Bi9393 Analytical cytometry
Lesson 2
Department of Cytokinetics
Institute of Biophysics, CAS,
v.v.i.
Kralopolska 135
612 65 Brno
E-mail: ksoucek@ibp.cz
tel.: 541 517 166
Karel Souček, Ph.D.
K. Souček Bi9393 Analytical cytometry
a, Representative flow cytometry plots (left) and
percentage of degranulating NK cells isolated from the
blood of ten healthy donors (right), as measured by
surface CD107a, in response to uninfected and ZIKVinfected
JEG-3 cells (8 h coculture, E:T ratio 1:3). b,
NK cell-specific killing of uninfected and ZIKV-infected
JEG-3 cells. c, ER stress, as assessed by XBP1 splicing
(left) and increases in BIP (middle) and CHOP (right)
mRNA, in JEG-3 cells that were uninfected or infected
with ZIKV, HSV-2 or human cytomegalovirus (HCMV) for
1–2 days or treated with tunicamycin (Tu) for 1 day.
Indicated samples were pretreated with the ER stress
inhibitor salubrinal (n = 3 samples). mRNA levels, as
assayed by quantitative PCR with reverse transcription
(RT–qPCR), were normalized to ACTB. d, Effect of
salubrinal pretreatment of target cells on NK cell killing
of ZIKV-infected (top) and tunicamycin-treated (bottom)
JEG-3 cells (n = 6 samples). e, Effect of NKR-blocking
antibodies (Ab) on NK cell killing of uninfected or ZIKVinfected
JEG-3 cells (n = 3–7 samples). Ctrl, control. f,
Specific killing of the classical NK cell target
722.221 cells, or of uninfected or ZIKV-infected JEG-
3 cells by human NK cell line YT cells knocked out
for NCR1 or NCR3 or treated with control single-guide
RNAs (n = 3–6 samples). g, Representative flow
cytometry histogram (left) and mean fluorescence
intensity (MFI) of NKR–Ig fusion protein (NKp46–Ig and
NKG2D–Ig) binding to uninfected or ZIKV-infected JEG-
3 cells (right) (n = 3 samples). b,d–f, Specific killing
assessed by 8 h 51Cr release assay using an E:T ratio of
10:1 unless otherwise indicated. Data are mean ± s.e.m.
of at least three independent experiments or technical
replicates. Statistics were performed using two-tailed,
nonparametric, unpaired t-test (a,b), one-way analysis
of variance (ANOVA) (c), two-way ANOVA (e–g) or area
under the curve followed by one-way ANOVA (d).
*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
-CD56 is a single transmembrane glycoprotein also known
as N-CAM (Neural Cell Adhesion Molecule), Leu-19, or
NKH1. It is a member of the Ig superfamily. The 140 kD
isoform is expressed on NK cells and NK-T cells. CD56 is
also expressed in the brain (cerebellum and cortex) and at
neuromuscular junctions.
-lysosomal-associated membrane protein-1 (LAMP-1 or
CD107a) has been described as a marker of CD8+ T-cell
degranulation following stimulation.
Flow cytometry
For surface staining, cells were stained for 30 min on ice in the dark
with LIVE/DEAD-Violet stain (1:1,000) and then with primary
antibodies for 15–30 min in PBS and 2% FCS (followed by secondary
antibodies, when applicable, for 20 min). For protein–Ig staining, cells
were incubated with 50 µg ml–1 fusion protein for 1 h at 4 °C and then
stained with fluorescent-anti-human IgG for 1 h. Cells were fixed in 1%
paraformaldehyde (Affymetrix) for 10 min before flow cytometry. Flow
cytometry was assessed on gated live cells (Supplementary Fig. 1). For
intracellular staining, cells were fixed and permeabilized using the
CytoFix/CytoPerm kit. One of the treated samples was used for isotype
staining, and MFI of staining with the isotype control antibody was
subtracted from MFI of the specific antibody. Analysis was performed
on a FACSCanto II (BD). BD FACSDiva 8.0 (BD) software was used for
data collection, with analysis performed using FlowJo v.10.4.2
(TreeStar).
Reproducibility of results
Circ Res. 2015 Jan 2;116(1):116-26. doi: 10.1161/CIRCRESAHA.114.303819.
Reproducibility in science: improving the standard for basic and
preclinical research.
Begley CG1 , Ioannidis JP2 .
Nature. 2015 Feb 5;518(7537):27-9. doi: 10.1038/518027a.
