SCYM ASCP CORRECT EXAMS QUESTIONS AND
ANSWERS SURE A+
✔✔argon laser - ✔✔Air-cooled argon-ion laser producing blue light at 488 nm. This
wavelength is convenient for the excitation of fluorescein, the first immunofluorescent
label to be used. Other air-cooled lasers in general use include He-Ne (633 nm) and
He-CD (325 nm).
✔✔solid state lasers - ✔✔Solid state lasers producing light at 355, 405, 488, 530, 594,
635 and 780 nm are available. Most solid state lasers produce between 10 and 25 mW.
There is at least one diode laser giving 200 mW at 488 nm.
✔✔lenses - ✔✔As the lasers interact with particles and cells at the observation point or
the interrogation point, scattered and fluorescence light is generated. In order to
measure this light, the cytometer needs to collect as much of it as possible.
✔✔What is the job of the lenses? - ✔✔The optical collection system of a cytometer
must accomplish two goals. First, it must gather as much light as possible from the
interrogation point. Second, it must collimate that light so that all rays propagate parallel
to each other and can travel through the collection path without diverging.
✔✔Dichroic Filters - ✔✔Dichroic filters (sometimes called beam splitters) are used in
the flow cytometer at an angle often of 45°. Short wavelength pass (SWP) filters
transmit light below a given wavelength and reflect light of longer wavelengths. Long
wavelength pass (LWP) filters work in the reverse fashion. Their important parameters
are the wavelength for 50% transmission (the cut off for LWP or the cut-on wavelength
for SWP), the peak transmission and the slope at the cut-on or cut-off wavelength. Their
properties depend on the angle at which they are used.
✔✔Optical Pathyway with fibers - ✔✔Optical fibers are used to deliver lasers to the
interrogation point on some cytometers. This strategy also provides a space-saving
benefit in terms of where the lasers can be positions in the instrument. However, a
downside to this approach is that there can be significant power loss between the laser
,output and the interrogation point as laser light travels through the fiber. Additionally,
fibers are not compatible with higher energy light, especially UV wavelengths, which can
degrade the material of the fiber over time and require frequent replacement.
✔✔Optical Pathyway with Lens and Fibers using optical gel - ✔✔Some cytometers use
the lens and the fibers, which are directly coupled using an optical gel which may
minimize light loss due to refraction. As light passes through different types of mediums
(water, quartz, and air), it bends at the media interfaces. The degree to which this
occurs depends on the difference in refractive index between the two mediums: the
greater the difference, the more refraction occurs. By coupling the lens, which is
typically glass or quartz, to material with a similar refractive index, like gel, there may be
less loss as light transitions between the mediums. The downside of gels is that they
can crack and uncouple the lens from the fibers, which will prevent most collected light
from entering the fibers and require a service engineer to repair.
✔✔Photon are emitted when? - ✔✔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 - ✔✔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 - ✔✔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) - ✔✔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 - ✔✔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 - ✔✔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 - ✔✔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 - ✔✔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 - ✔✔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) - ✔✔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
ANSWERS SURE A+
✔✔argon laser - ✔✔Air-cooled argon-ion laser producing blue light at 488 nm. This
wavelength is convenient for the excitation of fluorescein, the first immunofluorescent
label to be used. Other air-cooled lasers in general use include He-Ne (633 nm) and
He-CD (325 nm).
✔✔solid state lasers - ✔✔Solid state lasers producing light at 355, 405, 488, 530, 594,
635 and 780 nm are available. Most solid state lasers produce between 10 and 25 mW.
There is at least one diode laser giving 200 mW at 488 nm.
✔✔lenses - ✔✔As the lasers interact with particles and cells at the observation point or
the interrogation point, scattered and fluorescence light is generated. In order to
measure this light, the cytometer needs to collect as much of it as possible.
✔✔What is the job of the lenses? - ✔✔The optical collection system of a cytometer
must accomplish two goals. First, it must gather as much light as possible from the
interrogation point. Second, it must collimate that light so that all rays propagate parallel
to each other and can travel through the collection path without diverging.
✔✔Dichroic Filters - ✔✔Dichroic filters (sometimes called beam splitters) are used in
the flow cytometer at an angle often of 45°. Short wavelength pass (SWP) filters
transmit light below a given wavelength and reflect light of longer wavelengths. Long
wavelength pass (LWP) filters work in the reverse fashion. Their important parameters
are the wavelength for 50% transmission (the cut off for LWP or the cut-on wavelength
for SWP), the peak transmission and the slope at the cut-on or cut-off wavelength. Their
properties depend on the angle at which they are used.
✔✔Optical Pathyway with fibers - ✔✔Optical fibers are used to deliver lasers to the
interrogation point on some cytometers. This strategy also provides a space-saving
benefit in terms of where the lasers can be positions in the instrument. However, a
downside to this approach is that there can be significant power loss between the laser
,output and the interrogation point as laser light travels through the fiber. Additionally,
fibers are not compatible with higher energy light, especially UV wavelengths, which can
degrade the material of the fiber over time and require frequent replacement.
✔✔Optical Pathyway with Lens and Fibers using optical gel - ✔✔Some cytometers use
the lens and the fibers, which are directly coupled using an optical gel which may
minimize light loss due to refraction. As light passes through different types of mediums
(water, quartz, and air), it bends at the media interfaces. The degree to which this
occurs depends on the difference in refractive index between the two mediums: the
greater the difference, the more refraction occurs. By coupling the lens, which is
typically glass or quartz, to material with a similar refractive index, like gel, there may be
less loss as light transitions between the mediums. The downside of gels is that they
can crack and uncouple the lens from the fibers, which will prevent most collected light
from entering the fibers and require a service engineer to repair.
✔✔Photon are emitted when? - ✔✔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 - ✔✔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 - ✔✔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) - ✔✔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 - ✔✔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 - ✔✔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 - ✔✔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 - ✔✔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 - ✔✔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) - ✔✔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
