CA2050758A1 - Flow imaging cytometer - Google Patents
Flow imaging cytometerInfo
- Publication number
- CA2050758A1 CA2050758A1 CA002050758A CA2050758A CA2050758A1 CA 2050758 A1 CA2050758 A1 CA 2050758A1 CA 002050758 A CA002050758 A CA 002050758A CA 2050758 A CA2050758 A CA 2050758A CA 2050758 A1 CA2050758 A1 CA 2050758A1
- Authority
- CA
- Canada
- Prior art keywords
- light
- image
- image capturing
- light source
- flow
- 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
Classifications
-
- 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
- G01N15/1429—Electro-optical investigation, e.g. flow cytometers using an analyser being characterised by its signal processing
-
- 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
- G01N2021/1789—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 white-light image and a fluorescent image are capable of being captured by a single video camera, and a single light source for producing exciting light is used to pick up the fluorescent image and monitor arrival of particles to the image capturing area. Specifically, besides a strobe light source, a light source, the output of which normally is low, is provided for exciting fluo-rescence and for monitoring cell flow-through. An image intensifier is used in the light-receiving system of the fluorescent image and is supplied with a voltage the size and timing of which are controlled in such a manner that the irradiation of a cell with the strobe light when the white-light image is captured will not have an adverse effect upon the aforementioned light-receiving system.
This makes it possible to pick up two images each in a different zone on the light-receiving surface of image capturing means.
ABSTRACT OF THE DISCLOSURE:
A white-light image and a fluorescent image are capable of being captured by a single video camera, and a single light source for producing exciting light is used to pick up the fluorescent image and monitor arrival of particles to the image capturing area. Specifically, besides a strobe light source, a light source, the output of which normally is low, is provided for exciting fluo-rescence and for monitoring cell flow-through. An image intensifier is used in the light-receiving system of the fluorescent image and is supplied with a voltage the size and timing of which are controlled in such a manner that the irradiation of a cell with the strobe light when the white-light image is captured will not have an adverse effect upon the aforementioned light-receiving system.
This makes it possible to pick up two images each in a different zone on the light-receiving surface of image capturing means.
Description
FLOW I~AGI~G CYTOMETER
BACKGROUND OF THE INVENTION:
ield of the In ention This invention relates to a ~low imaging cytometer.
s ~ore particularly, the in~en-tion rela~es to a flow-imaging particle analyzing system in which cells fluorescentlY
stained in a manner suitable for the ~particular cells of interest are introduced to a flow cell to be ~ormed into a planar sheathed flow and irradia~ed with white light (strobe light) to obtain a white-light image and e~Yci-ted with laser light to obtain a fluorescent image in a highly efficient manner, and in which the two types of images can be captured simultaneously by a single video camera and subject to analysis.
BACKGROUND OF THE INVENTION:
ield of the In ention This invention relates to a ~low imaging cytometer.
s ~ore particularly, the in~en-tion rela~es to a flow-imaging particle analyzing system in which cells fluorescentlY
stained in a manner suitable for the ~particular cells of interest are introduced to a flow cell to be ~ormed into a planar sheathed flow and irradia~ed with white light (strobe light) to obtain a white-light image and e~Yci-ted with laser light to obtain a fluorescent image in a highly efficient manner, and in which the two types of images can be captured simultaneously by a single video camera and subject to analysis.
2. Description of the Prior Art Attempts have been made to irradia~e cells, which have been stained and smeared on a glass slide, with light such as visible light or ultraviolet light under a micro-scope, capture a fluorescent image of cells of interest, analyze the resulting image and obtain physiological information relati.ng to the cells. However, a method of this kind is not suited to the analytical processing of a large number of cells in a short time, and analysis using fluorescent images has only limited application.
In another example of the conventional ~low ima~ing cytometer, the cell information is obtained using a gross value of the fluorescence emitted erom the fluorescently stained cell. In other ~ords, the fluorescence emitted from each portion of the cell is integrated over the entirety of the cell, and the cell information is obtained in the ~eorm O-e such an integrated value. Though such a me-thod lends itsel~ to analysis of a large number of cells in a short period o~ time, it ls not possible to acquire detailed in-formation as to which por-tions o-f individual cells have been stained and caused to emit fluorescence. Consequentl~, this method is 1imited in terms of analytical performance.
