arranged on a second side o~ the flow cell ~or capturing images of particle components in the specimen solution irradiated continuously with low-luminance light from the first light sourcei processing means for executing prescribed analysis based upon image data from the first and second image capturing means, and control means for : detecting the particle components based upon the image data from the second image capturing means, and on the basis o~ such detection, for switching the ~irst light source over to irradiation with the high-luminance light, wherein the particle components to be sensed are treated with a ~luorescent stain and the first light source is for e~citing fluorescence.
2~76:L
.~
Other features and advantages of the present invention will be apparent from the following description ~aken in conjunction with the accompanying drawings, in which like reference characters designate the same or similar parts throughout the ~igures thereof.
BRIEF DESCRIPTION OF THE DRAWINGS-.
Fig. 1 is a block diagram illustrating the con-struction of a ~low imaging cytometer according to a first embodiment o~ th~ present invention;
Fig. 2 is a diagram for describing an exciting-light irradiated area and an image capturing area in a flow cell of the cytometer;
Fig. 3 is a diagram showing the characteristic o~
a ~ilter for eliminating exciting light;
Fi.g. 4 is a diagram illustrating an example of the characteristic of a dlchroic mirror;
Fig. 5(a) :Is a diagram showlng an e~ample of a luminance curve in particle detection;
Fig. 5(b) is a diagram showing an example of a light-emission pulse for image capturing, the pulse being emitteda~ter detection of a particle;
Fig. 6 is a measurement flowchart associated with the ~low imaging cytometer o~ the present invention;
Fig. 7 is a block diagram illustrating the con-struction of a flow imaging cytometer according to a second embodiment of the present invention;
Fig. 8 is a diagram for describing e~amples of image capturing areas and irradiation areas in a flow cell associated w1th the cytometer of Fig. 7;
Fig. 9 is a block diagram illustrating the construction of a ~low imaging cytometer according to a third embodiment of the present invention;
Fîg. lO is a diagram for describing examples of image capturing areas and irradiation areas in a flow cell associated with the cytometer o~ Fig. 9;
Fig. 11 i.s a diagram illustrating an example o~ thecharacteristic o~ a dichroic mirror associated with the cytometer o~ Fig. 9;
2~7~ ~
Fig. 12 is a diagram illustrating another e,Yample of the characteristic of a dichroic mirror associated vith the cytometer of Fig. 9;
Fig. 13 is a diagram :illustrating an e~ample of the characteristic of a filter, associated with the cytometer of Fig. 9, for filtering out e~citing light;
Fig. 14 is a diagram illustrating an example of a pulse ~or image capturin~ at the time o~ particle detection:
Fig. 15 is a bloc~ diagram illustrating the construction o-f a flow imaging cytometer according to a fourth embodiment of the present invention;
Fig. 16 is a diagram for describing examples of image capturing and irradiation areas in a flow cell associated with the cytometer of Fig. 15;
Fig. 17 is a bloc~ diagram illustrating the con-struction of a flow imaging cytometer accordlng to a ~ifth embodiment of the present invention;
Fig, 18 is a diagram exempli~ying the characteristic of an exciting-light selecting (transmltting) filter; and Fig. 19 is a block diagram illustrating the con-struction o~ a ~low imaging cytometer according to a si~th embodiment of the present invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS:
First Embodiment Pre~erred embodim~n~s o~ the present in~ention will now be described in detail with re~erence to the accompanying drawings.
Fig. 1 is a block diagram illustrating the basic construction o~ a flow imaging cytometer according to a first embodiment o~ the present invention, and Fig. 6 is a me,asurement flowchart associated with this cytometer. As shown in Fig. 1, the flow imaging cytometer includes a flow cell 28 in which a stained specimen solution (o~ blood or urine, etc.) is enveloped by a sheathing liquid and formed into a ~lat flow. The flow cell 28 has a ~lat flow path 30 of small thickness and comparatively great width. (As seen in Fig. 1, the flow path 30 is narrow in the horizontal direction and wide in the vertical direction.) The specimen ., -6- 2~7~
solution alon~ vi~h its sheathing liquid flows through ~he flat flow path 30 in a direction perpendicular to the plane of the drawing.
The apparatus fur~her includes a laser (e.g, an He-Cd laser or Ar laser) 10, which serves as a -~irst light source, for e~citing fluorescence, and a white-light strobe light source 16, which serves as a third light source. ~or imaging with white light. The light ~rom each o~ these light sourees enters to a hal~-mirror 24. The single laser 10 performs a dual function, one o-~ which i5 to monitor the passage of cell particles through the flow cell 28, and the other of which is to provide light ~or capturing still pictures o~ the cells. The apparatus ineludes first, second and third image capturing means, namely a video camera 56, a CCD line sensor 40 and a video camera 46, respectively.
The seeond image capturing means 40 is ~or particLe detec-tion, and the ~irst and third image capturing means 56, 46 are used in fluoreseent image capturing and white-light image capturing. The laser 10 is so adapted that the quantity o~ light emitted thereby can be varied as required.
As indieated by a luminanee curve C1 in Fig. 5(a), particle detection ordinarily is performed using a continuous light emission of low luminanee. When a partiele has been deteet-ed, a light pulse is emitted at a high luminance i~ order to aequire an still pieture of the partiele, as indieated by eurve C3 in Fig. 5(b).
Fig. 2 is an enlarged view of a speeimen solution flow region as seen ~rom the direetion of arrow A in Fig. 1.
A linear image eapturing area A2 o~ the seeond image eapturing means 40 is formed so as to eross both an image eapturing area Al of the ~irst image eap~uring means 56 and an image capturing area A3 o~ the third image capturing means 47 in a direetion substantially perpendieular to the ~low of~partieles.
Light 12 from the laser 10 is aeted upon by a light-forming unit 14. whieh eomprises a cylindrical lens, and a eondenser lens 26, whereby the flow zone of the speeimen solution is lrradiated over a small width in the flow 2 ~
direction (though the width is slightl~ larger than the outer diameter of the particles of interest) and a large width in a direction orthogonal to the flow direction so as to cover the aforesaid image capturing area A2.
5 ( In Fig. 1, the specimen ~low zone is irradiated over a small width in the direction perpendicular to the plane o~
the drawing and over a large width in the ~ertical direction of the drawing.) When the linear image capturing area (also referred to as an "line sensor area) A2 is irradia-ted with light, the beam spot has the shape of an elongated ellipse.
