FLUORESCENT CELL VIABILITY COUNTER
United States Patent 3586859
Method and apparatus for automatically ascertaining the number of dead cells in a batch. All of the cells in the batch are stained with a specific fluorescent dye in a reservoir, then the cells are passed through a capillary tube by means of a peristaltic pump under a microscope while illuminated by an ultraviolet light. The microscope is coupled to a photomultiplier which in turn has its output applied to an electronic counter with a readout. After the dead cells are counted, the batch is immersed in a heating bath in which all the viable cells are killed and another count is obtained by repassing the batch beneath the microscope. The difference in the counts is the number of original viable cells while the first count is the original number of dead cells.
US Patent References:
Method and apparatus for detecting cancer cells
Kamentsky - June 1967 - 3327119
Method for measuring the nucleic acid in biological cells after enhancement in an acidic solution
Kamentsky - November 1968 - 3413464
METHOD AND APPARATUS FOR CLASSIFYING BIOLOGICAL CELLS BY MEASURING THE SIZE AND FLUORESCENT RESPONSE THEREOF
Wheeless, Jr. et al. - February 1970 - 3497690
Method and apparatus for detecting cancer cells
Kamentsky - June 1967 - 3327119
Method for measuring the nucleic acid in biological cells after enhancement in an acidic solution
Kamentsky - November 1968 - 3413464
METHOD AND APPARATUS FOR CLASSIFYING BIOLOGICAL CELLS BY MEASURING THE SIZE AND FLUORESCENT RESPONSE THEREOF
Wheeless, Jr. et al. - February 1970 - 3497690
Inventors:
Katz, Irwin J. (Brooklyn, NY)
Satriano, Nicholas M. (Rego Park, NY)
Benjaminson, Morris A. (New York, NY)
Satriano, Nicholas M. (Rego Park, NY)
Benjaminson, Morris A. (New York, NY)
Application Number:
05/011628
Publication Date:
06/22/1971
Filing Date:
02/16/1970
View Patent Images:
Export Citation:
Primary Class:
Other Classes:
436/63, 436/172, 435/808, 435/39
International Classes:
C12M1/34; G01N15/14; G06M1/10; G06M1/00; G01N21/26; G01N21/22
Field of Search:
250/71,71.5,83.3UV 356/36,39 424/7 23/23B
Primary Examiner:
Borchelt, Archie R.
Claims:
We claim
1. An apparatus for determining the number of viable and nonviable cells in a 5mg./ml. saline solution of 4 acetamido 440 isothiocyanostilbene 2,2 disulfonic acid Disodium which comprises:
2. The apparatus according to claim 1 wherein said restricted passageway is a capillary tube.
3. The apparatus according to claim 2 wherein said circulating means is a peristaltic pump.
4. The apparatus according to claim 3 wherein said means for killing is a heating module through which the cells are passed.
5. The apparatus according to claim 4 wherein said amplifying and detecting means is a photomultiplier circuit and tube.
6. The apparatus according to claim 5 further including a specimen reservoir disposed in line with and proximate said open end of said passageway.
7. A method for ascertaining the number of viable and nonviable cells within a specimen which comprises the steps of:
8. The method according to claim 7 wherein said dye is 4 acetamido 4' isothiocyanostilbene 2, 2 disulfonic acid Disodium.
9. The method according to claim 8 wherein said prepared solution consists of said specimen cells added to 5 mg. of said dye for every ml. of saline solution.
1. An apparatus for determining the number of viable and nonviable cells in a 5mg./ml. saline solution of 4 acetamido 440 isothiocyanostilbene 2,2 disulfonic acid Disodium which comprises:
2. The apparatus according to claim 1 wherein said restricted passageway is a capillary tube.
3. The apparatus according to claim 2 wherein said circulating means is a peristaltic pump.
4. The apparatus according to claim 3 wherein said means for killing is a heating module through which the cells are passed.
5. The apparatus according to claim 4 wherein said amplifying and detecting means is a photomultiplier circuit and tube.
6. The apparatus according to claim 5 further including a specimen reservoir disposed in line with and proximate said open end of said passageway.
7. A method for ascertaining the number of viable and nonviable cells within a specimen which comprises the steps of:
8. The method according to claim 7 wherein said dye is 4 acetamido 4' isothiocyanostilbene 2, 2 disulfonic acid Disodium.
9. The method according to claim 8 wherein said prepared solution consists of said specimen cells added to 5 mg. of said dye for every ml. of saline solution.
Description:
STATEMENT OF GOVERNMENT INTEREST
The invention described herein may be manufactured and used by or for the Government of the United States of America for governmental purposes without the payment of any royalties thereon or therefor.