2~7~
On ~he other hand, a cell classifying appara~us tha~
has been ~u~ into practical use employs a nechnique in ~vhich cells s~ained in a manner suitable for a particular cell of interest are introduced to a flow cell to be formed into a planar sheathed flow and irradiated with strobe light, a still picture is obtained b~ a video camera and image processing is applied. However, the state o~ the art is such that the capturing and analysis o~ ~`luorescent images of individual cells using this method have still not reached a practical stage because o~ problems related to fluorescent imaging sensitivity. The present invention makes use of the technology employed in a flow imaging cytometer of the type having a high image cap-turing ef~iciency, as previously proposed in the specification o-f Japanese Patent Application ~o. 185794/l~9o~
SUMMARY OF THE INVENTION:
Thus, the art still lacks a definitlve ~low-imaging particle analyzing system for sensing cells that pass through an image capturing area and irradiating the cells with concentrated exciting light, thereby to assure the required ~luorescent intensity and obtain a fluorescent image, and for subjecting the ~luorescent image, as well as a white-light image of the cells derived from the conventional white-light source, to highly ef-ficient image capturing and analysis using a single video camera.
Accordingly, an object of the presen-t invention is to provide a flow imaging cytometer which expands upon the idea o-f the previously proposed (the aforementioned Japanese Patent Application No. 185794/1990, hereina~ter referred to as "the earlier application'') flow imaging cytometer of the type having a high :Lmage capturing ef~iciency, wherein ~luorescence emitted by a ~luorescently stained ceLl as a result Oe irradiating the cell with laser light is obtained, by single video camera, as a two~dimensional i~age at the same time as a white-light image acquired by conventional strobe-llght (white-light) lrradiation.
.~ccording to the present invention, the foregoing object is attained by providing a ~low imaging cyto~neter 7 ~ 8 , comprising a ~low cell formed ~o include a fla~ ~low 2ath for causing ~ specimen solu~ion con~ainlng particle components to be sensed to flow as a ~lat s~ream~ a first ligh-~ source arranged on a first side of the flow cell s for irradiating the specimen solution in the flow cell with light the quanti-ty of ~hich is switched, a second light source arranged on the first side o~ the flow cell for irradiating the specimen solution in the flow cell with pulsed light, first image capturing means arranged on a second side of the ~low cell for capturing s~ill pictures of particle components in the specimen solution irradiated with high-luminance pulsed ligh-t from the first light source particle components irradiated with the light ~rom the second light source, second image capturing means arranged on the second side of the -flow cell for capturing still pictures o~ particle components in the specimen solution irradiated continuousl~ with low-luminance light from the first light source, processing means for e~YecUting 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 of such detection, first for switching the first light source over to irradiation with the high-luminance light. and -then operating the second light source -~ollowing a prescribed delay, within an image capturing period of the first image capturing means. wherein the first light source is a light source ~or e4~citing fluorescence, and the image resulting from the first light source and the image resulting from the second light source are each captured in a different area on a light-receiving sur~ace o-~ the i~irst image capturing means .
The flow imaging cytometer o~ the present invention is ~urther characterized in that the eirst image capturing means has a two-dimensional image capturin~ area on the flow of the specimen solution, the second image capturing means has a linear image capturing area on the -~low of the specimen solution, the image cap-~uring area of the second 2~ 7~8 image capturing means is formed so as ~o cross the flow of the specimen solution within the image capturing area o~
the first image capturing means, the image capturing area of the firs~ image capturing means is divided into a zone which includes, and a zone which does not includ0, the image capturing area of the second image capturing ~eans, and an image in one of these zones resulting from irradiation with the high-luminance light from the first li~ht source and an image in the other of these zones resultin~ from irradiation by the second light source are captured by the first image capturing means.
The flow imaging cytometer of the present invention is further characterized by having masking means for interrupting light on the optic path of the -first image capturing means in such a manner that the two images do not overlap each other on the light-receiving surface of the first image capturing means.
The flow imaging cytometer o-f the present invention is further characterized by having means for -forming the irradiating light ~rom the first light source into an elongated elliptical shape, and in that the light-receiving system of a fluorescent image is provided with an image intensifier, and the image intensifier is operated only when the fluorescsnt image is captured.