Reproducibility: standardize antibodies used in research.
Bradbury A1 , Plückthun A2 .
Nature 533, 452-454 (26 May 2016)
doi:10.1038/533452a
“I don’t know any
case yet of a
retracted paper that
used synthetic
creation to illustrate
the results in that
paper,” says Rocha,
who is working with
the blog Retraction
Watch on this issue.
“But it’s just a matter
of time, I guess.”
CST Antibody Validation Principles
https://www.cellsignal.at/about-us/cst-
antibody-validation-principles
Available technology
https://www.bosterbio.com/protocol-and-troubleshooting/flow-cytometry-principle
new automatic cell cloning assay (ACCA) for
determination of clonogenic capacity of CSCs
single cell/well
up to 384 well plate
re-culture after sorting (2D, 3D)
analysis: CyQuant, ATP, xCelligence, microscopy
Principles of flow cytometry and
sorting
◼ Light
◼ Fluorescence
◼ Excitation sources, optical systems and
fluorescence detection methods
◼ Fluid systems
K. Souček Bi9393 Analytical cytometry
Concepts
Photometry:
◼ Light - electromagnetic radiation visible to the human eye (400-750
nm, most sensitive ~ 550 nm). Measurements below 400 nm (UV, IF)
are radiation detection (radiometry).
◼ The energy of radiation is expressed in joules
◼ Radiant flux is given as the value of energy over time in watts (1 watt
= 1 joule/second)
◼ photon - elementary particle. They are described by their wavelength,
frequency, energy and momentum. The lifetime of a photon is infinite
(yet they arise and disappear), they exist only in motion. It has zero
rest mass but non-zero energy, defined by the relation E = hν, where h
is Planck's constant and ν is the frequency. Because it has energy, it
is acted upon by gravity according to general relativity, and it
gravitationally acts on its surroundings. (http://cs.wikipedia.org/wiki/Foton)
◼ The photon energy is expressed as E=hn and E=hc/l [−frequency
(Hz), c - speed of light (3x108 m/s), −wavelength (nm), h-Planck's
constant (6.63 x 10-34 J/s)]
◼ The energy is higher at shorter wavelengths and lower at longer
wavelengths.
Electromagnetic Spectrum
Light scattering
◼ Matter scatters light of wavelengths it is unable to
absorb
◼ The visible spectrum is 350-850 nm therefore
small particles and molecules (< 1/10 ) tend to
scatter visible light
◼ For small particles, the so-called Rayleigh
scattering (scatter) has been described whose
intensity is ~ the same in all directions
◼ The scattering of larger particles is characterised
by Mie scattering. Its quantity is greater in the
direction in which the light falls on the irradiated
particle.
Shapiro p.106Shapiro p.106http://hyperphysics.phy-astr.gsu.edu/hbase/atmos/blusky.html
Rayleigh and Mie scattering
◼ Rayleigh scattering - molecules
and very small particles do not
absorb, but scatter light that has
a smaller wavelength than their
size (blue sky - air scatters
shorter wavelengths better)
◼ Mie scattering is characteristic
of particles larger than the
wavelength of light (white glow
around the sun's disk, fog light)
kj
kj
http://hyperphysics.phy-astr.gsu.edu/hbase/atmos/blusky.html
K. Souček Bi9393 Analytical cytometry
Bending and decay of light
Short wavelengths are "bent"
more than long wavelengths
kj
original from Purdue University Cytometry Laboratories; modified by R.F. Murphy
Fluorescence
George Gabriel Stokes (1819 - 1903)
English physicist and mathematician
based at the University of Cambridge
http://www.nndb.com/people/131/000097837/
1852 - described fluorescence
The name is derived from the English word
fluospar (fluorite, fluorspar = CaF mineral2 )
- for his observations he used a quinine
solution, sunlight as a light source, a dark
blue window glass as an excitation filter and
a glass of white wine as an emission filter
G. C. Stokes "On the Change of Refrangibility of
Light" Philosophical Transactions of the Royal
Society of London, 1852, vol. 142, p. 463.)
K. Souček Bi9393 Analytical cytometry
The principle of fluorescence
Fluorescence (belongs to photoluminescence radiation, which is caused either by
the effect of other incident radiation or by the effect of incident particles) is the result
of a three-phase phenomenon of some chemical substances - fluorochromes,
fluorescent dyes.