As a result. the light intensity in the image capturing area A2 is rendered uniform to s~abilize par~icle detection.
In addition, the light intensity is raised to improve the S/N ratio at fluorescent image capturing.
Of the light which leaves the flow cell 28, exciting light is imaged on the CCD line sènsor 40 via an ob~ective lens 32, a half-mirror 34, a dichroic mirror 36 and a pro~ectin~ lens 38. Voltage con~orming to the quantity o~
light incident upon the CCD line sensor 40 is successi~ely extracted ~rom its output side. On the basis of this signal, a cell flo~-through discriminator 58 (which serves also as a pulse controller) determines in real-tlme whether a particIe has arrived for imaging. When a particle is detected, the discriminator/control~er 58 immediately .
outputs a light-emission triggering signal which causes the laser lO to emit a high-luminance pulse of light.
An image intensifier 52 also is rendered operable in response to the light-emission triggering signal ~rom the discriminator/controller 58. Further,-in synchronism with this signal,~a triggering signal which ~ires a white-light strobe 16 is produced.
Fig. 4 is a characteristic diagram o~ the dichroic mirror 36.
The high-luminance laser light from laser 10 irradiates the flow cell 28 in the manner described earlier.
If an irradiated particle emits fluorescence, the resulting fluorescent image is reflected by the dichroic mirror 36 (though the exciting light itself passes through the .
. .
.
, 7 ~ ~
dichroic mirror 36 wit~out reflection) and enters to a projecting lens ~0, whereby an image is formed on the input surface of the image intensifier 52, which serves as à photoelectric converting imaging device. The image intensifier ~2 rendered operable by the aforementioned signal ~'rom Ihe discriminator/controller 58. produces a fluorescen~ image photomultiplied by a ~actor of 103 - 10~
in comparison with the input image. The output fluorescent image is captured by the video camera ria a relay lens 54 whereby a.fluorescent image o~ a cell particle is obtained.
Since the cell flow-throu~h discriminator 58 produces neither the laser triggering signal nor the inten3ifier drive signal in the absence o~ particle detection, the image intensifier 52 is not rendered operational at such time and prevents incident light from reaching the video camera 56. For caution's sake, it is permissible to provide an exciting-light removal ~ilter between the dichr-oic mirror 36 and projecting lens 50.
A delay circuik 60 applies a fixed time delay to the aforementioned triggering signal to obtain the light-emission signal that causes the white-ligh* s-trobe 16 to -fire. Upon receiving this signal, the strobe light source 16 emits a light pulse which follows the high-luminance emission from strobe 10 by a fixed time delay, as indicated by luminance curve C3 in Fig. 5(b). This visible light from the white-light strobe 16 irradiates a portion of the particle-containing flow via a collimator lens 20, a condenser lens 22, a half-mirror 24 and the condenser lens 26. (The irradiation is such ~hat the image capturing areas Al and A3 in Fig. 2 are covered.) The resulting white-light image from flow cell 28 is formed on the video camera 46 via the objective lens 32, the half-mirror 34i an exciting-light removal filter 42 and an objective lens 44. Fig. 3 is a characteristic diagram of the exciting-light removal filter.
At the time of the high-luminance light pulse emission from laser 10, light in the wave-length region o~ the exciting light also reaches the CCD line sensor 40 corresponding to the image sensor area when strobe 16 emits ' :
. ~ ~
9 2 ~
a light pulse. However, if an inhibit signal is applied in this interval, the cell flow-through discriminator ~8 will not operate erroneously and will not produce an unnecessary signal. To this end, a high-speed electronic shutter can be provided in front of the line sensor 40, as will be described later.
Fur~her, the arrangement of Fi~. 1 is such tha-t under conditions set forth below, all particles which flow through the image capturing area A2 can be monitored (with the exception o~ particles through which the exciting light is transmitted intact). In this case, in the arrangement of Fig. 1, the exciting light emerging from the ~low cell 28 is intercepted and the fluorescent light from a particle is separated to form an image on the video camera 46 and on the CCD line sensor 40. As a result, the CCD line sensor 40 forms a fluorescent light image only. Thus, o~ the particles that pass through the image capturing area A2, only particles that emit fluorescence can be made the subject o~ monitoring. In order to select the fluorescent light, use can be made of an exciting-light removal filter or a dichroic mirror, by way of example. To e~fect separa-tion into fluorescent light, the half-mirror 34 can be employed.
In operation, the o~e-line scanning period o~ the line sensor 40 in the apparatus described above is several tens o-f microseconds. There~ore, if the flow velocity o-f the specimen solution is given an appropriate value, a fluorescent image and a white-light image can be captured in the respective image capturing areas A1 and A3. Assume that the width of the image capturing area A2 o~ the CCD
line sensor 40 is 1 ~m, the scanning period of the line sensor is 33 ~sec and size of a leukocyte is 15 ~m. I-~ the ~low velocity is set at, say, 0.1 m/sec, then the distance traveled by ~he cell in one scanning interval will be 3.3 ~m. This means that four scans will always be made during transit of the leukocyte through the image capturing area A2 o~ the line sensor. It is determined by such scanning whether the cell passing through the area A2 is a cell of interest. Thi.s determination is performed in real-time. If a ~ell is determined to be a cell of interest, a triggering pulse is applied to the e~Yciting light source. t~ssume that the length of time from the start o-f scanning by the line sensor 40 to the application of the triggering pulse i5 ~0 ~sec. In this period of time the sample will move by about ~ ym. By making the e~citing-light irradiation area about 30 ym in size, the entirety o-f the cell will be irradiated with the exciting light. Since it is preferred that the size of the irradiation area be the same as that of the cell of interest to the greatest de~ree possible, the exciting-light irradiation area and the image sensor capturing area are set depending upon a balance between the signal processing time of the image sensor and the flow velocity of the sheathing liquid. The excitin~
light and fluorescent light are acted upon by the objective lens 26, hal-~-mirror 34, dichroic mirror 36 and pro~ecting lens S0 in such a manner that only the fluorescent component has its ima~e formed on the photoelectric converting surface of the image intensifier 52. Thereafter, the ~luorescent image is intensi~ied by a ~actor of 103 - 10~ by the image intensifier 52 to form an image on the ~luorescent surface side of the intensi~ier. Thls image is formed on the video camera 56 vla the relay lens 54. At this time a ~luorescent image of the leukocyte appears on -the image capturing screen.
