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SCYM (ASCP) Exam Questions With
Correct Answers.
Hydrodynamic Focusing - 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 - 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 - 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 - 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) - 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 - 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 - 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 - 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
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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 - answer✔✔wavelength above 650nM
Shortpass Filter - answer✔✔wavelength below 488nM
dichroics mirrors - 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 - 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 - 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 - 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 - 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 - 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 - 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 - 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? - 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.
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Dichroic Filters - 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 - 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
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 - answer✔✔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? - answer✔✔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 - answer✔✔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 - answer✔✔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.