Stage 1: Excitation
- Radiation from an external source (e.g., laser) excites the
fluorophore.
- This creates an excited state (S1').
Stage 2: Excited-State Lifetime
- The excited state lasts briefly (1–10^-9 seconds).
- The fluorophore may undergo changes and interactions.
- It loses some energy, creating a relaxed excited state (S1).
- Some molecules don't return energy through fluorescence
emission.
- This affects the fluorescence quantum yield.
Stage 3: Fluorescence Emission
- The fluorophore emits a photon and returns to its ground state
(S0).
- The emitted photon's energy (hvEM) is lower and has a longer
wavelength than the excitation photon (hvEX).
- The difference is called the Stokes shift and is crucial for
fluorescence technique sensitivity.
Jablonski diagram
K. Souček Bi9393 Analytical cytometry
Fluorescence characteristics
• intensity - the number of photons passing in a given direction through a
unit area per unit time
• spectral composition - spectral density of photon flux per unit interval
of wavelengths or frequencies
• polarization - the direction of oscillation of the electric vector of an
electromagnetic wave
• the time of extinction - is determined by the internal lifetime of the
excited state from which the emission occurs; it is closely related to the
processes leading to the non-radiative deactivation of this state
• coherence properties - relationships between the phases of light waves
Fluorescence spectra
The fluorescence process is cyclic.
In addition to the fluorochrome irreversibly destroyed (photobleaching), it can be
repeatedly excited.
Excitation of a fluorophore at three different wavelengths (EX 1, EX
2, EX 3) does not change the emission profile but does produce
variations in fluorescenceemission intensity (EM 1, EM 2, EM 3) that
correspond to the amplitude of the excitation spectrum.
K. Souček Bi9393 Analytical cytometry
Fluorescent dyes
• Fluorescent dyes (fluorophores, fluorochromes) are chemical compounds that contain a reactive group in their molecule that is
able to react with nucleophilic groups (NH₂, OH, SH).
• In general, fluorophores are divided into intrinsic and extrinsic.
Internal fluorescence
• The internal fluorescence of cells is determined by the presence of internal fluorophores, which include proteins, reduced forms
of NADH and NADPH, vitamin A, cytochromes, peroxidase, hemoglobin, myoglobin, and chlorophyll.
• Proteins emit fluorescent radiation in the UV region of the spectrum. The main fluorophores in proteins are aromatic amino
acids (phenylalanine, tryptophan, tyrosine), whose absorption and emission bands lie between 240 and 300 nm.
• The other substances listed emit in the visible region of the spectrum (blue, yellow or red).
External fluorescence
• External fluorophores are used much more often than internal ones.
• They are added to the sample under study and are divided into fluorescent tags and fluorescent probes according to the type of
binding.
Fluorescent markers
• They are most commonly used to fluorescently label proteins to which they bind by covalent linkage.
• The best known fluorescent labels are FITC (fluorescein-5-isothiocyanate) and TRITC (tetramethylrhodamine-5-isothiocyanate,
tetramethylrhodamine-5-isothiocyanate).
Fluorescent probes
• external fluorophores, which bind to the structure by non-covalent bonding and often change their fluorescence properties in the
process. These fluorophores are used to study changes in protein conformation, membrane thickness, membrane potential, etc.
• A number of fluorescent probes (e.g., acridine orange, ethidium bromide, DAPI, etc.) are used to identify and visualize nucleic
acids.
• The best known and most widely used fluorescent probe for visualizing all nuclear DNA is DAPI. Chemically, it is 4',6Diamidino-2-phenylindole.
Its absorption maximum is at 345 nm, its maximum fluorescence is at 455 nm (blue fluorophore
• Another frequently used fluorophore is acridine orange. This is a fluorescent probe whose absorption and emission bands vary
according to the substrate to which the DNA/RNA is bound. Both of these are usually supplied in the form of chloride salts.
Fluorescence detection
Equipment for fluorescence
(1) excitation source
(2) fluorochrome
(3) wave filters for isolating emitted photons from excited photons
(4) detectors for registering emitted photons
Fluorescence instruments
- The spectrofluorometer measures the average properties of the sample volume in the
cuvette.
- fluorescence microscope describes fluorescence as a phenomenon in a spatial
coordinate system
- flow cytometer measures fluorescence in the flow stream, allowing detection and
quantification of subpopulations within a large sample
Fluorescent signal
- The spectrofluorometer is flexible, allowing you to measure
in a continuous excitation and emission spectrum
wavelengths
- flow cytometer needs fluorescent markers
Excitable
a certain wavelength.