Other ~eatures and advantages of the present invention will be apparent from the ~ollowing description taken in con~unction with the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS:
Fig. 1 is a block diagram illustrating the con-struction o~ a flow imaging cytometer according to -the present inventlon;
Fig. 2 is an e,Yplanatory view illustrating an e,Yample ot' light-irradiating areas and image cap~uri.ng areas of the -~low cell shown in Flg. 1;
Fig. 3 i9 a timing chart illustrating irradiation timing and the timing of a gating signal t'or an image intensi~ier;
~s~r~
Fig. 1 is a dia~r~m showing an e.Yample ol an imaged .rame o~ a eQll cap~ured by a video c~mera: and Fi~. ~ is a diagram showing e.Yamples o~ semicircular and rectangular mas~s associa~ed with the se~-up of Fig. 1.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS:
A preferred embodiment of a flow image cytometer according to the present invention will now be described with reference to the drawings.
~ s illustrated in Fig. 1, the flow imaging cytometer includes a laser light source 1 (e.g. an ~e-Cd laser) for e~Yciting fluorescence and also ~or monitoring the passage of cells through the cy-tometer, and a strobe 2 serving as a ligh-t source for white-light photography. Unlike the strobe light source 2, the laser light source 1 is adapted to irradiate an image capturing area constantly in order to monitor cell flow-through. The light from laser 1 is stopped down to a finely elongated beam spot perpendicular to the direction of cell travel by a cylindrical lens 3 and a condenser lens ~ and irradiates an image capturing area of a line sensor 14, as illustrated in Fig. 2. The reason for this is to obtain a.more uniform light intensity and to improve the S/N ratio when a fluorescent image is captured.
In this embodiment, the image capturing area of the line sensor 14 is provided slightly above mid-center of the upper half of the generally rectangular image capturing area of video camera 24, as shown in Fig. 2.
The light from the laser 1 leaving the image capturing area passes through an obJective lens 8 and is then split by a beam-splitter 11, Part^of the light -from the beam-splitter 11 passes through a dichroic mirror 12 and enters to a projecting lens 13, which proceeds to form an image on the llne sensor 1~. The li.ne sensor 14 successively produces voltage ou-tputs con-~orming to the accumulated amount of photoelectrical conversion of each piAYel e,Yposed for a scanning period (several tens of microseconds) of one line. By means of signal processing similar to that set forth in the earlier applicat~on, triggers irradiation by e~Yciting light and for -fluorescent image capturing are applied when a cell crosses the image capturing area of the line sensor 14 during even-numbered ~ield intervals of the video camera 24.
The time from the instant a cell crosses the image capturing area of the line sensor 14 until the cell is irradiated with the e,Yciting light ~s 100 - 200 ~sec i~ the scanning period of the line sensor 14 is 50 ~sec. On the assumption that the ~low velocity of cells in elow cell 6 is 30 mm/sec, a cell will move 3 - 6 ~m in this period of time. Accordingly, the image of a cell obtained by being irradiated with the e,YCiting light can always be acquired in the area located in the upper half of one imaged frame, as illustrated in Fig. 4. The laser 1 usually delivers a small output power to monitor passage of cells through the flow cell, but the laser is caused to generate high-luminance pulses when fluorescent images are captured.
Fluorescence emitted by a cell in response to irradiation with the elxciting ligh~ passes through the objective lens 8 and the beam-splitter 11 to be reflected by the dichroic mirrors 12. The exciting Light which has passed through the image capturing area is intercepted by a beam stopper 7, and stray light is removed by the filter 20.
Near infrared light constantly emitted in order to monitor cell ~low-through also is eliminated by a filter 20.
The ~luorescent light which has passed through . the filter 20 enters to a projecting lens 21, ~hereby an image of the cell is -eormed on the photoelec~ric sur-eace o~ an image intensi-eier ~2. At this time a high-voltage is applied to the image intensifier 22 so that the image is intensi-fied by a factor o~ 10~ - 106 by an internal MCP
(a mlcrochannel plate) to -eorm an image on the eluorescent output sur~ace Oe the intensi~ier. This image, hale Oe which ls masked by a semicircular mask 23, is ac-ted upon by a proJecting lens 27 and a hal~-mirror 19 so that an image is formed on only half of a CCD area sensor Oe the video camera 24.
Meanwhile, the eluorescent light reflec-ted by the beam-splitter ll impinges upon a projecting lens 1~, which 2~Q7~8 , forms an 1ma~r~ on ~ semicircular mask 16. ~his irnage.
however. is blocl~ed by the ~ask.
