CA1164992A - Method of analyzing particles in a dilute fluid sample - Google Patents
Method of analyzing particles in a dilute fluid sampleInfo
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- CA1164992A CA1164992A CA000387730A CA387730A CA1164992A CA 1164992 A CA1164992 A CA 1164992A CA 000387730 A CA000387730 A CA 000387730A CA 387730 A CA387730 A CA 387730A CA 1164992 A CA1164992 A CA 1164992A
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Abstract
ABSTRACT OF THE INVENTION
A method for analyzing particles and particularly sediments of urine is accomplished by distributing the sample over an extended area. A plurality of optical still images is taken of the sample, with each image representing a different portion of the area. Each optical image is converted into an electronic image. The plurality of electronic images are composited to form one resultant electronic image.
A method for analyzing particles and particularly sediments of urine is accomplished by distributing the sample over an extended area. A plurality of optical still images is taken of the sample, with each image representing a different portion of the area. Each optical image is converted into an electronic image. The plurality of electronic images are composited to form one resultant electronic image.
Description
The present invention relates to a method of analy~ing particles in a fluid sample and more particularly to a method of analyzing biological fluid samples, such as urine, that are dilute, but without the necessity of physically creating a concentrated sample for analysis.
Heretofore the method for urine sediment examination requires the following steps: (i) urine must be poured into a tube and spun down in a centrifuge to separate the sediment from its suspending fluid; (ii) most of the cleared suspending fluid must be poured owt; (iii) the sediment must be re-suspended in the remaining fluid; (iv) the suspension must be trans-ferred to and spread on a microscope slide; (v) a coverslip must be placed over the suspension on the slide; (vi) the slide must be focused under a microscope, and (vii) a number of fields of view must be searched and exam-ined for the presence of abnormal numbers of red and white blood cells, epithelial cells, casts, bacteria, yeast, parasites, mucoid threads, crystals, etc., which compose urine sediment in various proportions depending upon the presence of disease. The steps of centrifugation (i), decanta-tion (ii) and re-suspension (iii) are used because the fluid sample is dilute. All these steps are currently performed manually. The manipula-tions involved frequently make the method messy and unpleasant. Spreading of the sediment suspension on the microscope slide often is uneven. ~nlen numerous sediments are viewed, prolonged peering into the eyepieces of a microscope becomes tiring. All these factors contribute to imprecision.
Other apparatus for handling biological specimens include the so-called Coulter counter. In this counter blood cells are passed in single file through an orifice and detected and counted by the manner in whicb they change the electric properties at the orifice. However, informa-tion from the Coulter counter is limited to the analysis of a single type of measurement. Where multiple parameter information is desired, the standard commercial way of obtaining it is by preparing a microscope slide with the cells fi~ed on an image plane and having a human operator or pattern recognition machine count statistically significant numbers of the .
cells as the cells are observed one-at-a-time on the slide through a micro-scope.
Other attempts have been made in recent years to provide optical analysis of particles flowing in a flow stream. For instance, Kay, et al., Journal of Histochemistry and Cytochemistry, Volume 27, page 329 (1979) shows a Coulter type orifice for moving cells in single file with the cells magnified on a vidicon. Additionally, Kachel, et al., Journal of Histo-chemistry and Cytochemistry, Volume 27, page 335, shows a device for moving cells in single file through a microscopic area where they are photographed.
See also for instance Flow Cytometry and Sorting, Melaned et al.~ John Wiley ~ Sons 1979, Chapter 1.
However, none of the references cited heretofore teach or suggest a solution to the problem of analysis of particles in a dilute fluid sample, without the necessity of initially creating a concentrated sample through centrifugation, decantatlon and re-suspension.
SUMMARY OF THE INVENTION
The invention provides a method of displaying elec~ronically concentrated microscopic particles, from a dilute biological fluid sample containing said particles, comprising: distributing said fluid sample over an extended area with substantially no particle overlapping other particles;
forming a plurality of optical still images of said sample over said area, with each optical image representing a different portion of said area; con-verting each of said optical still images to an electronic image; co~positing the images of the different particles from said electronic images to form one resultant electronic image; processing the resultant image; and display-ing said processed image, whereby said processed image is an image of electronically concentrated microscopic particles.
