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
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).
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.
Generation of differential pressure (syringe pump, pressure based) - ANSWER Low differ-
ential 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 in-
strument.
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
1
,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 fla-
vors
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 band-
pass 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 direct-
ing and capturing of light by the detectors.
Neutral density filter - ANSWER filter that reduces or modifies the intensity of all wave-
lengths, or colors, of light equally, giving no changes in hue of color rendition
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 re-
directed laser light.
2
, Polarization filter - ANSWER Polarization of scatter and fluorescence signals in flow cy-
tometry. ... Depending on the light source(s), the optical layout, and the types of mirrors and
filters used.
Arc lamp laser - ANSWER Arc lamps need optical filters to select the appropriate wave-
length. 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 interroga-
tion 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 transmis-
sion and the slope at the cut-on or cut-off wavelength. Their properties depend on the angle
at which they are used.
3
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
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).
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.
Generation of differential pressure (syringe pump, pressure based) - ANSWER Low differ-
ential 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 in-
strument.
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
1
,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 fla-
vors
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 band-
pass 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 direct-
ing and capturing of light by the detectors.
Neutral density filter - ANSWER filter that reduces or modifies the intensity of all wave-
lengths, or colors, of light equally, giving no changes in hue of color rendition
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 re-
directed laser light.
2
, Polarization filter - ANSWER Polarization of scatter and fluorescence signals in flow cy-
tometry. ... Depending on the light source(s), the optical layout, and the types of mirrors and
filters used.
Arc lamp laser - ANSWER Arc lamps need optical filters to select the appropriate wave-
length. 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 interroga-
tion 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 transmis-
sion and the slope at the cut-on or cut-off wavelength. Their properties depend on the angle
at which they are used.
3