BACKGROUND OF THE INVENTION
1.
Field of the Invention
The present invention relates to a system for counting and discriminating between living and dead cells and more particularly pertains to staining all the cells with a specific fluorescent dye and automatically counting the dead cells, and thereafter killing the viable cells and then counting all the cells, providing a rapid count of each.
2.
Description of the Prior Art
In the field of counting and discriminating dead and viable cells, it has been the general practice to employ a standard technique of visually and manually counting the cells exposed to trypan blue. This method is both tedious and extremely time consuming. A more recent method contemplates the use of an automatic cell spectrophotometer on cells which have been stained with trypan blue. This instrument is based upon the principle of light absorption and scattering which requires highly complex instrumentation. The technique itself is less sensitive than other techniques. Such prior art devices have been unsatisfactory in that errors are introduced by undissolved dye particulates, fluids in which the cells are suspended and in counting small size cells. Erythrocytes must be eliminated since they refract light and do not take up trypan blue, thereby resulting in a false count of the living cells.
SUMMARY OF THE INVENTION
The general purpose of this invention is to provide a method and apparatus that has all the advantages of similarly employed prior art devices and has none of the above described disadvantages. To attain this, the present invention provides a unique technique in which all the cells are stained with 4 acetamido 4' isothiocyanostilbene 2,2 disulfonic acid Disodium (SITS) a fluorescent dye and then passed through a capillary, illuminated by an ultraviolet light, beneath a microscope. The microscope is connected to a photomultiplier which provides a count only for those cells which fluoresce and applies to to a counter. All the cells are then killed by heat and recounted, thus giving, first, a count of the nonviable cells and, thereafter, a total count of all the cells, whereby an accurate rapid count of both viable and nonviable cells is provided.
An object of the present invention is to provide a simple, accurate, reliable inexpensive and automatic device for ascertaining a count of both dead and living cells in a specimen.
Another object is to provide a method and technique which is direct, simple and automatic for counting both viable and nonviable cells.
Other objects and many of the attendant advantages of this invention will be readily appreciated as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWING
The FIGURE is a block diagram of an embodiment made in accordance with the principle of this invention and illustrates the method employed.
DESCRIPTION OF THE PREFERRED EMBODIMENT
In the illustrated embodiment, a small specimen reservoir 10 is provided at one end of the flow tube 11, which is disposed directly above a larger reservoir 12 whose inner wall 13 converges downwardly toward a small opening 14 at the base and is connected to a transparent capillary tube 15. The capillary tube 15, which is transparent to fluorescent light, and the reservoir 12 are supported by a clear plastic slide 16. In order to maintain balance and prevent distortion, a solid plastic block 17 is seated above the capillary tube. The inner diameter of tube 15 is selected for the particular size of the cells to be counted. It serves to restrict the multiple simultaneous passage of the cells therethrough in order to permit observation of the cells individually. The capillary tube extends directly between the objective 18 of a standard fluorescent microscope 19 and the light condenser 20. The eyepiece end 21 of the microscope is connected to a photomultiplier tube 22 in any of the standard methods so that the multiplier tube directly senses the ultraviolet light from source 20' impinging on the eyepiece 21 and provides an output count for each light burst detected at the microscope. The electric pulse from the multiplier is applied to an electronic counter and computer 23 which also includes an output indication as by a printout. This type of counter/computer is well known in the art and is commercially available.
The capillary 15 extends beyond the plastic block 17 and is connected to a circulating means or a peristaltic pump 24 via a flexible coupling 25 while a second flexible coupling 26 connects the opposite end of the pump to a valve diverter 27. The flexible couplings decouple the pump movement and vibrations from the flow system. This form of pump prevents damage or destruction of the cells while supplying the necessary motive force to circulate the cells. The valve 27 either directs the flow into the waste receptacle 28 or through the tube 29 to a heating module 30. Within the module is a solution 31 which is heated by element 32 connected to a source of electrical power 33 and the module may include a thermostat 34. The flow tube 35 is coiled within the heating module to permit the heating action to act on the flowing cells. After the heating module, the cells are directed back to the reservoir along flow tube 11.
Having fully described the apparatus employed, the process or technique will now be detailed. It has been discovered that 4 acetamido 4' isothiocyanostilbene 2, 2 disulfonic acid Disodium (SITS), a fluorescent dye, has the ability to distinguish between living and nonliving cells. Viable cells take up the dye and retain it in their vesicles and do not fluoresce brightly if at all, while nonviable cells fluoresce very brightly, since the dye escapes from their vesicles. This dye is not pH dependent, has an emission spectrum of 415--420 millimicrons and an activation spectrum of 345--360 millimicrons. Five milligrams of the dye are added to each milliliter of a buffered physiological saline solution to form a 5mg./ml. solution which is added to the unknown cell suspension. In the above concentration the dye (SITS) is nontoxic.