The invention also provides a method of displaying electronically concentrated microscopic particles, from a dilute biological fluid sample containing said particles, comprising: distributing said fluid sample over an extended area substantially with no particle overlapping other particles;
forming a plurality of optical still ima~es of said sample over said area, with each optical image representing a different portion of said area;
converting each of said optical still images to an electronic image; pro-cessing each of sa.id electronic images; compositing ~he images of the dif-ferent particles from said electronic images to form one resultant electronic image; and displaying said one resultant electronic image; whereby said resultant image is an image of electronically concentrated microscopic particles.
The i.nvention will further be described, by way of example only, with reference to the accompanying drawings9 wherein:
Figure 1 is a perspective view of an apparatus which can be used with the method of this invention.
Figure 2 is a plan view of the flow chamber in Figure 1.
: Figure 3 is a cross-sectional vi~w of the apparatus of Figure 2 taken on the plane indicated at 3-3.
; Figure ~ is a schematic diagram of the electronic processor employed by the apparatus of Figure 1.
The method of the present invention comprises distributing a fluid sample, such as urine, over an extended area, such as smearing the sample over a microscope slide. A plurality of optical still images of the sample are taken, with each image representing a different portion of the slide. Thus, for example, the slide with the sample thereon may be mounted in a f~
l ~ microscope and moved about s~lch that a portion of the slide is in
Heretofore the method for urine sediment examination requires the following steps: (i) urine must be poured into a tube and spun down in a centrifuge to separate the sediment from its suspending fluid; (ii) most of the cleared suspending fluid must be poured owt; (iii) the sediment must be re-suspended in the remaining fluid; (iv) the suspension must be trans-ferred to and spread on a microscope slide; (v) a coverslip must be placed over the suspension on the slide; (vi) the slide must be focused under a microscope, and (vii) a number of fields of view must be searched and exam-ined for the presence of abnormal numbers of red and white blood cells, epithelial cells, casts, bacteria, yeast, parasites, mucoid threads, crystals, etc., which compose urine sediment in various proportions depending upon the presence of disease. The steps of centrifugation (i), decanta-tion (ii) and re-suspension (iii) are used because the fluid sample is dilute. All these steps are currently performed manually. The manipula-tions involved frequently make the method messy and unpleasant. Spreading of the sediment suspension on the microscope slide often is uneven. ~nlen numerous sediments are viewed, prolonged peering into the eyepieces of a microscope becomes tiring. All these factors contribute to imprecision.
Other apparatus for handling biological specimens include the so-called Coulter counter. In this counter blood cells are passed in single file through an orifice and detected and counted by the manner in whicb they change the electric properties at the orifice. However, informa-tion from the Coulter counter is limited to the analysis of a single type of measurement. Where multiple parameter information is desired, the standard commercial way of obtaining it is by preparing a microscope slide with the cells fi~ed on an image plane and having a human operator or pattern recognition machine count statistically significant numbers of the .
cells as the cells are observed one-at-a-time on the slide through a micro-scope.
Other attempts have been made in recent years to provide optical analysis of particles flowing in a flow stream. For instance, Kay, et al., Journal of Histochemistry and Cytochemistry, Volume 27, page 329 (1979) shows a Coulter type orifice for moving cells in single file with the cells magnified on a vidicon. Additionally, Kachel, et al., Journal of Histo-chemistry and Cytochemistry, Volume 27, page 335, shows a device for moving cells in single file through a microscopic area where they are photographed.
See also for instance Flow Cytometry and Sorting, Melaned et al.~ John Wiley ~ Sons 1979, Chapter 1.
However, none of the references cited heretofore teach or suggest a solution to the problem of analysis of particles in a dilute fluid sample, without the necessity of initially creating a concentrated sample through centrifugation, decantatlon and re-suspension.
SUMMARY OF THE INVENTION
The invention provides a method of displaying elec~ronically concentrated microscopic particles, from a dilute biological fluid sample containing said particles, comprising: distributing said fluid sample over an extended area with substantially no particle overlapping other particles;
forming a plurality of optical still images of said sample over said area, with each optical image representing a different portion of said area; con-verting each of said optical still images to an electronic image; co~positing the images of the different particles from said electronic images to form one resultant electronic image; processing the resultant image; and display-ing said processed image, whereby said processed image is an image of electronically concentrated microscopic particles.
The invention also provides a method of displaying electronically concentrated microscopic particles, from a dilute biological fluid sample containing said particles, comprising: distributing said fluid sample over an extended area substantially with no particle overlapping other particles;
forming a plurality of optical still ima~es of said sample over said area, with each optical image representing a different portion of said area;
converting each of said optical still images to an electronic image; pro-cessing each of sa.id electronic images; compositing ~he images of the dif-ferent particles from said electronic images to form one resultant electronic image; and displaying said one resultant electronic image; whereby said resultant image is an image of electronically concentrated microscopic particles.