After the exciting light source is triggered, the strobe light source 16 is triggered following a suitable delay. In a case where a wavelength o~ the ~visible-light re~ion is used as the light source 10 for e~citing fluorescence, the time delay should be long enough for the cell of interest ~C in Fig. 2) to exit from the exci~ing-light irradiation area. This is processing ~or taking a still picture on the outer side of a portion of saturated luminance produced by the exciting light source in the image capturing area. To achie~e this, the time delaY
selected should range from 100 to 300 ~s after triggering of the exciting light. The strobe light from strobe 16 i5 7 ~ ~
collimated b~ the collimator lens 20 and then co~densed by the condenser lens 22. The condensed light is reflected by the half-mirror 24 so as to impin~e upon the condenser lens 26, whereby -the ima~e capturing area is irradiated. The transmitted light is acted upon by the objective lens 32, half-mirror 34, exciting-light removal filter 42 and projecting lens 44, whereby an image is formed on the CCD
sur~ace of the video camera 46. A white-light image of the leukocyte appears on the image capturing screen at ~his time.
By thus adopting the ~oregoing arrangement, ~oth a white-light image and fluorescent image of a cell of interest can be obtained. By providing a high-speed electronic shutter, which is synchronized to -the e~citing-light triggering pulse, ln ~ront o~ the image sensor, theinfluence o~ the strobe llght upon the fluorescent image can be reduced.
In this embodiment of the invention set forth above, only capturing o~ the fluQrescent image is described.
Howe~er, it is possible to obtain a three-dimensional distribution of the quantity of ~luorescence within a cell by using an image processor to process the image obtained from the video camera 56. In addition, a depolarized image of the fluorescent light from a cell can be obtained by using a linearly polarized laser as the exciting light source and arranging a polarizer, which passes a polarized component orthogonal to the polarizing direction of the exciting light, between the dichroic mirror 36 and project-ing lens 50 of the opt~cal system for fluorescent image photography. In such case, if visible light is used for the light source which induces ~luorescence in this embodiment, then the filter for removing the exciting light on the side of the white-light image capturlng system would need to be a filter, of the kind shown in Fig. 3, for eliminating wavelengths in the narrow-band region.
Second Embodiment Fig. 7 is a block diagram illustrating a second embodiment of the present invention, which does not possess 2~76~
the white~ ht image capturing function of the -first embodiment. With this arrangement it is still possible to apply particle detection to all particle~ or only to particles which emit fluorescence. As shown in Fig. 8, there is no area A3 for white-light image capturing.
Third embodiment Fig. 9 is a block diagram illustrating a third embodiment o~ the present invention. Here, un].ike the ~irst and second embodiments, the light sources are a light source (a second light source) for monitoring particles, and a light source (first light source) for use in image capturing. The second light source is a near infrared semiconductor laser 64, which is associa-ted with a collimator lens 66 and a dichroic mirror 68. The laser 64 emits light at all times. The ~irst light source is the laser 10 f'or e~citing fluorescence in the ~irst and second embodiments. When a particle is detected, a pulse -~or image capturing purposes is emitted, as shown in Fig. 14, and the fluorescent image is captured by the video camera 56.
Fig. 11 is a characteristic diagram of the dichroic mirror 68, which is capable o~ separating the e~citin~
light and the monitoring infrared light from each other.
Fig. 12 is a characteristic diagram of a dichroic mirror 70 (Fig. 9), which is capable of separating e~citing light, fluorescent light and infrared light. Fig. 13 is a characteristic diagram of exciting light removal filter 72 (Fig. 9) for separating exciting light and fluorescent light ~rom each other.
Fig. 10 illustrates image capturing areas. Here a -fluorescent-light irradiated area B1 and an in-~rared-light irradiated area B2 are shown to~be separated from each other, although they can overlap each other if desired.
It should be noted that an exciting laser can be used instead of the infrared semiconductor laser 64.
Fourth Embodiment Fig. 15 illustrates a ~ourth embodiment o~ the present invention. in which the -third embodiment is additionally provided~with a white-light image capturing -13- 2~
~unction. To -this end. a high-speed electronic shutter 72 synchronized to the e~citing light is disposed in ~ront of the image i~tensifier 52 of the video camera 56 for fluorescent image capturing.
Fifth Embodiment In this embodiment, as shown in Fig. 17, a whi~e-light strobe light source 11, a collimator lens 75 and an e~citing-light selecting filter 76 are employed instead o~
the exciting laser o~ the first light source in the third embodimen.t. This makes it possible to per~orm the same ~unctions. The filter can be exchanged for another when required so that exciting light or fluorescen~ light can be selected at will. This arrangement i9 highly versatile since there is no limitation imposed upon the ob~ect of measurement.
More speci~ically, ~ system capable of photograph-ing a ~luorescent image composed of any exciting-light wavelength can be provided by using the white-light source 11, such as a halogen lamp, as the exciting light source, and changing the exciting-light selecting ~ilter 76. In this syste~, means can be provided -~or changing an exciting-light removal filter 72 and the e~citing-light selecting ~ilter 76 manuallY or automatically depending upo~ the staining solution used to treat the measurement sample.
As a result, when the CCD llne sensor 40 has determined that a particle is a cell o~ interest, a ~Iuorescent image of this cell can be obtained by the video camera 56 by irradiating the cell with pulsed 11ght from the light source 11.
Sixth Embodiment In this embodiment, as shown in Fig. 19, the white-light strobe light source 1~1, a collimat~or lens 77 and an exciting-light transmitting fil-ter 78 are employed instead o~ the exciting laser o~ the first light source. This 3s makes it possible to perform~the same ~unctions.
It is possible to change the type of filter when necessary. In other words, exciting light or -fluorescent light can be selected at will, and there~ore the arrangement ~07~
is highly versatile since there is no limita~ion imposed upon the object of measurement.
