Coulter Counter

Coulter counter is a commercially available device for determining the size distribution of electrically nonconducting particles suspended in a conducting medium.

1. Coulter Counter Physiology Seminar Mrinmoy Pal

2. Graham, Marshall Don. "The Coulter principle: foundation of an industry."  Journal of the Association for Laboratory Automation  8.6 (2003): 72-81 . Wallace H. Coulter Joseph R. Coulter Wallace H Coulter discovered the Coulter Principle in the late 1940s (though a patent was not awarded until October 20, 1953). Coulter was influenced by the atomic bombs dropped on Hiroshima and Nagasaki. These events motivated Coulter to simplify and improve blood cell analysis so that large populations could be screened rapidly, as would be necessary in the event of a nuclear war. Remembering his visits to hospitals, where he observed lab workers hunched over microscopes manually counting blood cells smeared on glass, Wallace focused the first application on counting red blood cells . 1935, he joined General Electric X-Ray as a sales and service engineer, electronics companies

3. Coulter Counter Coulter counter is commercially available device for determining the size distribution of electrically nonconducting particles suspended in a conducting medium. Two electrodes passing constant current are placed on either side of a small hole or aperture through which the suspension is sucked. Because of the smallness of the aperture the major resistance in the circuit is at the aperture and when a nonconducting particle passes through , the resistance is changed giving rise to electrical pulse. . The number of pulses is equal to the number of cells counted and the strength of the signal (pulse height) is directly proportional to the cell volume . The electrical response of the instrument is essentially independent of the shape of particles with the same volume, an exception to this may occur with some extreme shapes. Color or refractive index of the particles does not affect the results. Coulter, Wallace H. "High speed automatic blood cell counter and cell size analyzer."  Proc Natl Electron Conf . Vol. 12. 1956.

4. Graham, Marshall Don. "The Coulter principle: foundation of an industry."  Journal of the Association for Laboratory Automation  8.6 (2003): 72-81 . Working Principle

5. Coulter, Wallace H. "High speed automatic blood cell counter and cell size analyzer."  Proc Natl Electron Conf . Vol. 12. 1956. The pulses produced at the orifice are amplified and displayed on the oscilloscope screen and appear as vertical lines o r spikes. The height of an individual pulse spike from the baseline is a measure of relative size of the cell. The threshold control dial enables the user to set a cut off. Only if the pulse exceeds the threshold, it gets counted. In addition to a display of relative cell size the oscilloscope also indicates the threshold control by brightening that portion above the threshold level . Electronic System & Output

6. The Model A Coulter Counter The round black object at the upper right of the stand is the stirrer motor used to keep heavy particles in suspension. The console contains, from left to right, the mechanical totalizer used for slowly accumulating high-value digits, the three decade counters used for the rapidly accumulating low-value digits, and the oscilloscope display tube. Controls for the single threshold and the aperture current appear below the display . Coulter, Wallace H. "High speed automatic blood cell counter and cell size analyzer."  Proc Natl Electron Conf . Vol. 12. 1956. Major Drawbacks Single threshold : M ultiple sample runs at successively increasing thresholds were required for generating a cumulative size distribution. The voltage source: Accurate counting and sizing was hampered by instrument’s sensitivity to aperture dimension and temperature-induced resistivity change of the electrolyte. Commercial Models

7. The Model B Coulter Counter FN Model Commercial Models Improvements: Dual-threshold current-sensitive amplifier Multi-channel Pulse Height A nalyzers Graham, Marshall Don. "The Coulter principle: foundation of an industry."  Journal of the Association for Laboratory Automation  8.6 (2003): 72-81 .

8. Experimental Considerations Coincidence: Anomalous electrical pulses can be generated when multiple particles enter the aperture simultaneously. This situation is known as coincidence . This occurs because there is no way to ensure that a single large pulse is the result of a single large particle or multiple small particles entering the aperture at once. To prevent this situation, samples must be fairly dilute. Primary Coincidence Secondary Coincidence

9. Experimental Considerations Porous particles The Coulter Principle measures the volume of an object , since the disturbance in the electric field is proportional to the volume of electrolyte displaced from the aperture. This leads to some confusion amongst those who are used to optical measurements from microscopes or other systems that only view two dimensions and also show the boundaries of an object. For porous particles the volume of pores that are parallel to the electric field lines is not measured , while the volume of pores perpendicular to these lines does contribute to the measured volume . Thus, Particles with interconnected pores produce a size related to their solid volume as shown below. Envelope volume Solid volume Porous p article

10. Experimental Considerations Particle path The shape of the generated electrical pulse varies with the particle path through the aperture . The use of the Edit facility ( signal processing algorithm ) eliminates the distorted pulses, so that the recovered size distribution is close to the correct one . Using a longer tunnel aperture may enhance the accuracy of the results for samples with a narrow size distribution. This behaviour of long tunnel apertures is mostly due to the greater approach to parabolic flow inside the bore. Still better engineering technique was required to correct for artifacts resulting from particle path .