The i.nvention will further be described, by way of example only, with reference to the accompanying drawings9 wherein:
Figure 1 is a perspective view of an apparatus which can be used with the method of this invention.
Figure 2 is a plan view of the flow chamber in Figure 1.
: Figure 3 is a cross-sectional vi~w of the apparatus of Figure 2 taken on the plane indicated at 3-3.
; Figure ~ is a schematic diagram of the electronic processor employed by the apparatus of Figure 1.
The method of the present invention comprises distributing a fluid sample, such as urine, over an extended area, such as smearing the sample over a microscope slide. A plurality of optical still images of the sample are taken, with each image representing a different portion of the slide. Thus, for example, the slide with the sample thereon may be mounted in a f~
l ~ microscope and moved about s~lch that a portion of the slide is in
2 ¦ the imaging area. Each image will be of a different portion of ¦ the slideO Each of the optical irnages is converted to an elec-4 ¦ tronic image. The plurality of electronic images are electronic-S ¦ ally composited to form one resultant electronic image. The one 6 ¦ resultant electronic image may be further processed.
7 ¦ The method of the present invention may be practiced 8 ¦ by using an apparatus 5, shown in ~IGV~E lo The apparatus 5 9 ¦ includes a body 10 containing a flow chamber having an inlet ¦ 12 for a fluid sample, such as urine, and an outlet 14 with a ll ¦ passageway 16 extending between them past an imaging area 18.
12 ¦ The passageway 16 has an inlet with a conduit 20 adapted to be 13 ¦ connected to a volume of saline solution 22. As illustrated in 14 ¦ Figs. 2 and 3, the inlet 12 for the urine sample has a needle 24 ¦ in the passageway 16 downstream from the conduit 20 with the 16 I needle 24 connected to a container 26 adapted to hold the urine 17 ¦ sample to be analyzed. The urine sample flows in a direction 18 ¦ from the inlet 12 to the outlet 14.
19 ¦ The cross-sectional area of the passageway 16 becomes ¦ progressively smaller as the passageway extends from the inlet 21 12 to the outlet 14 while at the same time the passageway 16 22 ¦ becomes much shallower and much wider. Thus, as illustrated in 23 ¦ Figs~ 2 and 3 the passageway 16 has a width and depth of about 24 5,000 microns at the inlet 12 and a width and depth of about 500 microns at midpoint 2~, and a depth of 100 rnicrons with a width 26 exceeding 5,000 microns at the examination area 18.
27 It will be appreciated that the fluid sample flowing 28 through the examination area 1~ is many times deeper than the 29 largest cells which have a maximum dimension of about 20 microns, but with the flow passageway shaped in this way the fluid sample ~ f.
1 entering through the openiny 12 is confined to a stable flow 2 path of minimum shear in the examination area 18, and the
7 ¦ The method of the present invention may be practiced 8 ¦ by using an apparatus 5, shown in ~IGV~E lo The apparatus 5 9 ¦ includes a body 10 containing a flow chamber having an inlet ¦ 12 for a fluid sample, such as urine, and an outlet 14 with a ll ¦ passageway 16 extending between them past an imaging area 18.
12 ¦ The passageway 16 has an inlet with a conduit 20 adapted to be 13 ¦ connected to a volume of saline solution 22. As illustrated in 14 ¦ Figs. 2 and 3, the inlet 12 for the urine sample has a needle 24 ¦ in the passageway 16 downstream from the conduit 20 with the 16 I needle 24 connected to a container 26 adapted to hold the urine 17 ¦ sample to be analyzed. The urine sample flows in a direction 18 ¦ from the inlet 12 to the outlet 14.
19 ¦ The cross-sectional area of the passageway 16 becomes ¦ progressively smaller as the passageway extends from the inlet 21 12 to the outlet 14 while at the same time the passageway 16 22 ¦ becomes much shallower and much wider. Thus, as illustrated in 23 ¦ Figs~ 2 and 3 the passageway 16 has a width and depth of about 24 5,000 microns at the inlet 12 and a width and depth of about 500 microns at midpoint 2~, and a depth of 100 rnicrons with a width 26 exceeding 5,000 microns at the examination area 18.
27 It will be appreciated that the fluid sample flowing 28 through the examination area 1~ is many times deeper than the 29 largest cells which have a maximum dimension of about 20 microns, but with the flow passageway shaped in this way the fluid sample ~ f.