In this embodiment, the light source 64 for monitor-ing cell flow-through in the third embodiment is made to serve also as the exciting light source. therebY simplifying the construction of the system. Passage of cells through the cytometer is monitored by the CCD line sensor 40 by using the e~citing light source to perform irradiation normally at low power. Then, when flow-through of a cell o~ interest has been detected, the cell is irradiated with pulsed light emitted by the exciting light source at high power, thereby makin~ it possible for the video camera to obtain a ~luorescent image.
The present invention has the following advantages:
(1) The flow imaging cytometer o~ the present invention constantly monitors the ima~e capturing area and takes a still picture o~ a particle when the particle reaches the image capturing area. This makes it possible to acquire ~luorescent images o~ particles accurately and e~iciently.
~2) In an arrangement where a white light source is provided, a white-light image can be obtained as well as a ~luorescent image. Particle analysis can be per~ormed with`greater precision as a result.
~3) The arrangement can be simpli~ied i-~ one light source is made to serve as both a monitoring light source and a photographic light source.
In another aspect of the present invention, the foregoing objects are attained by providing a flow imaging lS cytometer comprising a flow cell ~ormed to include a flat flow path for causing a specimen solution containlng particle components to be sensed to flow as a flat stream, a ~irst light source arranged on a first side o~ the flow cell ~or irradiating the specimen solution in the flow cell with light quantity of which is switched, first image capturing means arranged on a second side of the flow cell for capturing still pictures of particle components in the specimen solution irradiated with high-luminance pulsed light ~rom the first light source, second image capturing means arranged on a second side o~ the flow cell ~or capturing images of particle components in the specimen solution irradiated continuously with low-luminance light from the first light sourcei processing means for executing prescribed analysis based upon image data from the first and second image capturing means, and control means for : detecting the particle components based upon the image data from the second image capturing means, and on the basis o~ such detection, for switching the ~irst light source over to irradiation with the high-luminance light, wherein the particle components to be sensed are treated with a ~luorescent stain and the first light source is for e~citing fluorescence.
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Other features and advantages of the present invention will be apparent from the following description ~aken in conjunction with the accompanying drawings, in which like reference characters designate the same or similar parts throughout the ~igures thereof.
BRIEF DESCRIPTION OF THE DRAWINGS-.
Fig. 1 is a block diagram illustrating the con-struction of a ~low imaging cytometer according to a first embodiment o~ th~ present invention;
Fig. 2 is a diagram for describing an exciting-light irradiated area and an image capturing area in a flow cell of the cytometer;
Fig. 3 is a diagram showing the characteristic o~
a ~ilter for eliminating exciting light;
Fi.g. 4 is a diagram illustrating an example of the characteristic of a dlchroic mirror;
Fig. 5(a) :Is a diagram showlng an e~ample of a luminance curve in particle detection;
Fig. 5(b) is a diagram showing an example of a light-emission pulse for image capturing, the pulse being emitteda~ter detection of a particle;
Fig. 6 is a measurement flowchart associated with the ~low imaging cytometer o~ the present invention;
Fig. 7 is a block diagram illustrating the con-struction of a flow imaging cytometer according to a second embodiment of the present invention;
Fig. 8 is a diagram for describing e~amples of image capturing areas and irradiation areas in a flow cell associated w1th the cytometer of Fig. 7;
Fig. 9 is a block diagram illustrating the construction of a ~low imaging cytometer according to a third embodiment of the present invention;
Fîg. lO is a diagram for describing examples of image capturing areas and irradiation areas in a flow cell associated with the cytometer o~ Fig. 9;
Fig. 11 i.s a diagram illustrating an example o~ thecharacteristic o~ a dichroic mirror associated with the cytometer o~ Fig. 9;
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Fig. 12 is a diagram illustrating another e,Yample of the characteristic of a dichroic mirror associated vith the cytometer of Fig. 9;
Fig. 13 is a diagram :illustrating an e~ample of the characteristic of a filter, associated with the cytometer of Fig. 9, for filtering out e~citing light;
Fig. 14 is a diagram illustrating an example of a pulse ~or image capturin~ at the time o~ particle detection:
Fig. 15 is a bloc~ diagram illustrating the construction o-f a flow imaging cytometer according to a fourth embodiment of the present invention;
Fig. 16 is a diagram for describing examples of image capturing and irradiation areas in a flow cell associated with the cytometer of Fig. 15;
Fig. 17 is a bloc~ diagram illustrating the con-struction of a flow imaging cytometer accordlng to a ~ifth embodiment of the present invention;
Fig, 18 is a diagram exempli~ying the characteristic of an exciting-light selecting (transmltting) filter; and Fig. 19 is a block diagram illustrating the con-struction o~ a ~low imaging cytometer according to a si~th embodiment of the present invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS:
First Embodiment Pre~erred embodim~n~s o~ the present in~ention will now be described in detail with re~erence to the accompanying drawings.
Fig. 1 is a block diagram illustrating the basic construction o~ a flow imaging cytometer according to a first embodiment o~ the present invention, and Fig. 6 is a me,asurement flowchart associated with this cytometer. As shown in Fig. 1, the flow imaging cytometer includes a flow cell 28 in which a stained specimen solution (o~ blood or urine, etc.) is enveloped by a sheathing liquid and formed into a ~lat flow. The flow cell 28 has a ~lat flow path 30 of small thickness and comparatively great width. (As seen in Fig. 1, the flow path 30 is narrow in the horizontal direction and wide in the vertical direction.) The specimen ., -6- 2~7~
solution alon~ vi~h its sheathing liquid flows through ~he flat flow path 30 in a direction perpendicular to the plane of the drawing.
The apparatus fur~her includes a laser (e.g, an He-Cd laser or Ar laser) 10, which serves as a -~irst light source, for e~citing fluorescence, and a white-light strobe light source 16, which serves as a third light source. ~or imaging with white light. The light ~rom each o~ these light sourees enters to a hal~-mirror 24. The single laser 10 performs a dual function, one o-~ which i5 to monitor the passage of cell particles through the flow cell 28, and the other of which is to provide light ~or capturing still pictures o~ the cells. The apparatus ineludes first, second and third image capturing means, namely a video camera 56, a CCD line sensor 40 and a video camera 46, respectively.
The seeond image capturing means 40 is ~or particLe detec-tion, and the ~irst and third image capturing means 56, 46 are used in fluoreseent image capturing and white-light image capturing. The laser 10 is so adapted that the quantity o~ light emitted thereby can be varied as required.
