Photon are emitted when? Correct Answer The photons
that are emitted when the laser hits the cell as it passes
through the interrogation point are detected by the
photomultiplier (PMD) or photodiode (PD). These photons
can come from light being scattered by the cell or by
fluorescence emission of fluorophores associated with the
cell. Once in the detector, the photons are converted to
electrons, and the signal is multiplied proportionately. The
signal exits the detector as an electric current (also called
photocurrent), and this is the point where the signal enters
the electronics system. A simplification of the path of the
photons as they travel through the electronics system
amplifier Correct Answer The electrons, that were
converted from photons, exit the detector the electric
current travels to the amplifier (amp) where it is amplified
and converted to a voltage pulse. This pulse is then
converted to a digital number via the analog-to-digital
converter (ADC). The digital number is transferred to the
computer and becomes the data that you will analyze.
Voltage Pulse Correct Answer The voltage pulse (also
called a signal pulse) is created when the cell reaches the
interrogation point and crosses the path of the laser. The
number of photons emitted is proportional to the
,fluorescent light generated after the cell passes through
the laser. These photons are converted to electrons in the
detector, which is why these are called voltage pulses.
Photomultiplier Tube (PMT) Correct Answer The size of
the voltage pulse also depends on the PMT voltage or pre-
amplifier gain and the amplification factor (also called the
amplification gain). Signals can be amplified by applying a
voltage to the PMTs, thus creating a greater electrical
current, or by increasing the amplification gain. Amplifier
settings can be linear or logarithmic (Lin or Log). Log
amplification is often used to separate negative from dim
positive signals, whereas Lin amplification is often used to
amplify scatter and fluorescent parameters.
Noise Correct Answer Noise will come from small particles
and debris in our samples because, despite what we might
think, cytometers are stupid and they will measure
anything that goes through them
Anatomy of the voltage pulse Correct Answer The voltage
pulse has three attributes: The pulse height is the
maximum peak of light collected. The full pulse width is
the time from the start of the pulse to the end of the pulse.
The pulse area is the integral of the height over width
(time). Each of these three measurements provides
different information about the cell and is used to answer a
specific experimental question.
Voltage Pulse Scatter Correct Answer Forward scatter
light (FSC), also called low-angle light scatter, is the
,amount of light that is scattered in the forward direction as
a laser light strikes the cell. The magnitude of forward
scatter is roughly proportional to the size of the cell, which
means the voltage pulse of the forward scatter will reflect
the relative size of the cell. The amount of light scattered
in the forward direction is less intense with small cells than
the light scattered by larger cells and the resulting voltage
pulse will also be smaller.
Side scatter light (SSC) or light that is scattered at larger
angles, is caused by granularity and structural complexity
inside the cell or on the cell surface. Increased cell
complexity results in more light scatter and larger voltage
pulses.
Interpreting the voltage pulse of fluorescence emission
Correct Answer As with FSC and SSC, the fluorescent
light emitted by the cell as it crosses the laser beam will
result in a voltage pulse. The amount of fluorescence
emitted by the fluorophores associated with the cell is
dependent on a couple of factors. The first factor is the
number of fluorophores associated with the cell. For
example, the cell could have low expression of the surface
protein that you are detecting so that there are very few
fluorescent antibody conjugates bound to the surface.
Another example could be that a dead cell dye has bound
in high quantities because the cell is no longer alive. Both
of these examples would have different levels of
fluorescence associated with the cell.
, florescence emission Correct Answer Not all fluorophores
are created equal when it comes to how much
fluorescence is emitted. For example, some fluorophores
have structures that lend themselves to much brighter
fluorescence, like phycoerythrin (PE), which is a large
protein from red algae. Other fluorophores are very small
organic ring structures like fluorescein, which is
significantly less bright than PE.
The fate of the voltage pulse (or binning) Correct Answer
Once the voltage pulse has been created it will go through
an amplification process and a digitization process. The
exact order of these processes depends on the flow
cytometer you are using. Regardless of the flow
cytometer, however, the final fate of the voltage pulse data
will be the same. After amplification and digitization, the
voltage pulse data will go through a process called
binning. In this process the voltage pulse data (height, full
pulse width, and area) for each cell is assigned to a bin,
depending on its value. Every parameter detected for that
specific cell (i.e., FSC, SSC, fluorescence of each
channel) has a voltage pulse that will be assigned to a bin
for that parameter.
Population Distribution Correct Answer As the data for all
of the cells are collected and binned the distribution of the
data begins resemble a population distribution curve or a
histogram. The number of bins depends on the sampling
rate and resolution of the ADC.
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