SCYM (ASCP) UPDATED ACTUAL Exam
Questions and CORRECT Answers
Hydrodynamic Focusing - CORRECT ANSWER - Most modern flow cytometers tightly
position the sample for optical analysis via hydrodynamic focusing. Here, a carrier fluid called
the sheath fluid is used to position the sample of cells into a single file for optical interrogation.
Hydordynamic focusing and sheath fluids - CORRECT ANSWER - The central stream
(sample stream) is focused and surrounded by the secondary slower stream (sheath fluid). The
shape and size of the flow cell is crucial to hydrodynamic focusing, and traditionally the cell is
nozzle shaped. ... In a flow cytometer, the sheath fluid pressure is constant while the sample fluid
is adjusted
Sample Pressure and the Sheath Pressure - CORRECT ANSWER - The difference
between the sample pressure and the sheath pressure is the differential pressure. This controls the
width of the core stream and the total number of cells passing the laser intercept.
differential pressure based flow cytometers - CORRECT ANSWER - Differential pressure
based flow cytometers currently dominate the market. These systems have two pressure
regulators. The first is at a constant pressure that sets how fast the fluids runs at. The second is
regulated by the investigator (like on this LSR-II control panel).
Generation of differential pressure (syringe pump, pressure based) - CORRECT
ANSWER - Low differential pressure allows the cells to move past the interrogation point
one at a time. .... One kind involves generating pressure using a pump and regulator system ...
Differential pressure based fluidic system. ... peristaltic and/or syringe pumps to deliver the
sample into the instrument.
Characterization of syringe-pump-driven induced pressure - CORRECT ANSWER - In
syringe-pump-driven microfluidic systems, pressure fluctuations are observed in an elastic
microchannel. The syringe pump is driven by an electrical stepper motor, from which
mechanical oscillations are expected to generate flow-rate fluctuations and in turn leads to the
pressure fluctuations in the channel flow.
,Optical Filters - CORRECT ANSWER - Filters are pieces of glass coated on both sides
that allow light of a certain collection, or band, of wavelengths to pass through while absorbing
or interfering with photons of other wavelengths. These come in bandpass, longpass, and
shortpass flavors
Band Pass Optical Filter - CORRECT ANSWER - A filter that allows light between a set
wavelength to pass through and reflects light above and below the set wavelength. For example,
a bandpass filter with a wavelength of 550/40nm would allow light between 530nm and 570nm
to pass through, but reflect light below 530nm and above 570nm.
Longpass Filter - CORRECT ANSWER - wavelength above 650nM
Shortpass Filter - CORRECT ANSWER - wavelength below 488nM
dichroics mirrors - CORRECT ANSWER - Dichroic mirrors can block light by phased
reflection allowing certain light to pass through and interfering with other wavelengths. For
example, a 500LP dichroic mirror would transmit light above 500 nm and reflect the light below
500 nm in a different direction. A 525SP dichroic mirror would transmit all light below 525 nm
and reflect all light above 525 nm in a different direction. These dichroic mirrors are critical in
the directing and capturing of light by the detectors.
neutral density filter - CORRECT ANSWER - filter that reduces or modifies the intensity
of all wavelengths, or colors, of light equally, giving no changes in hue of color rendition
polarization filter - CORRECT ANSWER - Polarization of scatter and fluorescence
signals in flow cytometry. ... depending on the light source(s), the optical layout, and the types of
mirrors and filters used.
light source - CORRECT ANSWER - The light source can be a laser, an arc lamp or even
an LED. Today, the majority of instruments use a laser. Lasers illuminate the stream with
coherent, focused light of specific wavelength (energy) and power. This illumination facilitates
the generation of fluorescence signals from cells labeled with fluorophores and light scatter
signals from redirected laser light.
, arc lamp laser - CORRECT ANSWER - Arc lamps need optical filters to select the
appropriate wavelength. They do not give the sensitivity needed to observe weak fluorescence
but offer a cheaper alternative for observing strong fluorescences, for example, in DNA analysis.
argon laser - CORRECT ANSWER - 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 - CORRECT ANSWER - 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 - CORRECT ANSWER - 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? - CORRECT ANSWER - 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 - CORRECT ANSWER - 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 - CORRECT ANSWER - 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
Questions and CORRECT Answers
Hydrodynamic Focusing - CORRECT ANSWER - Most modern flow cytometers tightly
position the sample for optical analysis via hydrodynamic focusing. Here, a carrier fluid called
the sheath fluid is used to position the sample of cells into a single file for optical interrogation.