As indieated by a luminanee curve C1 in Fig. 5(a), particle detection ordinarily is performed using a continuous light emission of low luminanee. When a partiele has been deteet-ed, a light pulse is emitted at a high luminance i~ order to aequire an still pieture of the partiele, as indieated by eurve C3 in Fig. 5(b).
Fig. 2 is an enlarged view of a speeimen solution flow region as seen ~rom the direetion of arrow A in Fig. 1.
A linear image eapturing area A2 o~ the seeond image eapturing means 40 is formed so as to eross both an image eapturing area Al of the ~irst image eap~uring means 56 and an image capturing area A3 o~ the third image capturing means 47 in a direetion substantially perpendieular to the ~low of~partieles.
Light 12 from the laser 10 is aeted upon by a light-forming unit 14. whieh eomprises a cylindrical lens, and a eondenser lens 26, whereby the flow zone of the speeimen solution is lrradiated over a small width in the flow 2 ~
direction (though the width is slightl~ larger than the outer diameter of the particles of interest) and a large width in a direction orthogonal to the flow direction so as to cover the aforesaid image capturing area A2.
5 ( In Fig. 1, the specimen ~low zone is irradiated over a small width in the direction perpendicular to the plane o~
the drawing and over a large width in the ~ertical direction of the drawing.) When the linear image capturing area (also referred to as an "line sensor area) A2 is irradia-ted with light, the beam spot has the shape of an elongated ellipse.
As a result. the light intensity in the image capturing area A2 is rendered uniform to s~abilize par~icle detection.
In addition, the light intensity is raised to improve the S/N ratio at fluorescent image capturing.
Of the light which leaves the flow cell 28, exciting light is imaged on the CCD line sènsor 40 via an ob~ective lens 32, a half-mirror 34, a dichroic mirror 36 and a pro~ectin~ lens 38. Voltage con~orming to the quantity o~
light incident upon the CCD line sensor 40 is successi~ely extracted ~rom its output side. On the basis of this signal, a cell flo~-through discriminator 58 (which serves also as a pulse controller) determines in real-tlme whether a particIe has arrived for imaging. When a particle is detected, the discriminator/control~er 58 immediately .
outputs a light-emission triggering signal which causes the laser lO to emit a high-luminance pulse of light.
An image intensifier 52 also is rendered operable in response to the light-emission triggering signal ~rom the discriminator/controller 58. Further,-in synchronism with this signal,~a triggering signal which ~ires a white-light strobe 16 is produced.
Fig. 4 is a characteristic diagram o~ the dichroic mirror 36.
The high-luminance laser light from laser 10 irradiates the flow cell 28 in the manner described earlier.
If an irradiated particle emits fluorescence, the resulting fluorescent image is reflected by the dichroic mirror 36 (though the exciting light itself passes through the .
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.
, 7 ~ ~
dichroic mirror 36 wit~out reflection) and enters to a projecting lens ~0, whereby an image is formed on the input surface of the image intensifier 52, which serves as à photoelectric converting imaging device. The image intensifier ~2 rendered operable by the aforementioned signal ~'rom Ihe discriminator/controller 58. produces a fluorescen~ image photomultiplied by a ~actor of 103 - 10~
in comparison with the input image. The output fluorescent image is captured by the video camera ria a relay lens 54 whereby a.fluorescent image o~ a cell particle is obtained.
Since the cell flow-throu~h discriminator 58 produces neither the laser triggering signal nor the inten3ifier drive signal in the absence o~ particle detection, the image intensifier 52 is not rendered operational at such time and prevents incident light from reaching the video camera 56. For caution's sake, it is permissible to provide an exciting-light removal ~ilter between the dichr-oic mirror 36 and projecting lens 50.
A delay circuik 60 applies a fixed time delay to the aforementioned triggering signal to obtain the light-emission signal that causes the white-ligh* s-trobe 16 to -fire. Upon receiving this signal, the strobe light source 16 emits a light pulse which follows the high-luminance emission from strobe 10 by a fixed time delay, as indicated by luminance curve C3 in Fig. 5(b). This visible light from the white-light strobe 16 irradiates a portion of the particle-containing flow via a collimator lens 20, a condenser lens 22, a half-mirror 24 and the condenser lens 26. (The irradiation is such ~hat the image capturing areas Al and A3 in Fig. 2 are covered.) The resulting white-light image from flow cell 28 is formed on the video camera 46 via the objective lens 32, the half-mirror 34i an exciting-light removal filter 42 and an objective lens 44. Fig. 3 is a characteristic diagram of the exciting-light removal filter.
At the time of the high-luminance light pulse emission from laser 10, light in the wave-length region o~ the exciting light also reaches the CCD line sensor 40 corresponding to the image sensor area when strobe 16 emits ' :
. ~ ~
9 2 ~
a light pulse. However, if an inhibit signal is applied in this interval, the cell flow-through discriminator ~8 will not operate erroneously and will not produce an unnecessary signal. To this end, a high-speed electronic shutter can be provided in front of the line sensor 40, as will be described later.
Fur~her, the arrangement of Fi~. 1 is such tha-t under conditions set forth below, all particles which flow through the image capturing area A2 can be monitored (with the exception o~ particles through which the exciting light is transmitted intact). In this case, in the arrangement of Fig. 1, the exciting light emerging from the ~low cell 28 is intercepted and the fluorescent light from a particle is separated to form an image on the video camera 46 and on the CCD line sensor 40. As a result, the CCD line sensor 40 forms a fluorescent light image only. Thus, o~ the particles that pass through the image capturing area A2, only particles that emit fluorescence can be made the subject o~ monitoring. In order to select the fluorescent light, use can be made of an exciting-light removal filter or a dichroic mirror, by way of example. To e~fect separa-tion into fluorescent light, the half-mirror 34 can be employed.