Background fluorescence
- endogenous components - autofluorescence
- unbound or non-specifically bound marks
= reagent background
Multi-colour marking
- two or more brands, while monitoring various functions K. Souček Bi9393 Analytical cytometry
Fluorescence Output of Fluorophores
Comparing Different Dyes
F-actin ~
BODIPY FL phallacidin
anti-ß tubulin ~
Texas Red
goat anti-mouse IgG
DNA ~
DAPI
Mouse 3T3
 Hind III
propidium iodide
K. Souček Bi9393 Analytical cytometry
K. Souček Bi9393 Analytical cytometry
Processes interfering with and
detecting fluorescence
◼ Quenching - "quenching" fluorescence with polar
solvents, heavy ions.
◼ Bleaching - a change in the structure of a
fluorescent molecule leading to a loss of fluorescence
(by light or chemical interaction.
◼ Photon saturation - a state where the amount
of molecules in the excited state corresponds to the
amount of molecules in the basal level
K. Souček Bi9393 Analytical cytometry
Photobleaching
- irreversible destruction or photobleaching of the excited fluorophore
Oregon Green 514 goat anti-mouse IgG
fluorescein goat anti-mouse IgG
anti-human cytochrome oxidase subunit I
K. Souček Bi9393 Analytical cytometry
The basis of flow cytometryThe basis of flow cytometry
Cells in suspension
flow individually across
illuminated part where
scatter light and emit
fluorescence,
that is detected, filtered and
converted to digital values
saved to the computer
Fluidics
Optics
Electronics
original from Purdue University Cytometry Laboratories; modified by R.F. Murphy
Optics - light sourceOptics - light source
• the need to focus the light source at the same place where the cell flow is
focused
• Lasers
• produce a single wavelength of light (325, 488, ~630nm)
• provide mW - W of light
• provide a coherent light current
• Arc-lamps (Arc-lamps), not used in current systems
• produce a mixture of wavelengths that must be filtered
• provide mW of light
• cheap - air-cooled
• incoherent light current
- optical channels- optical channels
• the path of light from the point of cell irradiation to the detector
• optical parts separate certain wavelengths
original from Purdue University Cytometry Laboratories; modified by R.F. Murphy
LASER(s)
- coherent (continuous luminous flux)
- Monochrome
- Focused
http://hyperphysics.phy-astr.gsu.edu/hbase/hframe.html
http://www.ilt.fraunhofer.de/eng/100053.html
http://en.wikipedia.org/wiki/Helium-neon_laser
K. Souček Bi9393 Analytical cytometry
LASER vs. Arc lamp
http://en.wikipedia.org/wiki/Image:Helium_neon_laser_spectrum.png
H.M. Shapiro, Practical Flow Cytometry, 4th ed.
http://en.wikipedia.org/wiki/Helium-neon_laser
http://www.olympusmicro.com/primer/anatomy/sources.html
K. Souček Bi9393 Analytical cytometry
Optics - "Forward Scatter" channelOptics - "Forward Scatter" channel
• the portion of light scattered in the
same axis as the direction of the
light beam
• the intensity of the "forward scatter"
corresponds to the size, shape and
optical homogeneity of the cells
original from Purdue University Cytometry Laboratories; modified by R.F. Murphy
Forward Angle Light Scatter
FALS
Sensor
Laser
original from Purdue University Cytometry Laboratories; modified by R.F. Murphy
Optics - "Side Scatter"
channel
Optics - "Side Scatter"
channel
• the part of the light scattered
perpendicularly to the side of the axis of
light beam direction side (90o ) scatter
channel
• the intensity of the "side scatter"
corresponds to the size, shape and optical
homogeneity of the cells
original from Purdue University Cytometry Laboratories; modified by R.F. Murphy
90 Degree Light Scatter
FALS Sensor
90LS Sensor
Laser
original from Purdue University Cytometry Laboratories; modified by R.F. Murphy
Optics - Light ScatterOptics - Light Scatter
• "Forward scatter" captures surface
properties and particle size
• can be used to distinguish between living
and dead cells
• "Side scatter" corresponds to inclusions
within cells
• it is possible to distinguish between granular
and non-granular populations
original from Purdue University Cytometry Laboratories; modified by R.F. Murphy
Optics - fluorescent channelsOptics - fluorescent channels
• the fluorescence emitted from each
fluorochrome is detected by a specific
fluorescence channel
• specificity of detection is controlled by
the wave selectivity of the filter and
mirrors
original from Purdue University Cytometry Laboratories; modified by R.F. Murphy
Laser
Fluorescence Detectors
Freq
Fluorescence
FALS Sensor
Fluorescence detector
(PMT3, PMT4 etc.)