1 entering through the openiny 12 is confined to a stable flow 2 path of minimum shear in the examination area 18, and the
3 particles in the ~luid sample are oriented in that area with
4 their maximum cross-sectional area visible in the plane of Fig.
2. The flow characteristics in the passageway 16 may be con-6 trolled by adjusting the fluid pressure in containers 22 and 26 7 either automatically or by adjusting the static heights thereof.
8 Preferably the fluid sample flowing in the examination 9 area 18 has a cross-sectional area of minimum shear which is not substantially larger than the minimum cross-sectional area of 11 the particles. Hence the particles are aligned in the fluid 12 sample flowing in the examination area 18 with their minimum 13 cross-sectional area extended transverse to the direction of 14 flow. The term "minimum shear" is used herein to mean "minimum velocity gradient" so that a particle moving in the stream tends 16 to align itself with the direction of the stream much as a log 17 floating down a river will align itself with the direction of 18 flow where there is a flow gradient.
19 A microscope 30 is focused on the examination area 18 and the examination area 18 is illuminated from below by a 21 strobe light 32 which is preferably a U.S. Scientific Instrument 22 Corporation Model 3018 containing a 2UPl.5 lamp. The light 32 23 is directed at the microscope 30 in a direction substantially 24 parallel to the thickness of the body lO. The stroble light 32 operates, preferably, at one-sixtieth of a second, thereby 26 forming a series of still optical images at the microscope 30.
27 The output of the microscope 30 is focused on a CCD camera 34 28 which is preferably a CCD camera model number TCl160BD manu-29 factured by RCA. The CCD camera 34 converts each optical image into an electronic image. The CCD Camera 34 also segments each
2. The flow characteristics in the passageway 16 may be con-6 trolled by adjusting the fluid pressure in containers 22 and 26 7 either automatically or by adjusting the static heights thereof.
8 Preferably the fluid sample flowing in the examination 9 area 18 has a cross-sectional area of minimum shear which is not substantially larger than the minimum cross-sectional area of 11 the particles. Hence the particles are aligned in the fluid 12 sample flowing in the examination area 18 with their minimum 13 cross-sectional area extended transverse to the direction of 14 flow. The term "minimum shear" is used herein to mean "minimum velocity gradient" so that a particle moving in the stream tends 16 to align itself with the direction of the stream much as a log 17 floating down a river will align itself with the direction of 18 flow where there is a flow gradient.
19 A microscope 30 is focused on the examination area 18 and the examination area 18 is illuminated from below by a 21 strobe light 32 which is preferably a U.S. Scientific Instrument 22 Corporation Model 3018 containing a 2UPl.5 lamp. The light 32 23 is directed at the microscope 30 in a direction substantially 24 parallel to the thickness of the body lO. The stroble light 32 operates, preferably, at one-sixtieth of a second, thereby 26 forming a series of still optical images at the microscope 30.
27 The output of the microscope 30 is focused on a CCD camera 34 28 which is preferably a CCD camera model number TCl160BD manu-29 factured by RCA. The CCD camera 34 converts each optical image into an electronic image. The CCD Camera 34 also segments each
-5-~ f-1 of t;le ele~tronic image into a plurality of pixels, with each 2 pixel corresponding to a defined portion of each image. The 3 1plurality of electronic images (each optical image is converted 4 ¦into an electronic image) are then composited to create one 5 ¦ resultant electronic image. This may be done, Eor example, by
6 ~summing all the pixels that correspond to the same defined
7 ~ portion of each image. The one resultant electronic image is an
8 ¦ image of an apparent concentrated fluid sample, but without the
9 ¦ necessity of physically creating a concentrated fluid sample.
10 ¦ Moreover, the degree of apparent concentration is controlled by
11 ¦ the number of images that are composited. Thus, an image of an
12 ¦ apparent ten-fold concentration is accomplished by compositing
13 ¦ ten images to form one resultant image. Since the degree of
14 ¦ apparent concentration is controlled electronically, it should ¦ be obvious that with the method of the present invention, the 16 ¦ image of the apparent concentration of the fluid sample may be 17 ¦ varied with considerable ease.
18 ¦ The one resultant electronic image may be further 19 ¦ processedr electronically, and displayed. Alternatively, each 20 ¦ electronic image may be processed, electronically, prior to 21 ¦ being composited to form the one resultant electronic image.