11. The method consists of feeding the suspension from a 30 μ diameter hole positioned 2 to 4 mm from the aperture of the Coulter device. Both tubes are immersed in a bath of particle-free electrolyte. When the electrolyte is sucked through the sensing aperture, the stream tube containing the suspension is accelerated and thinned so that the particles all travel essentially the same streamline, and consequently have uniformity of approach and passage through the sensing aperture. Spielman , Lloyd, and Simon L. Goren. "Improving resolution in Coulter counting by hydrodynamic focusing."  Journal of Colloid and Interface Science  26.2 (1968): 175-182. Hydrodynamic Focusing

12. Sweet, R.G. 1965. High frequency recording with electrostatically deflected ink jets. Rev. Sci. Instr. 36: 131–136. Richard Sweet RG Sweet’s Ink Jet ‘‘Oscillograph’’ which was the basis for the oscillation for separating cells into single droplets in Fulwyler’s cell sorter. Cell Sorter

13. Fulwyler, Mack J. "Electronic separation of biological cells by volume."  Science  150.3698 (1965 ) : 910-911 . Mack Fulwyler Mack Fulwyler used Wallace Coulter’s Coulter principle as the basis for identifying different red blood cells in his cell sorter invention. Cell Sorter

14. Cell counting and sizing: A cell suspension enters the droplet generator (C) by way of a tube (D) and emerges as a high-velocity fluid jet (E). Cell volume is sensed as the cell passes through a Coulter aperture within the droplet generator (C). An electric pulse proportional to cell volume is obtained at J. Droplet formation: A piezoelectric crystal (A), driven at a frequency of 72,000 cy/sec, produces vibrations which pass down the rod (B) into the liquid within the droplet generator. The shape of the rod serves to amplify the magnitude of the vibrations within the liquid. The velocity fluctuations of the emerging liquid produce bunching of the liquid column. Cell Sorting: Now the isolated cell droplet arrives at the separation point (I ). The size of the charging pulse needed to deflect droplets into the proper vessel is electronically determined from the cell-volume pulse and the droplet is charged electronically at the charging collar F. The charged droplets are then deflected (H) on entering the electrostatic field between the deflection plates (G). Finally, a series of collection vessels (L) receive the deflected droplets. Fulwyler, Mack J. "Electronic separation of biological cells by volume."  Science  150.3698 (1965): 910-911 . Cell Sorter

15. FACS: Fluorescence-activated cell sorting  Becton Dickinson (1974) Source: http://docs.abcam.com/pdf/protocols/Introduction_to_flow_cytometry_May_10.pdf The Flow system (fluidics) The Optical system (light sensing) Once the sheath fluid is running at laminar flow, the cells are injected into the center of the stream, at a slightly higher pressure.  The principles of hydrodynamic focusing cause the cells to align in a single file. Advancement in technologies such as fluorescence tagging and light scattering The intensity of fs signal has been attributed to cell size and the intensity of ss signal is proportional to the amount of cytosolic structure in the cell.

16. FACS: Fluorescence-activated cell sorting Source: http://docs.abcam.com/pdf/protocols/Introduction_to_flow_cytometry_May_10.pdf Application: Immunophenotyping , DNA Ploidy and S-phase Fraction Analysis The Electronic system (signal processing)

17. Lab-on-a-chip Coulter Counter Mei, Zhe , et al. "Counting leukocytes from whole blood using a lab-on-a-chip Coulter counter."  2012 Annual International Conference of the IEEE Engineering in Medicine and Biology Society . IEEE, 2012.   A lab-on-a-chip Coulter counter allows cancer patients going through chemotherapy and patients with compromised immune systems to take less than 10uL of blood to measure lymphocyte concentration. The test can be self-administered or performed at the point-of-care clinics to reduce costs and risks of hospital infection . Instead of electroplated or deposited metal electrodes, off-the-shelf gold pins were used as electrodes to simplify fabrication process and to achieve superior uniformity in E-field distribution for improved signal quality.

18. A life tree showing the impact of Wallace Coulter’s inventions and discoveries up to the date of his death in 1998. On the right are some of the 85 patents issued to Wallace Coulter; technologies that made major contributions to the field are shown on the left side of the tree. In the centre are some of the most important technologies with which Wallace Coulter was directly involved or, as in the case of monoclonal antibodies, in which he immediately saw the opportunity and built significant businesses and technologies that capitalized on these inventions Robinson, J. Paul. "Wallace H. Coulter: decades of invention and discovery."  Cytometry Part A  83.5 (2013): 424-438.