original from Purdue University Cytometry Laboratories; modified by R.F. Murphy
Optics - filter propertiesOptics - filter properties
• are constructed of materials that
absorb a certain wavelength (and
transmit another)
• the transition between absorbance
and transmission is not exact; it is
necessary to specify the refraction
of light in the filter design
original from Purdue University Cytometry Laboratories; modified by R.F. Murphy
Chroma Technology Corporation
Optics - filter propertiesOptics - filter properties
• "Long pass" filter passes wavelength
above the "cut" length
• "Short pass" filter passes wavelength
below the "cut" length
• The "Band pass" filter passes a
wavelength in a narrow range
around the specific wavelength
original from Purdue University Cytometry Laboratories; modified by R.F. Murphy
Standard Long Pass FiltersStandard Long Pass Filters
Transmitted LightLight Source
520 nm Long Pass Filter
>520 nm
Light
Transmitted LightLight Source
575 nm Short Pass Filter
<575 nm
Light
Standard Short Pass FiltersStandard Short Pass Filters
original from Purdue University Cytometry Laboratories; modified by R.F. Murphy
Standard Band Pass Filters
Transmitted LightWhite Light Source
630 nm BandPass Filter
620 -640 nm Light
original from Purdue University Cytometry Laboratories; modified by R.F. Murphy
Optics - filter propertiesOptics - filter properties
• if the filter is placed at a 45o angle to
the light source, the light that is
supposed to pass through does so,
but the blocked light is reflected at
a 90o angle
• dichroic filters, dichroic mirrors
original from Purdue University Cytometry Laboratories; modified by R.F. Murphy
Dichroic Filter/MirrorDichroic Filter/Mirror
Filter placed at 45o
Reflected light
Transmitted LightLight Source
original from Purdue University Cytometry Laboratories; modified by R.F. Murphy
Optics - filter arrangementOptics - filter arrangement
• to measure more than one "scatter"
or fluorescence together, we use
multiple channels (and detectors)
• the multi-channel arrangement must
meet
• spectral properties of the fluorochrome
used
• the correct order of arrangement of
filters and mirrors
original from Purdue University Cytometry Laboratories; modified by R.F. Murphy
Optics - detectorsOptics - detectors
• two general types of detectors
• photodiode
•in the past especially for strong signal
(forward scatter detector)
•present - highly sensitive AVALANCHE"
photodiodes (APD)
• photomultiplier tube (PMT)
•more sensitive than a normal photodiode,
can be damaged by overexposure
original from Purdue University Cytometry Laboratories; modified by R.F. Murphy
Photomultiplier tubes (photomultipliers, PMTs)
http://en.wikipedia.org/wiki/Photomultiplier
http://hamamatsu.magnet.fsu.edu/articles/photomultipliers.html
Basic characteristics:
-high sensitivity detectors (single photon)
-high signal gain/low noise
-large detection area
-fast response frequency
-high working voltage (1000 - 2000 V)
K. Souček Bi9393 Analytical cytometry
"normal" photodiode
Comparison with PMT
Advantages:
1. excellent signal linearity
2. spectral detection range 190 nm to
1100 nm (silicon)
3. low noise
4. Resistance to mechanical influences
5. low price
6. small size and weight
7. long service life
8. High quantum efficiency (~80%)
9. Does not need high voltage
Disadvantages
1. Small area
2. Impossibility of integral amplification
3. Much lower sensitivity
4. Counting photons only for special
products
5. Shorter response time
http://en.wikipedia.org/wiki/Photodiode
K. Souček Bi9393 Analytical cytometry
https://en.wikipedia.org/wiki/Avalanche_photodiode
Present: the "AVALANCHE" photodiode (APD)
- Highly sensitive semiconductors - comparable
to PMT
PMT
PMT
PMT
PMT
Dichroic
Filters
Bandpass
Filters
Example Channel Layout for Laserbased
Flow Cytometry
Laser
1
2
3
4
Flow cell
original from Purdue University Cytometry Laboratories; modified by R.F. Murphy
BD FACSCalibur system
http://www.bdbiosciences.com/immunocytometry_systems/
http://www.bdbiosciences.com/immunocytometry_systems/
K. Souček Bi9393 Analytical cytometry
BD LSR II system
http://jcsmr.anu.edu.au/facslab/facs/LSR2.html
http://jcsmr.anu.edu.au/facslab/facs/LSR2.html