In operation, the o~e-line scanning period o~ the line sensor 40 in the apparatus described above is several tens o-f microseconds. There~ore, if the flow velocity o-f the specimen solution is given an appropriate value, a fluorescent image and a white-light image can be captured in the respective image capturing areas A1 and A3. Assume that the width of the image capturing area A2 o~ the CCD
line sensor 40 is 1 ~m, the scanning period of the line sensor is 33 ~sec and size of a leukocyte is 15 ~m. I-~ the ~low velocity is set at, say, 0.1 m/sec, then the distance traveled by ~he cell in one scanning interval will be 3.3 ~m. This means that four scans will always be made during transit of the leukocyte through the image capturing area A2 o~ the line sensor. It is determined by such scanning whether the cell passing through the area A2 is a cell of interest. Thi.s determination is performed in real-time. If a ~ell is determined to be a cell of interest, a triggering pulse is applied to the e~Yciting light source. t~ssume that the length of time from the start o-f scanning by the line sensor 40 to the application of the triggering pulse i5 ~0 ~sec. In this period of time the sample will move by about ~ ym. By making the e~citing-light irradiation area about 30 ym in size, the entirety o-f the cell will be irradiated with the exciting light. Since it is preferred that the size of the irradiation area be the same as that of the cell of interest to the greatest de~ree possible, the exciting-light irradiation area and the image sensor capturing area are set depending upon a balance between the signal processing time of the image sensor and the flow velocity of the sheathing liquid. The excitin~
light and fluorescent light are acted upon by the objective lens 26, hal-~-mirror 34, dichroic mirror 36 and pro~ecting lens S0 in such a manner that only the fluorescent component has its ima~e formed on the photoelectric converting surface of the image intensifier 52. Thereafter, the ~luorescent image is intensi~ied by a ~actor of 103 - 10~ by the image intensifier 52 to form an image on the ~luorescent surface side of the intensi~ier. Thls image is formed on the video camera 56 vla the relay lens 54. At this time a ~luorescent image of the leukocyte appears on -the image capturing screen.
After the exciting light source is triggered, the strobe light source 16 is triggered following a suitable delay. In a case where a wavelength o~ the ~visible-light re~ion is used as the light source 10 for e~citing fluorescence, the time delay should be long enough for the cell of interest ~C in Fig. 2) to exit from the exci~ing-light irradiation area. This is processing ~or taking a still picture on the outer side of a portion of saturated luminance produced by the exciting light source in the image capturing area. To achie~e this, the time delaY
selected should range from 100 to 300 ~s after triggering of the exciting light. The strobe light from strobe 16 i5 7 ~ ~
collimated b~ the collimator lens 20 and then co~densed by the condenser lens 22. The condensed light is reflected by the half-mirror 24 so as to impin~e upon the condenser lens 26, whereby -the ima~e capturing area is irradiated. The transmitted light is acted upon by the objective lens 32, half-mirror 34, exciting-light removal filter 42 and projecting lens 44, whereby an image is formed on the CCD
sur~ace of the video camera 46. A white-light image of the leukocyte appears on the image capturing screen at ~his time.
By thus adopting the ~oregoing arrangement, ~oth a white-light image and fluorescent image of a cell of interest can be obtained. By providing a high-speed electronic shutter, which is synchronized to -the e~citing-light triggering pulse, ln ~ront o~ the image sensor, theinfluence o~ the strobe llght upon the fluorescent image can be reduced.
In this embodiment of the invention set forth above, only capturing o~ the fluQrescent image is described.
Howe~er, it is possible to obtain a three-dimensional distribution of the quantity of ~luorescence within a cell by using an image processor to process the image obtained from the video camera 56. In addition, a depolarized image of the fluorescent light from a cell can be obtained by using a linearly polarized laser as the exciting light source and arranging a polarizer, which passes a polarized component orthogonal to the polarizing direction of the exciting light, between the dichroic mirror 36 and project-ing lens 50 of the opt~cal system for fluorescent image photography. In such case, if visible light is used for the light source which induces ~luorescence in this embodiment, then the filter for removing the exciting light on the side of the white-light image capturlng system would need to be a filter, of the kind shown in Fig. 3, for eliminating wavelengths in the narrow-band region.
Second Embodiment Fig. 7 is a block diagram illustrating a second embodiment of the present invention, which does not possess 2~76~
the white~ ht image capturing function of the -first embodiment. With this arrangement it is still possible to apply particle detection to all particle~ or only to particles which emit fluorescence. As shown in Fig. 8, there is no area A3 for white-light image capturing.
Third embodiment Fig. 9 is a block diagram illustrating a third embodiment o~ the present invention. Here, un].ike the ~irst and second embodiments, the light sources are a light source (a second light source) for monitoring particles, and a light source (first light source) for use in image capturing. The second light source is a near infrared semiconductor laser 64, which is associa-ted with a collimator lens 66 and a dichroic mirror 68. The laser 64 emits light at all times. The ~irst light source is the laser 10 f'or e~citing fluorescence in the ~irst and second embodiments. When a particle is detected, a pulse -~or image capturing purposes is emitted, as shown in Fig. 14, and the fluorescent image is captured by the video camera 56.
Fig. 11 is a characteristic diagram of the dichroic mirror 68, which is capable o~ separating the e~citin~
light and the monitoring infrared light from each other.
Fig. 12 is a characteristic diagram of a dichroic mirror 70 (Fig. 9), which is capable of separating e~citing light, fluorescent light and infrared light. Fig. 13 is a characteristic diagram of exciting light removal filter 72 (Fig. 9) for separating exciting light and fluorescent light ~rom each other.
Fig. 10 illustrates image capturing areas. Here a -fluorescent-light irradiated area B1 and an in-~rared-light irradiated area B2 are shown to~be separated from each other, although they can overlap each other if desired.
It should be noted that an exciting laser can be used instead of the infrared semiconductor laser 64.
Fourth Embodiment Fig. 15 illustrates a ~ourth embodiment o~ the present invention. in which the -third embodiment is additionally provided~with a white-light image capturing -13- 2~
~unction. To -this end. a high-speed electronic shutter 72 synchronized to the e~citing light is disposed in ~ront of the image i~tensifier 52 of the video camera 56 for fluorescent image capturing.
Fifth Embodiment In this embodiment, as shown in Fig. 17, a whi~e-light strobe light source 11, a collimator lens 75 and an e~citing-light selecting filter 76 are employed instead o~
the exciting laser o~ the first light source in the third embodimen.t. This makes it possible to per~orm the same ~unctions. The filter can be exchanged for another when required so that exciting light or fluorescen~ light can be selected at will. This arrangement i9 highly versatile since there is no limitation imposed upon the ob~ect of measurement.