22 ¦ One processor which may be employed, to process electronically 23 ¦ either each electronic image or the one resultant electronic 24 ¦ image, is the processor marketed as Image Analysis System Model 25 1 C~1285 by Hamamatsu Systems, Inc., Waltham/ Massachusetts.
26 ¦ Preferably, however, the output of the CCD camera 34 is connected 27 1 to an electronic processor 36 which is illustrated in greater 28 ¦ detail in Fig. 4 and includes a black and ~hite television 29 ¦ monitor 38 and a frame grabber 40 which stores still electronic 30 1 images of the subject viewed by the CCD camera 34. The frame l -6-r 11~99~
l grabber 40 is preferably a Model FG08 frame grabber made by the 2 ¦ Matrox Corporation of Montreal, the output of which is supplied 3 ¦ to a video refresh mernory 42 model RGs 256 rnade by Matrox 4 1 Corporation which are both coupled to the multibus 44 of the 5 ¦ central processing unit 46 which is preferably an Intel 80/20 6 ¦ computer. The multibus 44 is also coupled to a 48K random 7 ¦ access memory 48 of Electronic Solutions, Inc., and a 16K dual 8 ¦ port random access memory 50 model RM 117 of Data Cube Corpora-9 1 tion. The output of the video refresh memory 42 is also coupled ¦ to a color monitor 52 which may be used to provide digitally 11 ¦ enhanced video images of individual still frames for human 12 ¦ examination. -~13 ¦ The second output of the dual port ram 5a is connected 1~ 1 to a multibus 54 which is connected to an Applied Micro Devices ¦ central processing unit 5~, a 48K random access memory of 16 ¦ Electronic Solutions, Inc. 58 and removable storage in the form 17 ¦ of a floppy disc controller 60, such as an Advanced Micro Devices 18 1 Model 8/8 and two units of Shugart floppy disc storage 62.
19 ¦ ~ith the apparatus shown in FigO 4, a number of speci-I fic methods, for the creation of the one resultant electronic 21 ¦ image, is possible~
22 ¦ In the first alternative, fluid sample such as urine 23 ¦ is entered into the inlet 12. The fluid is illuminated by the 24 ¦ strobe light 32 and a plurality of optical still images of the 25 1 sample are taken by the microscope 30. secause the fluid is 26 translucent and the illumination is from below~ the optical image 27 will be of dark particles on a light background. The optical 28 images are converted into electronic images which are then 29 digitized and stored in memory 48. The one resultant electronic image, the composition of the plurality of electronic images, 32 1"' (~ 8~ ~
1 is formed by sum~ing the di~3itized data of each electronic image 2 with that data stored in memory 48.
3 In a variation of the above described method, prior 4 to the data of the digitized image being stored in memory 48, the background data of each image is first removed, electronic-6 ally. The pertinent information from each image may be collected 7 and stored in one resultant electronic image.
8 In yet another alternative, urine is injected into 9 the inlet 12 as before. The urine is illuminated by the strobe light 32. However, using the well known technique of dark field ll illumination or phase contrast illumination, the optical image 12 produced at the microscope 30 will be of light particles on a 13 dark background. Each optical image is converted into an elec-14 tronic image by the CCD camera 34. Due to the nature of the CCD
camera 34, it retains the electronic image in the camera, if the 16 electronic image is not read out. Thus, a subsequent electronic 17 image (converted from an optical image) will be composited to the 18 previous electronic imageO The one resultant electronic image 19 may therefore be formed at the CCD camera 34.
A wide variety of programming may be employed for 21 further processing the one resultant electronic image with the 22 apparatus of Fig. 4 depending upon the particular task which 23 user wishes to perform.
24 For example with urine, in the method of the prior art, if chemical particles, such as phosphates are in the 26 imaging area and obscure the view of the biological particles, 27 the phosphate particles are removed chemically through the 28 addition of hydrochloric acid. With the method of the present 29 invention, however, the chemical particles may be removed electronically, i~e. through image processing techniques. If 31 it is desired to remove particles of particular size, color or 32 ~ 3_ 1 shape from view, this may be done electronically without re-2 preparing the sample each time. Moreover, with the method of 3 the present invention, biological particles which heretofore 4 may not be removed chemically, may be similarly electronically removed from the image. Thus a greater degree of flexibility 6 is possible with the present invention.