BD FACSVerse system
http://www.bdbiosciences.com/instruments/facsverse/features/index.jsp
http://www.bdbiosciences.com/instruments/facsverse/features/index.jsp
Aria II
SP6800 spectral analyzer
Image Stream & Flowsight Amnis combination
of flow cytometry and image
analysis
Amnis - applications
CellStream, Luminex
ThermoFisherScientific: Attune
CytPix Flow Cytometer
BD FACSDiscover S8
SCIENCE 20 Jan 2022 Vol 375, Issue 6578 pp. 315-320 DOI: 10.1126/science.abj3013
Octagon Detection System
SSC
PE
PE-Cy7
FITC
PerCP-Cy5.5
PE-TxRed
"cube" for conventional fluorescence
microscope
Acousto Optical Beam Splitter AOBS®
Acousto Optical Beam Splitter
AOBS®
http://micro.magnet.fsu.edu/primer/java/filters/aotf/index.html
http://micro.magnet.fsu.edu/primer/java/filters/aotf/index.html
http://simple.wikipedia.org/wiki/Tellurium
Supercontinuum Generation
-a nonlinear process for strong spectral broadening of light
The benefits of AOBS®
•Adaptable to any new dye
•8 lines simultaneously
•Reflected light imaging
•High transmission
•Truly confocal - real optical sectioning
•Fast switching
•Freely tunable
•Fluorescence correlation spectroscopy
with multi-line lasers
https://www.bdbiosciences.com/en-us/applications/research-applications/multicolor-flow-cytometry/product-selection-tools/spectrum-viewer
https://www.bdbiosciences.com/en-
us/applications/research-applications/multicolor-flow-
cytometry/product-selection-tools/spectrum-viewer
Fluorescence Spectrum Viewers
K. Souček Bi9393 Analytical cytometry
https://www.thermofisher.com/cz/en/home/life-
science/cell-analysis/labeling-
chemistry/fluorescence-spectraviewer.html
http://www.biolegend.com/panelselector
http://www.biolegend.com/spectraanalyzer
http://www.biolegend.com/webtoolstab
https://fluorofinder.com
https://www.bosterbio.com/protocol-and-troubleshooting/flow-cytometry-principle
Fluidic systems and hydrodynamics
Fluidics Cart Cytometer
Fluid system : BD FACSAria II
AIR
PRESSURE
BULK
INJECTION
Sheath Tank
AIR
PRESSURE
ASPIRATED
WASTE
(VACUUM)
ASPIRATED
WASTE
(DEGAS)
SHEATH
FILTER
0” – 9”
R1
Sheath Regulator
R2
Sample Regulator
V17
V20
V5V6
Hydrodynamic focusing in the
cuvette
Sheath
Sample
Sheath
Sample
Sample pressure
low, small core
stream. Good
for DNA analysis
High sample
pressure, broader
core stream.
Bad for DNA
analysis
Laser Beams
ANd9GcSG6MDDEyKW2lrEy-4pYcbhalq94g7mws3iCujkWC8szVoIZvRtvw
Particle Delivery: Hydrodynamic Focusing
Intensities
Count
Narrow particle focus = Narrow distribution
Laser Cross
Sectional Area
• Sample core is ' pinched ' by fast flowing sheath
• Sample volume ratios of 100 – 1000
• Large ratios => low sample inputs
• Resolution of particle populations
sheath
sheath
Hydrodynamic core
Conventional Instrumentation: Low Flow Rates (12µL/min)
Particle Delivery: Hydrodynamic Focusing
Conventional Instrumentation: High Flow Rate (60µL/min)
Intensities
Count
Broad particle focus = Broad distribution
• Increased sample input = increased core size
• Particle distributions broadened, CVs increase
• Instrument resolution decreased
• Historically, low volumetric sample rates used (25  l/min
– 150  l/min)
sheath
sheath
Hydrodynamic core
Laser Cross
Sectional Area
Attune® Acoustic Focusing Cytometer
Acoustic Focusing = Better Precision
Acoustic focusing module
Narrow particle focus = Narrow
distribution
12 µL/min 1000 µL/min
Acoustic focusing of particles occurs
prior to mixing with sheath fluid
https://upload.wikimedia.org/wikipedia/commons/3/35/Kundt_tube.png
Attune NxT ( 2nd generation )
100 200 300 400 500 600 700 800 900 1000
Maximum Sample Input Rate (  l/min)
Instrument 1
Instrument 2
Instrument 6
Instrument 5
Instrument 4
Instrument 3
Attune®
Attune® Throughput Compared to Hydrodynamic
Focused Instruments
• Attune® can analyze at sample rates from 25µL/min to 1000µL/min without losing accuracy
• Traditional Flow Cytometers can only run at most 150µL/min and will sacrifice data quality
• Higher sample rates enable dilution of limited samples and analysis of Rare Events Faster
Hydrodynamic
Focused
Instruments
• the pressure of the carrier (sheathing) liquid drives the
buffer through the cuvette and the higher pressure in the
sample tube introduces the sample into the cuvette.