After ~ransmission of the signal for ~riggering irradiation wi~h ~he e.Yciting ligh~, strobe irradiation is triggered following a suitable time delay. This time delay should be so selected tha~ strobe irradia~ion is triggered when a cell of interest has reached the lower half of the image capturing area of video camera 24. The strobe triggering signal applied to a light-source power supply 26 is produced by a power-supply controller 28, which functions also a discriminator for cell flow-through.
In operation, the light from strobe ~ is collimated by a collimator lens 10, the collimated light passes through a condenser lens 9 and a dichroic mirror 4 and enters to the condenser lens ~, by virtue of which substantially the entirety of the image capturing area of video camera ~4 is irradiated with the strobe light uniformly. This strobe light which has passed through the image capturing area is reflected by the beam-splitter 11 upon being acted upon by the objective lens 8. The reflected ligh~ impinges upon the projecting lens 15. The latter forms an ima~e upon the semicircular mask 16, shown in Fig. 5. The upper half of the image is blocked by the mask 16, as a result of which an image is formed on only half of a CCD area sensor of the video camera 24 via a projecting lens 17, a mirror 18 and the half-mirror 19.
The part of the light reflected by the dichr-oic mirror 12 upon passage through the beam-splitter 11 passes through the filter 20 and the projecting lens 21, whereby an image is formed on the photoelectric surface of ima~e intensit`ier '~2. However, since gating ls appl.ied l.n such a manner that a nega-tive voltage is impressed upon the lmage .in-tensi~'ier 22 a-t this point ln time, an image does not appear on its fluorescent surface. (In other words, the shutter o~ the in-tensieier Ls closed.) A gating signal for this purpose is produced by the discriminator/power-supply controller ~8 which, as mentioned above, is for 2~07~8 ~judging ~vhen a celL has passed through the image cap~uring area, and lor controlling ~he li~ht sources.
Fig. 3 is an e.~ample illustrating the timing of e.Yciting-light emission, strobe-light emission and the timing of gating signals for the image intensi~ier 2~ after detection of a cell passing through the image capturing area. The signals for controlling such timing are produced by the discriminator/controller ~8 shown in Fig. 1.
In accordance with the above sequence, the fluores-cent image and white-light image of a cell can be captured by a single video camera after the cell has passed through the image capturing area.
It will suffice if the time delay from the moment the cell flows through the image capturing area of line sensor 14 to the moment the cell is irradiated with the strobe light is fiYed so long as the flow velocity of the cell can be made constant. If there is the possibility that flow velocity will fluctuate, however, the following expedient can be adopted. Specifically, the position at which the white-light image of the cell appears in the ima~e capturing area can readily be determined by image processing e~Yecuted by an image analyzer 25. Therefore, if this position shifts from the expected position, feedback control is applied so as to correct the time delay which elapses until irradiation with the light from strobe 2 is performed.
In another embodiment, the positions at which the white-light image 1.ight-receiving system and fluorescent image light-receiving system are disposed in ~ig. 1 can be interchanged if desired.
The lnvention as descri.bed above affords the eollowin~ advantages:
(1) Since passage oE cells through the image capturing area is monitored all times, the images of cells can be obtained eEficiently and with excellent selectivity even if the cell of interest has a low concentration in the speci.men lmder e.~amination.
(2) The irradiating light for obtaining the fluorescent image of a cell need not irradiate the entire 2~7~
image cap~uring area of the video camera; it can be stopped down to a specific area ins~ead. This makes it 2oSsible to raise the intensity of the irradiating light per unit area so that e,Yposure time can be shortened.
(3) Two images, namely the white-light image and the fluorescent image, can be acquired in one imaged frame simultaneously by a single video camera. This ~acilitates image analytical processing and has advantages in terms of cost.
In another example of the conventional ~low ima~ing cytometer, the cell information is obtained using a gross value of the fluorescence emitted erom the fluorescently stained cell. In other ~ords, the fluorescence emitted from each portion of the cell is integrated over the entirety of the cell, and the cell information is obtained in the ~eorm O-e such an integrated value. Though such a me-thod lends itsel~ to analysis of a large number of cells in a short period o~ time, it ls not possible to acquire detailed in-formation as to which por-tions o-f individual cells have been stained and caused to emit fluorescence. Consequentl~, this method is 1imited in terms of analytical performance.
