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SCYM (ASCP) QUESTIONS AND ANSWERS WITH SOLUTIONS 2024
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SCYM (ASCP) QUESTIONS AND ANSWERS WITH SOLUTIONS 2024
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SCYM (ASCP) QUESTIONS ANDANSWERS WITH SOLUTIONS 2024
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 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.
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.
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