More speci~ically, ~ system capable of photograph-ing a ~luorescent image composed of any exciting-light wavelength can be provided by using the white-light source 11, such as a halogen lamp, as the exciting light source, and changing the exciting-light selecting ~ilter 76. In this syste~, means can be provided -~or changing an exciting-light removal filter 72 and the e~citing-light selecting ~ilter 76 manuallY or automatically depending upo~ the staining solution used to treat the measurement sample.
As a result, when the CCD llne sensor 40 has determined that a particle is a cell o~ interest, a ~Iuorescent image of this cell can be obtained by the video camera 56 by irradiating the cell with pulsed 11ght from the light source 11.
Sixth Embodiment In this embodiment, as shown in Fig. 19, the white-light strobe light source 1~1, a collimat~or lens 77 and an exciting-light transmitting fil-ter 78 are employed instead o~ the exciting laser o~ the first light source. This 3s makes it possible to perform~the same ~unctions.
It is possible to change the type of filter when necessary. In other words, exciting light or -fluorescent light can be selected at will, and there~ore the arrangement ~07~
is highly versatile since there is no limita~ion imposed upon the object of measurement.
In this embodiment, the light source 64 for monitor-ing cell flow-through in the third embodiment is made to serve also as the exciting light source. therebY simplifying the construction of the system. Passage of cells through the cytometer is monitored by the CCD line sensor 40 by using the e~citing light source to perform irradiation normally at low power. Then, when flow-through of a cell o~ interest has been detected, the cell is irradiated with pulsed light emitted by the exciting light source at high power, thereby makin~ it possible for the video camera to obtain a ~luorescent image.
The present invention has the following advantages:
(1) The flow imaging cytometer o~ the present invention constantly monitors the ima~e capturing area and takes a still picture o~ a particle when the particle reaches the image capturing area. This makes it possible to acquire ~luorescent images o~ particles accurately and e~iciently.
~2) In an arrangement where a white light source is provided, a white-light image can be obtained as well as a ~luorescent image. Particle analysis can be per~ormed with`greater precision as a result.
~3) The arrangement can be simpli~ied i-~ one light source is made to serve as both a monitoring light source and a photographic light source.
(4) When the second image capturing area ~or particle detection is made linear in shape, a restriction is placed upon the positions which particles can occupy in an imaged frame. The simplifies subsequent image processing.
(S) In the monitoring o~ particles; it is posslble to s~itch between the monitoring o~ all particles and the monitoring soleIy of particles which emit ~luorescence.
(6) Since ~ilters can be interchanged dep~nding upon the subject o~ measuremen~, the apparatus can be used with greater universality.
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As many apparently widely different embodiments of the present invention can be made without departlng from the spirit and scope thereof, it is to be understood that the invention is not limited to the specific embodiments thereof e~cept as de~ined in the appendecl claims.
:
(S) In the monitoring o~ particles; it is posslble to s~itch between the monitoring o~ all particles and the monitoring soleIy of particles which emit ~luorescence.
(6) Since ~ilters can be interchanged dep~nding upon the subject o~ measuremen~, the apparatus can be used with greater universality.
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As many apparently widely different embodiments of the present invention can be made without departlng from the spirit and scope thereof, it is to be understood that the invention is not limited to the specific embodiments thereof e~cept as de~ined in the appendecl claims.
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Claims (12)
1. A flow imaging cytometer comprising:
a flow cell formed to include a flat flow path for causing a specimen solution containing particle components to be sensed to flow as a flat stream;
a first light source arranged on a first side of said flow cell for irradiating the specimen solution in said flow cell with pulsed light;
first image capturing means arranged on a second side of said flow cell for capturing still pictures of the particle components in the specimen solution irradiated by said first light source;
a second light source arranged on the first side of said flow cell for irradiating the specimen solution in said flow cell with light constantly;
second image capturing means arranged on the second side of said flow cell for capturing images of the particle components irradiated by said second light sourcs;
processing means for executing prescribed analysis based upon image data from said first and second image capturing means; and control means for detecting the particle components based upon the image data from said second image capturing means, and on the basis of such detection, for actuating said first light source within an image capturing period of said first image capturing means, wherein the particle components to be sensed are treated with a fluorescent stain and said first light source is for exciting fluorescence.
a flow cell formed to include a flat flow path for causing a specimen solution containing particle components to be sensed to flow as a flat stream;
a first light source arranged on a first side of said flow cell for irradiating the specimen solution in said flow cell with pulsed light;
first image capturing means arranged on a second side of said flow cell for capturing still pictures of the particle components in the specimen solution irradiated by said first light source;
a second light source arranged on the first side of said flow cell for irradiating the specimen solution in said flow cell with light constantly;
second image capturing means arranged on the second side of said flow cell for capturing images of the particle components irradiated by said second light sourcs;
processing means for executing prescribed analysis based upon image data from said first and second image capturing means; and control means for detecting the particle components based upon the image data from said second image capturing means, and on the basis of such detection, for actuating said first light source within an image capturing period of said first image capturing means, wherein the particle components to be sensed are treated with a fluorescent stain and said first light source is for exciting fluorescence.
2. The flow imaging cytometer according to claim 1, wherein said second light source emits infrared light.
3. The flow imaging cytometer according to claim 1, wherein said second light source emits exciting light for exciting fluorescence.
4. The flow imaging cytometer according to any of claims 1 through 3, further comprising:
a third light source arranged on the first side of said flow cell for irradiating the specimen solution in said flow cell with pulsed white light; and third image capturing means arranged on the second side of said flow cell for capturing still pictures of the particle components in the specimen solution irradiated by said third light source;
said control means detecting the particle components based upon the image data from said second image capturing means, and on the basis of such detection, for actuating said first and third light sources after respective prescribed time delays within image capturing periods of said first and third image capturing means respectively.
a third light source arranged on the first side of said flow cell for irradiating the specimen solution in said flow cell with pulsed white light; and third image capturing means arranged on the second side of said flow cell for capturing still pictures of the particle components in the specimen solution irradiated by said third light source;
said control means detecting the particle components based upon the image data from said second image capturing means, and on the basis of such detection, for actuating said first and third light sources after respective prescribed time delays within image capturing periods of said first and third image capturing means respectively.
