SCYM (ASCP) (Latest )
Questions with Revised Rationalized
Answers, (A+ Guarantee)
Hydrodynamic Focusing - CORRECT ANSWERS 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 ANSWERS 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 ANSWERS 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 ANSWERS 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 ANSWERS 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 ANSWERS 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 ANSWERS 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 ANSWERS 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 ANSWERS wavelength above 650nM
Shortpass Filter - CORRECT ANSWERS wavelength below 488nM
dichroics mirrors - CORRECT ANSWERS 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 ANSWERS 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 ANSWERS 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 ANSWERS 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 ANSWERS 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 ANSWERS 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 ANSWERS 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 ANSWERS 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 ANSWERS 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 ANSWERS 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 ANSWERS 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
Questions with Revised Rationalized
Answers, (A+ Guarantee)
Hydrodynamic Focusing - CORRECT ANSWERS 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 ANSWERS 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 ANSWERS 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 ANSWERS 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 ANSWERS 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 ANSWERS 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 ANSWERS 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 ANSWERS 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 ANSWERS wavelength above 650nM
Shortpass Filter - CORRECT ANSWERS wavelength below 488nM
dichroics mirrors - CORRECT ANSWERS 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 ANSWERS 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 ANSWERS 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 ANSWERS 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 ANSWERS 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 ANSWERS 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 ANSWERS 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 ANSWERS 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 ANSWERS 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 ANSWERS 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 ANSWERS 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