7 It should be appreciated that there are many advan-8 tages to the method of the present invention. The first and 9 foremost is that the analysis of particles of a dilute sample may be made without first physically creating a concentrated ~~
11 sample, with its attending problems of centrifugation, decanta-12 tion and resuspension. The method of resuspension of the prior 13 art results in overlap of the various particles or results in a 14 biased image. With the method of the present invention, the fluid is more statistically representative of the particles with 16 less likelihood of overlap of the particles, and there is no bias 17 of the image. Next, it should be appreciated that the degree of 18 apparent concentration may be varied electronically. In addi-19 tion, the elimination of manual handling steps saves time, potential sources of error and offers biological safeguards 21 (potentially infectious samples are analyzed w~th a minimum o~
22 human handling). Then too, consumable items, such as tubes, 23 pipettes and microscope slides, are not used resulting in 24 economic savings. Finally, with the image in electronic form, a number of imaging techniques may be used to further process 26 the image, including the electronic remo~al of chemical and 27 biological particles.
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32 ~ 9_
18 ¦ The one resultant electronic image may be further 19 ¦ processedr electronically, and displayed. Alternatively, each 20 ¦ electronic image may be processed, electronically, prior to 21 ¦ being composited to form the one resultant electronic image.
22 ¦ One processor which may be employed, to process electronically 23 ¦ either each electronic image or the one resultant electronic 24 ¦ image, is the processor marketed as Image Analysis System Model 25 1 C~1285 by Hamamatsu Systems, Inc., Waltham/ Massachusetts.
26 ¦ Preferably, however, the output of the CCD camera 34 is connected 27 1 to an electronic processor 36 which is illustrated in greater 28 ¦ detail in Fig. 4 and includes a black and ~hite television 29 ¦ monitor 38 and a frame grabber 40 which stores still electronic 30 1 images of the subject viewed by the CCD camera 34. The frame l -6-r 11~99~
l grabber 40 is preferably a Model FG08 frame grabber made by the 2 ¦ Matrox Corporation of Montreal, the output of which is supplied 3 ¦ to a video refresh mernory 42 model RGs 256 rnade by Matrox 4 1 Corporation which are both coupled to the multibus 44 of the 5 ¦ central processing unit 46 which is preferably an Intel 80/20 6 ¦ computer. The multibus 44 is also coupled to a 48K random 7 ¦ access memory 48 of Electronic Solutions, Inc., and a 16K dual 8 ¦ port random access memory 50 model RM 117 of Data Cube Corpora-9 1 tion. The output of the video refresh memory 42 is also coupled ¦ to a color monitor 52 which may be used to provide digitally 11 ¦ enhanced video images of individual still frames for human 12 ¦ examination. -~13 ¦ The second output of the dual port ram 5a is connected 1~ 1 to a multibus 54 which is connected to an Applied Micro Devices ¦ central processing unit 5~, a 48K random access memory of 16 ¦ Electronic Solutions, Inc. 58 and removable storage in the form 17 ¦ of a floppy disc controller 60, such as an Advanced Micro Devices 18 1 Model 8/8 and two units of Shugart floppy disc storage 62.
19 ¦ ~ith the apparatus shown in FigO 4, a number of speci-I fic methods, for the creation of the one resultant electronic 21 ¦ image, is possible~
22 ¦ In the first alternative, fluid sample such as urine 23 ¦ is entered into the inlet 12. The fluid is illuminated by the 24 ¦ strobe light 32 and a plurality of optical still images of the 25 1 sample are taken by the microscope 30. secause the fluid is 26 translucent and the illumination is from below~ the optical image 27 will be of dark particles on a light background. The optical 28 images are converted into electronic images which are then 29 digitized and stored in memory 48. The one resultant electronic image, the composition of the plurality of electronic images, 32 1"' (~ 8~ ~
1 is formed by sum~ing the di~3itized data of each electronic image 2 with that data stored in memory 48.
3 In a variation of the above described method, prior 4 to the data of the digitized image being stored in memory 48, the background data of each image is first removed, electronic-6 ally. The pertinent information from each image may be collected 7 and stored in one resultant electronic image.
8 In yet another alternative, urine is injected into 9 the inlet 12 as before. The urine is illuminated by the strobe light 32. However, using the well known technique of dark field ll illumination or phase contrast illumination, the optical image 12 produced at the microscope 30 will be of light particles on a 13 dark background. Each optical image is converted into an elec-14 tronic image by the CCD camera 34. Due to the nature of the CCD
camera 34, it retains the electronic image in the camera, if the 16 electronic image is not read out. Thus, a subsequent electronic 17 image (converted from an optical image) will be composited to the 18 previous electronic imageO The one resultant electronic image 19 may therefore be formed at the CCD camera 34.