The cell suspension in the dye solution is placed in the specimen reservoir 10, the ultraviolet source 20' activated and the pump 24 started. When the cell suspension solution reaches flow through the capillary tube 15 and passes beneath the objective 18 of the ultraviolet microscope 19, the photomultiplier 22 will respond only to those cells with a high fluorescent output and provide a count at computer 23 only of the dead or nonviable cells per unit volume.
After all the dead cells are counted, the solution proceeds into the heating module 30, where all the viable cells are killed, resulting in their releasing the SITS from their vesicles and providing bright fluorescent staining. The solution then is recirculated to the specimen reservoir 10 and held until all the initial or original contents therein have been depleted.
With all the cells now dead, the solution is again recirculated and counted as previously described. This count is of the total number of cells initially in solution. The computer 23 is also programmed to subtract the number of nonviable cells counted in the first passage from the total number of cells counted in the second passage so as to print out the following information:
1. number of viable cells
2. number of nonviable cells
3. total number of cells Bypass or diverting valve 27 is opened to the waste receptacle 28 and the specimen solution discarded by being pumped therein. The system is then flushed by adding washing fluid to the specimen reservoir and circulating as necessary and is thereafter discarded into the waste receptacle.
The method of this invention comprises the following steps:
a. applying the specimen cells to be counted to a saline solution of a fluorescent dye SITS;
b. passing the cell solution through a restricted passage;
c. exposing said cell solution in said passage to an ultraviolet light;
d. detecting and counting the fluorescent light pulses thereof;
e. killing any viable cells in said solution; and then
f. detecting and recounting said fluorescent pulses.
The above described invention is not limited as to the cell type which may be counted or examined and the dye is specific for biological material, and the problem of false counts is eliminated by the excellent solubility of the dye. It should be understood, of course, that the foregoing disclosure relates to only a preferred embodiment of the invention and that numerous modifications or alterations may be made therein without departing from the spirit and the scope of the invention as set forth in the appended claims.
The invention described herein may be manufactured and used by or for the Government of the United States of America for governmental purposes without the payment of any royalties thereon or therefor.
BACKGROUND OF THE INVENTION
1.
Field of the Invention
The present invention relates to a system for counting and discriminating between living and dead cells and more particularly pertains to staining all the cells with a specific fluorescent dye and automatically counting the dead cells, and thereafter killing the viable cells and then counting all the cells, providing a rapid count of each.
2.
Description of the Prior Art
In the field of counting and discriminating dead and viable cells, it has been the general practice to employ a standard technique of visually and manually counting the cells exposed to trypan blue. This method is both tedious and extremely time consuming. A more recent method contemplates the use of an automatic cell spectrophotometer on cells which have been stained with trypan blue. This instrument is based upon the principle of light absorption and scattering which requires highly complex instrumentation. The technique itself is less sensitive than other techniques. Such prior art devices have been unsatisfactory in that errors are introduced by undissolved dye particulates, fluids in which the cells are suspended and in counting small size cells. Erythrocytes must be eliminated since they refract light and do not take up trypan blue, thereby resulting in a false count of the living cells.
SUMMARY OF THE INVENTION
The general purpose of this invention is to provide a method and apparatus that has all the advantages of similarly employed prior art devices and has none of the above described disadvantages. To attain this, the present invention provides a unique technique in which all the cells are stained with 4 acetamido 4' isothiocyanostilbene 2,2 disulfonic acid Disodium (SITS) a fluorescent dye and then passed through a capillary, illuminated by an ultraviolet light, beneath a microscope. The microscope is connected to a photomultiplier which provides a count only for those cells which fluoresce and applies to to a counter. All the cells are then killed by heat and recounted, thus giving, first, a count of the nonviable cells and, thereafter, a total count of all the cells, whereby an accurate rapid count of both viable and nonviable cells is provided.
An object of the present invention is to provide a simple, accurate, reliable inexpensive and automatic device for ascertaining a count of both dead and living cells in a specimen.
Another object is to provide a method and technique which is direct, simple and automatic for counting both viable and nonviable cells.
Other objects and many of the attendant advantages of this invention will be readily appreciated as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWING
The FIGURE is a block diagram of an embodiment made in accordance with the principle of this invention and illustrates the method employed.