2~7~
On ~he other hand, a cell classifying appara~us tha~
has been ~u~ into practical use employs a nechnique in ~vhich cells s~ained in a manner suitable for a particular cell of interest are introduced to a flow cell to be formed into a planar sheathed flow and irradiated with strobe light, a still picture is obtained b~ a video camera and image processing is applied. However, the state o~ the art is such that the capturing and analysis o~ ~`luorescent images of individual cells using this method have still not reached a practical stage because o~ problems related to fluorescent imaging sensitivity. The present invention makes use of the technology employed in a flow imaging cytometer of the type having a high image cap-turing ef~iciency, as previously proposed in the specification o-f Japanese Patent Application ~o. 185794/l~9o~
SUMMARY OF THE INVENTION:
Thus, the art still lacks a definitlve ~low-imaging particle analyzing system for sensing cells that pass through an image capturing area and irradiating the cells with concentrated exciting light, thereby to assure the required ~luorescent intensity and obtain a fluorescent image, and for subjecting the ~luorescent image, as well as a white-light image of the cells derived from the conventional white-light source, to highly ef-ficient image capturing and analysis using a single video camera.
Accordingly, an object of the presen-t invention is to provide a flow imaging cytometer which expands upon the idea o-f the previously proposed (the aforementioned Japanese Patent Application No. 185794/1990, hereina~ter referred to as "the earlier application'') flow imaging cytometer of the type having a high :Lmage capturing ef~iciency, wherein ~luorescence emitted by a ~luorescently stained ceLl as a result Oe irradiating the cell with laser light is obtained, by single video camera, as a two~dimensional i~age at the same time as a white-light image acquired by conventional strobe-llght (white-light) lrradiation.
.~ccording to the present invention, the foregoing object is attained by providing a ~low imaging cyto~neter 7 ~ 8 , comprising a ~low cell formed ~o include a fla~ ~low 2ath for causing ~ specimen solu~ion con~ainlng particle components to be sensed to flow as a ~lat s~ream~ a first ligh-~ source arranged on a first side of the flow cell s for irradiating the specimen solution in the flow cell with light the quanti-ty of ~hich is switched, a second light source arranged on the first side o~ the flow cell for irradiating the specimen solution in the flow cell with pulsed light, first image capturing means arranged on a second side of the ~low cell for capturing s~ill pictures of particle components in the specimen solution irradiated with high-luminance pulsed ligh-t from the first light source particle components irradiated with the light ~rom the second light source, second image capturing means arranged on the second side of the -flow cell for capturing still pictures o~ particle components in the specimen solution irradiated continuousl~ with low-luminance light from the first light source, processing means for e~YecUting 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 of such detection, first for switching the first light source over to irradiation with the high-luminance light. and -then operating the second light source -~ollowing a prescribed delay, within an image capturing period of the first image capturing means. wherein the first light source is a light source ~or e4~citing fluorescence, and the image resulting from the first light source and the image resulting from the second light source are each captured in a different area on a light-receiving sur~ace o-~ the i~irst image capturing means .
The flow imaging cytometer o~ the present invention is ~urther characterized in that the eirst image capturing means has a two-dimensional image capturin~ area on the flow of the specimen solution, the second image capturing means has a linear image capturing area on the -~low of the specimen solution, the image cap-~uring area of the second 2~ 7~8 image capturing means is formed so as ~o cross the flow of the specimen solution within the image capturing area o~
the first image capturing means, the image capturing area of the firs~ image capturing means is divided into a zone which includes, and a zone which does not includ0, the image capturing area of the second image capturing ~eans, and an image in one of these zones resulting from irradiation with the high-luminance light from the first li~ht source and an image in the other of these zones resultin~ from irradiation by the second light source are captured by the first image capturing means.
The flow imaging cytometer of the present invention is further characterized by having masking means for interrupting light on the optic path of the -first image capturing means in such a manner that the two images do not overlap each other on the light-receiving surface of the first image capturing means.
The flow imaging cytometer o-f the present invention is further characterized by having means for -forming the irradiating light ~rom the first light source into an elongated elliptical shape, and in that the light-receiving system of a fluorescent image is provided with an image intensifier, and the image intensifier is operated only when the fluorescsnt image is captured.