A wide variety of programming may be employed for 21 further processing the one resultant electronic image with the 22 apparatus of Fig. 4 depending upon the particular task which 23 user wishes to perform.
24 For example with urine, in the method of the prior art, if chemical particles, such as phosphates are in the 26 imaging area and obscure the view of the biological particles, 27 the phosphate particles are removed chemically through the 28 addition of hydrochloric acid. With the method of the present 29 invention, however, the chemical particles may be removed electronically, i~e. through image processing techniques. If 31 it is desired to remove particles of particular size, color or 32 ~ 3_ 1 shape from view, this may be done electronically without re-2 preparing the sample each time. Moreover, with the method of 3 the present invention, biological particles which heretofore 4 may not be removed chemically, may be similarly electronically removed from the image. Thus a greater degree of flexibility 6 is possible with the present invention.
7 It should be appreciated that there are many advan-8 tages to the method of the present invention. The first and 9 foremost is that the analysis of particles of a dilute sample may be made without first physically creating a concentrated ~~
11 sample, with its attending problems of centrifugation, decanta-12 tion and resuspension. The method of resuspension of the prior 13 art results in overlap of the various particles or results in a 14 biased image. With the method of the present invention, the fluid is more statistically representative of the particles with 16 less likelihood of overlap of the particles, and there is no bias 17 of the image. Next, it should be appreciated that the degree of 18 apparent concentration may be varied electronically. In addi-19 tion, the elimination of manual handling steps saves time, potential sources of error and offers biological safeguards 21 (potentially infectious samples are analyzed w~th a minimum o~
22 human handling). Then too, consumable items, such as tubes, 23 pipettes and microscope slides, are not used resulting in 24 economic savings. Finally, with the image in electronic form, a number of imaging techniques may be used to further process 26 the image, including the electronic remo~al of chemical and 27 biological particles.
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32 ~ 9_
Claims (11)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A method of displaying electronically concentrated microscopic particles, from a dilute biological fluid sample con-taining said particles, comprising:
distributing said fluid sample over an extended area with substantially no particle overlapping other particles;
forming a plurality of optical still images of said sample over said area, with each optical image representing a different portion of said area;
converting each of said optical still images to an electronic image;
compositing the images of the different particles from said electronic images to form one resultant elec-tronic image;
processing the resultant image; and displaying said processed image, whereby said processed image is an image of elec-tronically concentrated microscopic particles.
distributing said fluid sample over an extended area with substantially no particle overlapping other particles;
forming a plurality of optical still images of said sample over said area, with each optical image representing a different portion of said area;
converting each of said optical still images to an electronic image;
compositing the images of the different particles from said electronic images to form one resultant elec-tronic image;
processing the resultant image; and displaying said processed image, whereby said processed image is an image of elec-tronically concentrated microscopic particles.
2. A method of displaying electronically concentrated microscopic particles, from a dilute biological fluid sample con-taining said particles, comprising:
distributing said fluid sample over an extended area substantially with no particle overlapping other particles;
forming a plurality of optical still images of said sample over said area, with each optical image representing a different portion of said area;
converting each of said optical still images to an electronic image;
processing each of said electronic images;
compositing the images of the different particles from said electronic images to form one resultant elec-tronic image; and displaying said one resultant electronic image;
whereby said resultant image is an image of elec-tronically concentrated microscopic particles.
distributing said fluid sample over an extended area substantially with no particle overlapping other particles;
forming a plurality of optical still images of said sample over said area, with each optical image representing a different portion of said area;
converting each of said optical still images to an electronic image;
processing each of said electronic images;
compositing the images of the different particles from said electronic images to form one resultant elec-tronic image; and displaying said one resultant electronic image;
whereby said resultant image is an image of elec-tronically concentrated microscopic particles.
3. The method of Claim 1 or 2 wherein said processing step is:
electronically removing images of particles that are not desired for display.
electronically removing images of particles that are not desired for display.
4. The method of Claim 1 or 2 further comprising the steps of:
segmenting each of said electronic images into a plurality of pixels, with each pixel corresponding to a defined portion of each image; and summing all the pixels that correspond to the same defined portion of each image.
segmenting each of said electronic images into a plurality of pixels, with each pixel corresponding to a defined portion of each image; and summing all the pixels that correspond to the same defined portion of each image.
