1


2

 Electrons and protons posses equal and opposite charge
 Charge is measured in coulombs (C) is 6 x 10^{18} times the
charge on an electron which is 1 x 10^{19} C

3

 The potential energy of a charged particle in an electric field is
proportional to its charge  the electrical potential difference between
two points in the field of which a potential energy of 1 C charge
changes by 1 Joule, is defined as 1 volt (V).
 1 Amp represents the transfer of 1 coulomb of charge per second

4

 All materials offer resistance to the flow of electrons
 Based on Ohm’s Law, the flow of a current of 1 Amp through a material of
resistance of R ohms (W) produces a
drop in electrical potential or a voltage difference of E volts across
the resistance such that E=IR

5

 DC  direct current  the polarity of a current source remains the same
when the current is DC
 AC  Alternative current  this is generated by using a magnetic field
(generator) to convert mechanical into electrical energy  the polarity
changes with motion
 AC is characterized by its frequency (f) measured in hertz (Hz) (cycles
per second)
 The AC output of a generator is frequently in the form of a sine wave 
thus the voltage V(t) at any given time (t) is related to the maximum
voltage V_{max} by
 V(t) = V_{max} sin (2mft)

6

 A wire loop or coil exhibits inductance and responds to alternative
current in a frequency dependent fashion.
 AC produces a changing magnetic field  generates a voltage opposite in
polarity to the applied voltage

7

 In an inductance of 1 Henry (H) a voltage of 1 volt is induced by a
current changing at the rate of 1 Amp/second  this property is called reactance
 Reactance like resistance provides an impediment to the flow of current,
but unlike resistance is dependent on the frequency of the current

8

 A capacitor is a device with 2 conductors separated by an insulator
 If a DC current is applied to a capacitor a transient current flows but
stops when the potential difference between the conductors equals the
potential of the source
 If the source is removed the charge remains and can be release as
current
 The capacitance measured in Farads (F) is equal to the amount of charge
on either electrode in Coulombs divided by the potential difference
between the electrodes in volts  1 Farad = 1 coulomb/volt
 DC current will not flow “through” a capacitor  AC current will and the
higher the frequency the better the conduction

9

 In a circuit that contains both inductance and capacitance, one cancels
the other out
 The combined effect of resistance, inductive reactance and capacitive
reactance is referred to as impedance (Z) of the circuit
 Impedance is not the sum of resistance and reactance
 z=(R^{2}+(X_{l}X_{c})^{2})^{˝} (X_{l} = inductive reactance,
X_{c} = capacitive reactance)

10

 Cells are relatively poor conductors
 Blood is a suspension of cells in plasma which is a relatively good
conductor
 Previously it was known that the cellular fraction of blood could be
estimated from the conductance of blood
 As the ratio of cells to plasma increases the conductance of blood
decreases

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12

 This is similar to impedance, except that you use an AC current across
the electrodes of a coulter cell
 When the frequency used is in the radio frequency range (RF) the
parameter measured is known as electrical opacity
 This reflects the AC impedance of cells and is dependent on cellular
structure and less on size

13

 Electrical properties of cells and fluids
 Impedance: inductive reactance and capacitive reactance
 Coulter principle

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16

 Pulses collected in flow cytometers are analog events detected by analog
devices
 These pulses have a duration of no more than a few microseconds
 If you can’t digitize this pulse in that time you have to deal with a
combination of analog and digital pulse processing
 Until recently it took several microseconds to digitize a pulse so this
was not fast enough for high speed collection
 New systems which have all digital electronics can digitize the pulse
directly at rates of several megahertz allowing all digital computation

17

 Since the analog pulse is very short (microseconds typically) the only
way to retain this pulse is to charge a capacitor
 The capacitor serves as a storage device for a signal
 For peak detection, the capacitor is charged from a circuit that allows
only a build up of signal (a diode achieves this) and the final or peak
signal represents the maximum signal obtained

18

 In an integrator the charge on the capacitor represents the signal
integral between the reset and the hold signals
 A pulse width is collected by charging a capacitor from the output of a
linear ramp generator which starts at a preset time and ends when a
signal reached a predetermined minimum – the voltage stored in the
capacitor is proportional to the duration (time length) of the pulse

19

 The purpose of a trigger signal is to be sure that the measurement made
is made on an appropriate signal not noise or an unwanted signal
 Frequently we use larger rather than smaller signal – example include
light scatter or fluorescence
 The key component is called the comparator circuit which is designed
around an analog and a digital input.
 The circuit is designed to have a constant voltage (set by the operator)
and a signal from the sample – by comparing the preset signal with the
sample signal, a cell is collected if it meets the criterion (digital
signal is 1, or rejected (digital signal is 0).
 Noise of no cells passing through the observation point means that the
comparator circuit output is a logical  so no signal is collected or
passed onto the rest of the detection circuit (this means the computer
does not have to waste valuable time)

20

 Of course it takes time to set and reset the comparator circuit so this
added to the time required for a complete measurement cycle
 The length of time it takes to complete a full cycle of analysis will
determine the analysis rate

21

 Coincidence occurs if a second cells arrives before the circuit has been
reset – this could mean both cells are aborted, however with more
sophisticated electronics, all the signals can be collected in “pipeline” which can be interrogated
to resolve the conflict without losing either signal

22

 Since the analog signal only lasts for a few microseconds at maximum, it
must be converted to a digital pulse for longer term processing
 This is achieved by an ADC – which has an analog input but a digital
output
 The circuit divides the signal into a preset number of channels based on
the number of bits collected
 8 bits will have 256 channels
 10 bits will have 1024 channels
 The higher the number of bits the more complex the computation and
higher cost

23

 ADCs generate a comparison voltage using a digital to analog (DAC)
converter using a process of successive approximation
 Essentially this process converts each bit and compares it with the
original until all the bits are converted.
 Because of design specifications the lower the number of bits the higher
the inaccuracy
 Thus to increase accuracy it is useful (and more costly) to collect more
bits and throw the least significant bits (LSBs) away.
 Thus an ADC with 12 bits might only have the bottom 8 bits used

24

 Electrical properties of cells and measurement systems
 Beam Geometry
 Pulses and their characteristics
 Coincidence detection
 ADC
 WEB http://www.cyto.purdue.edu
