SCYM (ASCP) QUESTIONS WITH CORRECT SOLUTIONS|
ALREADY GRADED A| ACE YOUR GRADES.
Hydrodynamic Focusing - --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 - --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 - --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 - --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) - --
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 - --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 - --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 - --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 - --Answers----wavelength above 650nM
Shortpass Filter - --Answers----wavelength below 488nM
,dichroics mirrors - --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 - --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 - --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 - --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 - --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 - --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 - --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 - --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? - --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 - --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
ALREADY GRADED A| ACE YOUR GRADES.
Hydrodynamic Focusing - --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 - --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 - --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 - --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) - --
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 - --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 - --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 - --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 - --Answers----wavelength above 650nM
Shortpass Filter - --Answers----wavelength below 488nM
,dichroics mirrors - --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 - --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 - --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 - --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 - --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 - --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 - --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 - --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? - --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 - --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