5. The method of Claim 1 or 2 further comprising the steps of:
segmenting each of said electronic images into a plurality of pixels, with each pixel corresponding to a defined portion of each image;
summing all the pixels that correspond to the same defined portion of each image;
removing the background data from each electronic image; and collecting the pertinent information from each electronic image to form one resultant image.
segmenting each of said electronic images into a plurality of pixels, with each pixel corresponding to a defined portion of each image;
summing all the pixels that correspond to the same defined portion of each image;
removing the background data from each electronic image; and collecting the pertinent information from each electronic image to form one resultant image.
6. The method of Claim 1 or 2 wherein said compositing step is formed by a CCD camera.
7. The method of Claim 1 or 2 wherein said fluid sample is urine and said particles are sediments.
8. A method of displaying electronically concentrated microscopic particles, from a moving dilute biological fluid sample containing said particles, comprising:
moving said sample in a direction of flow;
distributing said fluid sample over an extended area having a width and a thickness both measured perpendicular to the direction of flow, with the width many times the thickness, said sample distributed with substantially no particle overlapping other particles;
illuminating said fluid at a predetermined loca-tion in the direction of flow, with said illumination directed in a direction substantially perpendicular to the direction of flow;
forming a plurality of optical still images of said fluid sample, at said location with each optical image representing a different portion of said sample, converting each of said optical still images to an electronic image;
compositing the images of the different particles from said plurality of electronic images to form one resultant electronic image;
processing said one resultant image; and displaying said processed image;
whereby said processed image is an image of elec-tronically concentrated microscopic particles.
moving said sample in a direction of flow;
distributing said fluid sample over an extended area having a width and a thickness both measured perpendicular to the direction of flow, with the width many times the thickness, said sample distributed with substantially no particle overlapping other particles;
illuminating said fluid at a predetermined loca-tion in the direction of flow, with said illumination directed in a direction substantially perpendicular to the direction of flow;
forming a plurality of optical still images of said fluid sample, at said location with each optical image representing a different portion of said sample, converting each of said optical still images to an electronic image;
compositing the images of the different particles from said plurality of electronic images to form one resultant electronic image;
processing said one resultant image; and displaying said processed image;
whereby said processed image is an image of elec-tronically concentrated microscopic particles.
9. A method of displaying electronically concentrated microscopic particles, from a moving dilute biological fluid sample containing said particles, comprising:
moving said sample in a direction of flow;
distributing said fluid sample over an extended area having a width and thickness both measured perpen-dicular to the direction of flow, with the width many times the thickness, said samples being distributed with substantially no particle overlapping other particles;
illuminating said fluid at a predetermined loca-tion in the direction of flow, with said illumination directed in a direction substantially perpendicular to the direction of flow;
forming a plurality of optical still images of said fluid sample, at said location, with each optical image representing a different portion of said sample;
converting each of said optical still images to an electronic image;
processing each of said electronic images;
compositing the images of the different particles from said plurality of electronic images to form one resultant electronic image; and displaying said one resultant electronic image;
whereby said resultant image is an image of electronically concentrated microscopic particles.
moving said sample in a direction of flow;
distributing said fluid sample over an extended area having a width and thickness both measured perpen-dicular to the direction of flow, with the width many times the thickness, said samples being distributed with substantially no particle overlapping other particles;
illuminating said fluid at a predetermined loca-tion in the direction of flow, with said illumination directed in a direction substantially perpendicular to the direction of flow;
forming a plurality of optical still images of said fluid sample, at said location, with each optical image representing a different portion of said sample;
converting each of said optical still images to an electronic image;
processing each of said electronic images;
compositing the images of the different particles from said plurality of electronic images to form one resultant electronic image; and displaying said one resultant electronic image;
whereby said resultant image is an image of electronically concentrated microscopic particles.
10, The method of Claim 8 or 9 wherein said fluid sample is urine and the particles are sediments,
11. The method of Claim 8 or 9 wherein said fluid sample is urine and the particles are sediments and wherein said illuminating step is by stroboscopic illumination.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000387730A CA1164992A (en) | 1981-10-09 | 1981-10-09 | Method of analyzing particles in a dilute fluid sample |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000387730A CA1164992A (en) | 1981-10-09 | 1981-10-09 | Method of analyzing particles in a dilute fluid sample |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1164992A true CA1164992A (en) | 1984-04-03 |
Family
ID=4121144
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000387730A Expired CA1164992A (en) | 1981-10-09 | 1981-10-09 | Method of analyzing particles in a dilute fluid sample |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1164992A (en) |
-
1981
- 1981-10-09 CA CA000387730A patent/CA1164992A/en not_active Expired
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