Hydordynamic focusing and sheath fluids - CORRECT ANSWER - The central stream
(sample stream) is focused and surrounded by the secondary slower stream (sheath fluid). The
shape and size of the flow cell is crucial to hydrodynamic focusing, and traditionally the cell is
nozzle shaped. ... In a flow cytometer, the sheath fluid pressure is constant while the sample fluid
is adjusted
Sample Pressure and the Sheath Pressure - CORRECT ANSWER - The difference
between the sample pressure and the sheath pressure is the differential pressure. This controls the
width of the core stream and the total number of cells passing the laser intercept.
differential pressure based flow cytometers - CORRECT ANSWER - Differential pressure
based flow cytometers currently dominate the market. These systems have two pressure
regulators. The first is at a constant pressure that sets how fast the fluids runs at. The second is
regulated by the investigator (like on this LSR-II control panel).
Generation of differential pressure (syringe pump, pressure based) - CORRECT
ANSWER - Low differential pressure allows the cells to move past the interrogation point
one at a time. .... One kind involves generating pressure using a pump and regulator system ...
Differential pressure based fluidic system. ... peristaltic and/or syringe pumps to deliver the
sample into the instrument.
Characterization of syringe-pump-driven induced pressure - CORRECT ANSWER - In
syringe-pump-driven microfluidic systems, pressure fluctuations are observed in an elastic
microchannel. The syringe pump is driven by an electrical stepper motor, from which
mechanical oscillations are expected to generate flow-rate fluctuations and in turn leads to the
pressure fluctuations in the channel flow.
,Optical Filters - CORRECT ANSWER - Filters are pieces of glass coated on both sides
that allow light of a certain collection, or band, of wavelengths to pass through while absorbing
or interfering with photons of other wavelengths. These come in bandpass, longpass, and
shortpass flavors
Band Pass Optical Filter - CORRECT ANSWER - A filter that allows light between a set
wavelength to pass through and reflects light above and below the set wavelength. For example,
a bandpass filter with a wavelength of 550/40nm would allow light between 530nm and 570nm
to pass through, but reflect light below 530nm and above 570nm.
Longpass Filter - CORRECT ANSWER - wavelength above 650nM
Shortpass Filter - CORRECT ANSWER - wavelength below 488nM
dichroics mirrors - CORRECT ANSWER - Dichroic mirrors can block light by phased
reflection allowing certain light to pass through and interfering with other wavelengths. For
example, a 500LP dichroic mirror would transmit light above 500 nm and reflect the light below
500 nm in a different direction. A 525SP dichroic mirror would transmit all light below 525 nm
and reflect all light above 525 nm in a different direction. These dichroic mirrors are critical in
the directing and capturing of light by the detectors.
neutral density filter - CORRECT ANSWER - filter that reduces or modifies the intensity
of all wavelengths, or colors, of light equally, giving no changes in hue of color rendition
polarization filter - CORRECT ANSWER - Polarization of scatter and fluorescence
signals in flow cytometry. ... depending on the light source(s), the optical layout, and the types of
mirrors and filters used.
light source - CORRECT ANSWER - The light source can be a laser, an arc lamp or even
an LED. Today, the majority of instruments use a laser. Lasers illuminate the stream with
coherent, focused light of specific wavelength (energy) and power. This illumination facilitates
the generation of fluorescence signals from cells labeled with fluorophores and light scatter
signals from redirected laser light.
, arc lamp laser - CORRECT ANSWER - Arc lamps need optical filters to select the
appropriate wavelength. They do not give the sensitivity needed to observe weak fluorescence
but offer a cheaper alternative for observing strong fluorescences, for example, in DNA analysis.
argon laser - CORRECT ANSWER - 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 - CORRECT ANSWER - 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 - CORRECT ANSWER - 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? - CORRECT ANSWER - 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 - CORRECT ANSWER - 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 - CORRECT ANSWER - 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