Other ~eatures and advantages of the present invention will be apparent from the ~ollowing description taken in con~unction with the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS:
Fig. 1 is a block diagram illustrating the con-struction o~ a flow imaging cytometer according to -the present inventlon;
Fig. 2 is an e,Yplanatory view illustrating an e,Yample ot' light-irradiating areas and image cap~uri.ng areas of the -~low cell shown in Flg. 1;
Fig. 3 i9 a timing chart illustrating irradiation timing and the timing of a gating signal t'or an image intensi~ier;
~s~r~
Fig. 1 is a dia~r~m showing an e.Yample ol an imaged .rame o~ a eQll cap~ured by a video c~mera: and Fi~. ~ is a diagram showing e.Yamples o~ semicircular and rectangular mas~s associa~ed with the se~-up of Fig. 1.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS:
A preferred embodiment of a flow image cytometer according to the present invention will now be described with reference to the drawings.
~ s illustrated in Fig. 1, the flow imaging cytometer includes a laser light source 1 (e.g. an ~e-Cd laser) for e~Yciting fluorescence and also ~or monitoring the passage of cells through the cy-tometer, and a strobe 2 serving as a ligh-t source for white-light photography. Unlike the strobe light source 2, the laser light source 1 is adapted to irradiate an image capturing area constantly in order to monitor cell flow-through. The light from laser 1 is stopped down to a finely elongated beam spot perpendicular to the direction of cell travel by a cylindrical lens 3 and a condenser lens ~ and irradiates an image capturing area of a line sensor 14, as illustrated in Fig. 2. The reason for this is to obtain a.more uniform light intensity and to improve the S/N ratio when a fluorescent image is captured.
In this embodiment, the image capturing area of the line sensor 14 is provided slightly above mid-center of the upper half of the generally rectangular image capturing area of video camera 24, as shown in Fig. 2.
The light from the laser 1 leaving the image capturing area passes through an obJective lens 8 and is then split by a beam-splitter 11, Part^of the light -from the beam-splitter 11 passes through a dichroic mirror 12 and enters to a projecting lens 13, which proceeds to form an image on the llne sensor 1~. The li.ne sensor 14 successively produces voltage ou-tputs con-~orming to the accumulated amount of photoelectrical conversion of each piAYel e,Yposed for a scanning period (several tens of microseconds) of one line. By means of signal processing similar to that set forth in the earlier applicat~on, triggers irradiation by e~Yciting light and for -fluorescent image capturing are applied when a cell crosses the image capturing area of the line sensor 14 during even-numbered ~ield intervals of the video camera 24.
The time from the instant a cell crosses the image capturing area of the line sensor 14 until the cell is irradiated with the e,Yciting light ~s 100 - 200 ~sec i~ the scanning period of the line sensor 14 is 50 ~sec. On the assumption that the ~low velocity of cells in elow cell 6 is 30 mm/sec, a cell will move 3 - 6 ~m in this period of time. Accordingly, the image of a cell obtained by being irradiated with the e,YCiting light can always be acquired in the area located in the upper half of one imaged frame, as illustrated in Fig. 4. The laser 1 usually delivers a small output power to monitor passage of cells through the flow cell, but the laser is caused to generate high-luminance pulses when fluorescent images are captured.
Fluorescence emitted by a cell in response to irradiation with the elxciting ligh~ passes through the objective lens 8 and the beam-splitter 11 to be reflected by the dichroic mirrors 12. The exciting Light which has passed through the image capturing area is intercepted by a beam stopper 7, and stray light is removed by the filter 20.
Near infrared light constantly emitted in order to monitor cell ~low-through also is eliminated by a filter 20.
The ~luorescent light which has passed through . the filter 20 enters to a projecting lens 21, ~hereby an image of the cell is -eormed on the photoelec~ric sur-eace o~ an image intensi-eier ~2. At this time a high-voltage is applied to the image intensifier 22 so that the image is intensi-fied by a factor o~ 10~ - 106 by an internal MCP
(a mlcrochannel plate) to -eorm an image on the eluorescent output sur~ace Oe the intensi~ier. This image, hale Oe which ls masked by a semicircular mask 23, is ac-ted upon by a proJecting lens 27 and a hal~-mirror 19 so that an image is formed on only half of a CCD area sensor Oe the video camera 24.
Meanwhile, the eluorescent light reflec-ted by the beam-splitter ll impinges upon a projecting lens 1~, which 2~Q7~8 , forms an 1ma~r~ on ~ semicircular mask 16. ~his irnage.
however. is blocl~ed by the ~ask.