DESCRIPTION OF THE PREFERRED EMBODIMENT
In the illustrated embodiment, a small specimen reservoir 10 is provided at one end of the flow tube 11, which is disposed directly above a larger reservoir 12 whose inner wall 13 converges downwardly toward a small opening 14 at the base and is connected to a transparent capillary tube 15. The capillary tube 15, which is transparent to fluorescent light, and the reservoir 12 are supported by a clear plastic slide 16. In order to maintain balance and prevent distortion, a solid plastic block 17 is seated above the capillary tube. The inner diameter of tube 15 is selected for the particular size of the cells to be counted. It serves to restrict the multiple simultaneous passage of the cells therethrough in order to permit observation of the cells individually. The capillary tube extends directly between the objective 18 of a standard fluorescent microscope 19 and the light condenser 20. The eyepiece end 21 of the microscope is connected to a photomultiplier tube 22 in any of the standard methods so that the multiplier tube directly senses the ultraviolet light from source 20' impinging on the eyepiece 21 and provides an output count for each light burst detected at the microscope. The electric pulse from the multiplier is applied to an electronic counter and computer 23 which also includes an output indication as by a printout. This type of counter/computer is well known in the art and is commercially available.
The capillary 15 extends beyond the plastic block 17 and is connected to a circulating means or a peristaltic pump 24 via a flexible coupling 25 while a second flexible coupling 26 connects the opposite end of the pump to a valve diverter 27. The flexible couplings decouple the pump movement and vibrations from the flow system. This form of pump prevents damage or destruction of the cells while supplying the necessary motive force to circulate the cells. The valve 27 either directs the flow into the waste receptacle 28 or through the tube 29 to a heating module 30. Within the module is a solution 31 which is heated by element 32 connected to a source of electrical power 33 and the module may include a thermostat 34. The flow tube 35 is coiled within the heating module to permit the heating action to act on the flowing cells. After the heating module, the cells are directed back to the reservoir along flow tube 11.
Having fully described the apparatus employed, the process or technique will now be detailed. It has been discovered that 4 acetamido 4' isothiocyanostilbene 2, 2 disulfonic acid Disodium (SITS), a fluorescent dye, has the ability to distinguish between living and nonliving cells. Viable cells take up the dye and retain it in their vesicles and do not fluoresce brightly if at all, while nonviable cells fluoresce very brightly, since the dye escapes from their vesicles. This dye is not pH dependent, has an emission spectrum of 415--420 millimicrons and an activation spectrum of 345--360 millimicrons. Five milligrams of the dye are added to each milliliter of a buffered physiological saline solution to form a 5mg./ml. solution which is added to the unknown cell suspension. In the above concentration the dye (SITS) is nontoxic.
The cell suspension in the dye solution is placed in the specimen reservoir 10, the ultraviolet source 20' activated and the pump 24 started. When the cell suspension solution reaches flow through the capillary tube 15 and passes beneath the objective 18 of the ultraviolet microscope 19, the photomultiplier 22 will respond only to those cells with a high fluorescent output and provide a count at computer 23 only of the dead or nonviable cells per unit volume.
After all the dead cells are counted, the solution proceeds into the heating module 30, where all the viable cells are killed, resulting in their releasing the SITS from their vesicles and providing bright fluorescent staining. The solution then is recirculated to the specimen reservoir 10 and held until all the initial or original contents therein have been depleted.
With all the cells now dead, the solution is again recirculated and counted as previously described. This count is of the total number of cells initially in solution. The computer 23 is also programmed to subtract the number of nonviable cells counted in the first passage from the total number of cells counted in the second passage so as to print out the following information:
1. number of viable cells
2. number of nonviable cells
3. total number of cells Bypass or diverting valve 27 is opened to the waste receptacle 28 and the specimen solution discarded by being pumped therein. The system is then flushed by adding washing fluid to the specimen reservoir and circulating as necessary and is thereafter discarded into the waste receptacle.
The method of this invention comprises the following steps:
a. applying the specimen cells to be counted to a saline solution of a fluorescent dye SITS;
b. passing the cell solution through a restricted passage;
c. exposing said cell solution in said passage to an ultraviolet light;
d. detecting and counting the fluorescent light pulses thereof;
e. killing any viable cells in said solution; and then
f. detecting and recounting said fluorescent pulses.
The above described invention is not limited as to the cell type which may be counted or examined and the dye is specific for biological material, and the problem of false counts is eliminated by the excellent solubility of the dye. It should be understood, of course, that the foregoing disclosure relates to only a preferred embodiment of the invention and that numerous modifications or alterations may be made therein without departing from the spirit and the scope of the invention as set forth in the appended claims.