5. The flow imaging cytometer according to any of claims 1 through 3, wherein said first image capturing means has a two-dimensional image capturing area on the flow of the specimen solution, said second image capturing means has a linear image capturing area on the flow of the specimen solution, and the image capturing area of said second image capturing means is formed so as to cross the flow of the specimen solution within the image capturing area of said first image capturing means.
6. The flow imaging cytometer according to claim 4, wherein said first and third image capturing means each has a two-dimensional image capturing area on the flow of the specimen solution, said second image capturing means has a linear image capturing area on the flow of the specimen solution, and the image capturing area of said second image capturing means is formed so as to cross the flow of the specimen solution within the image capturing area of said first and third image capturing means.
7. The flow imaging cytometer according to claim 5 or 6, further comprising means for forming the irradiating light from said second light source into an elongated elliptical shape.
8. A flow imaging cytometer comprising:
a flow cell formed to include a flat flow path for causing a specimen solution containing particle components to be sensed to flow as a flat stream;
a first light source arranged on a first side of said flow cell for irradiating the specimen solution in said flow cell with light the quantity of which is switched;
first image capturing means arranged on a second side of said flow cell for capturing still pictures of particle components in the specimen solution irradiated with high-luminance pulsed light from said first light source;
second image capturing means arranged on a second side of said flow cell for capturing images of the particle components in the specimen solution irradiated continuously with low-luminance light from said first light source;
processing means for executing prescribed analysis based upon image data from said first and second image capturing means; and control means for detecting the particle components based upon the image data from said second image capturing means, and on the basis of such detection, for switching said first light source over to irradiation with the high-luminance light, wherein the particle components to be sensed are treated with a fluorescent stain and said first light source is for exciting fluorescence.
a flow cell formed to include a flat flow path for causing a specimen solution containing particle components to be sensed to flow as a flat stream;
a first light source arranged on a first side of said flow cell for irradiating the specimen solution in said flow cell with light the quantity of which is switched;
first image capturing means arranged on a second side of said flow cell for capturing still pictures of particle components in the specimen solution irradiated with high-luminance pulsed light from said first light source;
second image capturing means arranged on a second side of said flow cell for capturing images of the particle components in the specimen solution irradiated continuously with low-luminance light from said first light source;
processing means for executing prescribed analysis based upon image data from said first and second image capturing means; and control means for detecting the particle components based upon the image data from said second image capturing means, and on the basis of such detection, for switching said first light source over to irradiation with the high-luminance light, wherein the particle components to be sensed are treated with a fluorescent stain and said first light source is for exciting fluorescence.
9. The flow imaging cytometer according to claim 8, further comprising:
a third light source arranged on a first side of said flow cell for irradiating the specimen solution in said flow cell with pulsed white light; and third image capturing means arranged on a. second side of said flow cell for capturing still pictures of the particle components in the specimen solution irradiated by said third light source;
said control means for detecting the particle com-ponents based upon the image data from said second image capturing means, and on the basis of such detection, for switching said first light source over to irradiation with the high-luminance light and actuating said third light source after respective prescribed time delays.
a third light source arranged on a first side of said flow cell for irradiating the specimen solution in said flow cell with pulsed white light; and third image capturing means arranged on a. second side of said flow cell for capturing still pictures of the particle components in the specimen solution irradiated by said third light source;
said control means for detecting the particle com-ponents based upon the image data from said second image capturing means, and on the basis of such detection, for switching said first light source over to irradiation with the high-luminance light and actuating said third light source after respective prescribed time delays.
10. The flow imaging cytometer according to claim 8, wherein said first image capturing means has a two-dimensional image capturing area on the flow of the specimen solution, said second image capturing means has a linear image capturing area on the flow of the specimen solution, and the image capturing area of said second image capturing means is formed so as to cross the flow of the specimen solution within the image capturing area of said first image capturing means.
11. The flow imaging cytometer according to claim 9, wherein said first and third image capturing means each has a two-dimensional image capturing area on the flow of the specimen solution, said second image capturing means has a linear image capturing area on the flow of the specimen solution, and the image capturing area of said second image capturing means is formed so as to cross the flow of the specimen solution within the image capturing areas of said first and third image capturing means.
12. The flow imaging cytometer according to claim 10 or 11, further comprising means for forming the irradiating light from said first light source into an elongated elliptical shape.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP33151/1991 | 1991-02-27 | ||
| JP03033151A JP3121849B2 (en) | 1991-02-27 | 1991-02-27 | Flow image cytometer |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2050761A1 true CA2050761A1 (en) | 1992-08-28 |
Family
ID=12378582
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002050761A Abandoned CA2050761A1 (en) | 1991-02-27 | 1991-09-05 | Flow imaging cytometer |
Country Status (5)
| Country | Link |
|---|---|
| US (1) | US5247339A (en) |
| EP (1) | EP0501005B1 (en) |
| JP (1) | JP3121849B2 (en) |
| CA (1) | CA2050761A1 (en) |
| DE (1) | DE69131844T2 (en) |
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| US5733739A (en) * | 1995-06-07 | 1998-03-31 | Inphocyte, Inc. | System and method for diagnosis of disease by infrared analysis of human tissues and cells |
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-
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- 1991-02-27 JP JP03033151A patent/JP3121849B2/en not_active Expired - Fee Related
- 1991-09-04 DE DE69131844T patent/DE69131844T2/en not_active Expired - Fee Related
- 1991-09-04 EP EP91114920A patent/EP0501005B1/en not_active Expired - Lifetime
- 1991-09-05 US US07/755,302 patent/US5247339A/en not_active Expired - Fee Related
- 1991-09-05 CA CA002050761A patent/CA2050761A1/en not_active Abandoned
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5733739A (en) * | 1995-06-07 | 1998-03-31 | Inphocyte, Inc. | System and method for diagnosis of disease by infrared analysis of human tissues and cells |
Also Published As
| Publication number | Publication date |
|---|---|
| DE69131844D1 (en) | 2000-01-20 |
| JPH04270963A (en) | 1992-09-28 |
| JP3121849B2 (en) | 2001-01-09 |
| EP0501005A2 (en) | 1992-09-02 |
| US5247339A (en) | 1993-09-21 |
| DE69131844T2 (en) | 2000-05-18 |
| EP0501005A3 (en) | 1992-12-09 |
| EP0501005B1 (en) | 1999-12-15 |
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| Date | Code | Title | Description |
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| FZDE | Discontinued |