After ~ransmission of the signal for ~riggering irradiation wi~h ~he e.Yciting ligh~, strobe irradiation is triggered following a suitable time delay. This time delay should be so selected tha~ strobe irradia~ion is triggered when a cell of interest has reached the lower half of the image capturing area of video camera 24. The strobe triggering signal applied to a light-source power supply 26 is produced by a power-supply controller 28, which functions also a discriminator for cell flow-through.
In operation, the light from strobe ~ is collimated by a collimator lens 10, the collimated light passes through a condenser lens 9 and a dichroic mirror 4 and enters to the condenser lens ~, by virtue of which substantially the entirety of the image capturing area of video camera ~4 is irradiated with the strobe light uniformly. This strobe light which has passed through the image capturing area is reflected by the beam-splitter 11 upon being acted upon by the objective lens 8. The reflected ligh~ impinges upon the projecting lens 15. The latter forms an ima~e upon the semicircular mask 16, shown in Fig. 5. The upper half of the image is blocked by the mask 16, as a result of which an image is formed on only half of a CCD area sensor of the video camera 24 via a projecting lens 17, a mirror 18 and the half-mirror 19.
The part of the light reflected by the dichr-oic mirror 12 upon passage through the beam-splitter 11 passes through the filter 20 and the projecting lens 21, whereby an image is formed on the photoelectric surface of ima~e intensit`ier '~2. However, since gating ls appl.ied l.n such a manner that a nega-tive voltage is impressed upon the lmage .in-tensi~'ier 22 a-t this point ln time, an image does not appear on its fluorescent surface. (In other words, the shutter o~ the in-tensieier Ls closed.) A gating signal for this purpose is produced by the discriminator/power-supply controller ~8 which, as mentioned above, is for 2~07~8 ~judging ~vhen a celL has passed through the image cap~uring area, and lor controlling ~he li~ht sources.
Fig. 3 is an e.~ample illustrating the timing of e.Yciting-light emission, strobe-light emission and the timing of gating signals for the image intensi~ier 2~ after detection of a cell passing through the image capturing area. The signals for controlling such timing are produced by the discriminator/controller ~8 shown in Fig. 1.
In accordance with the above sequence, the fluores-cent image and white-light image of a cell can be captured by a single video camera after the cell has passed through the image capturing area.
It will suffice if the time delay from the moment the cell flows through the image capturing area of line sensor 14 to the moment the cell is irradiated with the strobe light is fiYed so long as the flow velocity of the cell can be made constant. If there is the possibility that flow velocity will fluctuate, however, the following expedient can be adopted. Specifically, the position at which the white-light image of the cell appears in the ima~e capturing area can readily be determined by image processing e~Yecuted by an image analyzer 25. Therefore, if this position shifts from the expected position, feedback control is applied so as to correct the time delay which elapses until irradiation with the light from strobe 2 is performed.
In another embodiment, the positions at which the white-light image 1.ight-receiving system and fluorescent image light-receiving system are disposed in ~ig. 1 can be interchanged if desired.
The lnvention as descri.bed above affords the eollowin~ advantages:
(1) Since passage oE cells through the image capturing area is monitored all times, the images of cells can be obtained eEficiently and with excellent selectivity even if the cell of interest has a low concentration in the speci.men lmder e.~amination.
(2) The irradiating light for obtaining the fluorescent image of a cell need not irradiate the entire 2~7~
image cap~uring area of the video camera; it can be stopped down to a specific area ins~ead. This makes it 2oSsible to raise the intensity of the irradiating light per unit area so that e,Yposure time can be shortened.
(3) Two images, namely the white-light image and the fluorescent image, can be acquired in one imaged frame simultaneously by a single video camera. This ~acilitates image analytical processing and has advantages in terms of cost.
(4) Since the same laser can be used ~or monitor-ing cell flow-shrough and for e.Yciting fluorescence, the design of the optical system can be simplified and cos-ts reduced.
~5) By using flow imaging cytometry, a high processing capability not feasible with conventional microscopic measurement can be obtained.
As many apparently widely different embodiments of the present invention can be made without departing from the spirit and scope thereof, i~ is to be understood that the invention is not limited to the specific embodiments thereof e.Ycept as defined in the appended claims.
