SCYM ASCP QUESTIONS AND ANSWERS SURE A+
✔✔dichroics mirrors - ✔✔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 - ✔✔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 - ✔✔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 - ✔✔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 - ✔✔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 - ✔✔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
✔✔dichroics mirrors - ✔✔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 - ✔✔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 - ✔✔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 - ✔✔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 - ✔✔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 - ✔✔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
