Photomultiplier tubes (PMT’s)The PMTs in an Elite. 3 PMTs are shown, the other 2 have been removed to show their positions. A diode detector is used for forward scatter and a PMT for side scatter.
The Bio-Rad Bryte cytometer uses PMTs for forward and wide angle light scatter as well as fluorescence
Review of Electronics• Based on Ohm’s Law, the flow of a current of 1 Amp through a
material of resistance of R ohms () produces a drop in electrical potential or a voltage difference of V volts across the resistance such that V=IR
• 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
V(t) = Vmax sin (2ft)• 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• In an inductance of 1 Henry (H) on a voltage of 1 volt is induced by a
current changing at the rate of 1 Amp/second - this property is called reactance
Review of Electronics• Reactance like resistance provides an impediment to the flow of current, but
unlike resistance is dependent on the frequency of the current
• 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
• 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
• 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=(R2+(Xl-Xc)2)½ (Xl = inductive reactance, Xc = capacitive reactance)
The Coulter Principle•2 chambers filled with a conductive saline fluid are separated by a small orifice (100m or less)
•Thus, most of the resistance or impedance is now in the orifice.
•By connecting a constant DC current between 2 electrodes (one in each chamber), the impedance remains constant. If a cell passes through the orifice, it displaces an equivalent volume of saline and so increases the impedance.
• The problem with compensation is that it needs to be performed on linear data, not logarithmic data. Thus, either the entire electronics must be built in linear electronics, which requires at least 16 bit A-D converters, or a supplementary system must be inserted between the preamp and the display.
• We need the dynamic range for immunologic type markers, but we can’t calculate the compensation easily using log amps - certainly not without complex math.
• Flow cytometers amplify signals to values ranging between 0-10V before performing a digital conversion.
• Assuming this, with 4 decades and a maximum signal of 10 V we have:
Why use linear amps?• The problem with compensation is that it needs to be performed on
linear data, not logarithmic data. Thus, either the entire electronics must be built in linear electronics, which requires at least 16 bit A-D converters, or a supplementary system must be inserted between the preamp and the display.
• We need the dynamic range for immunologic type markers, but we can’t calculate the compensation easily using log amps - certainly not without complex math.
• Flow cytometers amplify signals to values ranging between 0-10V before performing a digital conversion.
• Assuming this, with 4 decades and a maximum signal of 10 V we have:
Compare the data plotted on a linear scale (above) and a 4 decade log scale (below). The date are identical, except for the scale of the x axis. Note the data compacted at the lower end of the the linear scale are expanded in the log scale.
Ratio circuits• Ratio circuits are analog circuits which produce an output
proportional to the ratio of the 2 input signals.• They are usually made from modules called analog multipliers. • Examples are calculation of surface density or antigenic receptor
sites by dividing the number of bound molecules by the cell surface area.
• e.g. Could use 2/3 power of volume to obtain surface area - but few cytometers make this parameter so can use the square of the cell diameter of scatter instead to approximate.
• pH can also be measured using ratio circuits• Calcium ratio (using Indo-1 we can ratio the long and short
We compare histograms to determine if there is a difference between them. If there is, we can make a statement of difference based on statistics. Since we are usually measuring biological phenomena, our conclusion will be related to the biological difference perhaps.
The question here might be:Is there a difference between these two data sets?
Basic Histogram OperationsGating or Region of Interest (ROI) selection• 1. A gate is a region of interest• Gates can be applied to any histogram• Gates or ROI can also be applied to mult-
parameter plots• Gates are applied to select out cells with a desired
characteristic.• Gates can be additive – this means the results are
Any number of gates can be applied to a histogram. Gates can be inclusive, exclusive or “either or”.
For example, you could select all cells that satisfy gate M6, excluding gate M3 – (M6-M3) would give you the same result as adding gates M1 and M4 (M1+M4).
Note: TIP – Tube identifier Parameter – allows the display of data points for multiple samplesTIG: Time Interval Gating – allow the display of multiple samples over time.
A: Color of dots gives an indication of the identify of subpopulations. e.g. in the above plot the green dots are high density and the mauve are low density areas (FS is Forward Scatter and 90ls is Ninety Degree light scatter or orthogonal light scatter.)
B: The color of lines in each contour provides an indication of the number of events in that level of the plot. e.g. in the above plot the green are high density and the mauve are low density with proper contour lines. The data sets of A and B are identical.
In this display, each population has been identified by a different color
Here, the multiple colors are in the lymphocyte gate. All of the se cells are identified on the left plot. When applied to the scatter plot, there is a region with multiple colors.
Figure 9.3.4 This figure shows an example of stimulation of neutrophils by PMA (50 nm/ml). On the left the unstimulated cells show no increase in DCF fluorescence . On the right, activatedcells increase the green DCF fluorescence at least 10 times the initial fluorescence.
Multiple histograms displayed in a combination format
This is the “Phenogram” format which displays all of the possible binary combinations of a set of fluorochromes – in this case there are 3 colors (n) so there are 2n =8 combinations.
Robinson, J. Paul, Durack, Gary & Kelley, Stephen: "An innovation in flow cytometry data collection & analysis producing a correlated multiple sample analysis in a single file". Cytometry 12:82-90,1991.
J. Paul Robinson, K.Ragheb, G. Lawler,S.Kelley, & G. Durack: Rapid Multivariate Analysis and Display of cross-reacting antibodies on Human Leukocytes. Cytometry 13:75-82,1992
The first distribution demonstrates forward gating. Cell fluorescence is gated based on their scatter characteristics. Below fluorescence is used to “backgate” the fluorescence signal onto the scatter dotplot
This example shows how complex the analysis can become for a large set of data with many variables. Represented are the number of dual plots that would have to be displayed to represent the possible number of combinations. It should be noted of course that you cannot display 3 or more dimensions in 2 dimensional space!!
• There are 2 primary types of detectors used in flow cytometers
• These have different sensitivities and applications
• We collect data in log space mostly because we need a large dynamic range (this is difficult to do in linear space because of limits and costs of hardware)
• Data acquisition and analysis
• Types of data formats and presentation formats
• Data analysis techniques such as gating, forward and back gating