~5) By using flow imaging cytometry, a high processing capability not feasible with conventional microscopic measurement can be obtained.
As many apparently widely different embodiments of the present invention can be made without departing from the spirit and scope thereof, i~ is to be understood that the invention is not limited to the specific embodiments thereof e.Ycept as defined in the appended claims.
Claims (5)
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 light the quantity of which is switched;
a second 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 particle components in the specimen solution irradiated with high-luminance pulsed light from said first light source and particle components irradiated with the light from said second light source;
second image capturing means arranged on the second side of said flow cell for capturing still pictures of 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 first for switching said first light source over to irradiation with the high-luminance light, and then operating said second light source following a prescribed delay, within an image capturing period of said first image capturing means;
wherein said first light source is a light source for exciting fluorescence, and the image resulting from said first light source and the image resulting from said second light source are each captured in a different area on a light-receiving surface of said first image capturing means.
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;
a second 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 particle components in the specimen solution irradiated with high-luminance pulsed light from said first light source and particle components irradiated with the light from said second light source;
second image capturing means arranged on the second side of said flow cell for capturing still pictures of 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 first for switching said first light source over to irradiation with the high-luminance light, and then operating said second light source following a prescribed delay, within an image capturing period of said first image capturing means;
wherein said first light source is a light source for exciting fluorescence, and the image resulting from said first light source and the image resulting from said second light source are each captured in a different area on a light-receiving surface of said first image capturing means.
The flow imaging cytometer according to claim 1.
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 an the flow of the specimen solution, 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, the image capturing area of said first image capturing means is divided into a zone which includes, and a zone which does not include, the image capturing area of the second image capturing means, and an image in one of these zones resulting from irradiation with the high-luminance light from said first light source and an image in the other of these zones resulting from irradiation by said second light source are captured by said first image capturing means.
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 an the flow of the specimen solution, 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, the image capturing area of said first image capturing means is divided into a zone which includes, and a zone which does not include, the image capturing area of the second image capturing means, and an image in one of these zones resulting from irradiation with the high-luminance light from said first light source and an image in the other of these zones resulting from irradiation by said second light source are captured by said first image capturing means.
3. The flow imaging cytometer according to claim 2, further comprising masking means for interrupting light on the optic path of said first image capturing means in such a manner that the two images do not overlap each other on the light-receiving surface of said first image capturing means.
4. The flow imaging cytometer according to claim 2, further comprising means for forming the irradiating light from said first light source into an elongated elliptical shape.
5. The flow imaging cytometer according to any one of claims 1 through 4, wherein a light-receiving system of a fluorescent image is provided with an image intensifier.
and said image intensifier is operated only when the fluorescent image is captured.
and said image intensifier is operated only when the fluorescent image is captured.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP03033138A JP3084295B2 (en) | 1991-02-27 | 1991-02-27 | Flow image cytometer |
| JP33138/1991 | 1991-02-27 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2050758A1 true CA2050758A1 (en) | 1992-08-28 |
Family
ID=12378238
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002050758A Abandoned CA2050758A1 (en) | 1991-02-27 | 1991-09-05 | Flow imaging cytometer |
Country Status (5)
| Country | Link |
|---|---|
| US (1) | US5159398A (en) |
| EP (1) | EP0501007B1 (en) |
| JP (1) | JP3084295B2 (en) |
| CA (1) | CA2050758A1 (en) |
| DE (1) | DE69116126T2 (en) |
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-
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- 1991-02-27 JP JP03033138A patent/JP3084295B2/en not_active Expired - Fee Related
- 1991-09-05 EP EP91115006A patent/EP0501007B1/en not_active Expired - Lifetime
- 1991-09-05 US US07/755,549 patent/US5159398A/en not_active Expired - Lifetime
- 1991-09-05 CA CA002050758A patent/CA2050758A1/en not_active Abandoned
- 1991-09-05 DE DE69116126T patent/DE69116126T2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| EP0501007A3 (en) | 1992-12-02 |
| EP0501007A2 (en) | 1992-09-02 |
| JPH04270962A (en) | 1992-09-28 |
| DE69116126D1 (en) | 1996-02-15 |
| DE69116126T2 (en) | 1996-07-04 |
| EP0501007B1 (en) | 1996-01-03 |
| US5159398A (en) | 1992-10-27 |
| JP3084295B2 (en) | 2000-09-04 |
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