• The principle of hydrodynamic focusing aligns the cells in
the cuvette "like pearls on a string" before they reach the
point where the laser beam intersects.
•Hydrodynamic focusing cannot dissociate cell aggregates.
Flow cytometry requires a suspension of single cells!
Fluidics – summary
Principles of flow cytometry and
sorting
◼ sorting
K. Souček Bi9393 Analytical cytometry
Frequency
Charge
Drop Delay
Amplitude
SORTING
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+++++-----
- - - - - - - - - -
http://www.cyto.purdue.edu/cdroms/cyto10a/seminalcontributions/fulwyler.html
_
Sorting - Sort Masks
Cells are randomized
distributed over the stream
Sorting - Sort Masks
Trailing
Interrogated
Leading
Mask
◼ A region of the stream monitored for the
presence of cells
◼ Determines how drops will be deflected if a
sorting conflict occurs
◼ Measured in 1/32 drop increments
Mask = 0 Mask = 8 Mask = 16 Mask = 32
4
4
8
8
16
16
Conflict Resolution
◼ Precision modes include three types of
masks
– Yield
– Purity
– Phase
Sorting - Sort Masks
Sort decisions are determined by sort masks
Target particles in a drop with
1/32-drop resolution
Sorting - Yield Mask
The yield mask defines how many drops will be sorted Yield mask of
8/32 indicated in blue; target particle shown in green
Yield Mask
Sorting - Purity Mask
Purity mask of 8/32 in blue, 4/32 in each adjacent drop;
target particles in green, non-target particles in red
Purity Mask
Purity Mask
Cell sorting - trends
◼ Easy operation
◼ Careful handling
– On-chip technology
◼ Size  and security 
◼ Microfluidic-based cell sorting
◼ Spectral cell sorting
◼ Image-based sorting
◼ Buoyancy Activated Cell Sorting (BACS )
– a method that uses low-density particles (microbubbles) for
flotation separation.
Electronics and data
Laser
Laser
Laser
Creation of a Voltage Pulse
Tim
Voltage TimVoltage
Tim
Voltage
1
2
3
Height, Area, and Width
Time (µs)
Voltage
Pulse area (A)
PulseHeight(H)
Pulse Width
(W)
0
Signal processing
team
analogsignalintensity
VOLTAGE
FSC ~ cell size
FL-1 (530/30nm) ~ green fluo.
FL-2 (585/42nm) ~ red fluo .
Analog/digital
conversion
Height
Width
Area ( ∫ )
FL- (H, W, A)
FL-1(H)
FL-2 (H)
dot fence
0 1000
1000
Signal
amplificatio
n (!)
lin or log
K. Souček Bi9393 Analytical cytometry
Voltage In
PMT
Power Supply
Levels 0–1000 Volts
Photon
In
Signal
Out
Digital data
to memory
Analog to Digital
Conversion
Digitization
the pulse
16,384 levels
Sample
the pulse
10 MHz
Analog to Digital Converter
Parameters
• Area: Sum of all height values
• Height: Maximum digitized value X 16
• Width: Area/Height X 64K
Data is displayed on a 262,144 scale
282
3060
10270
358
4004
9568
14524
AD converters
Number of bits # channels distinction
8 256 39.1 mV
10 1024 9.77 mV
12 4096 2.44 mV
14 16384 610  asl
16 65536 153  E
18 262144 38.1  E
20 1048576 9.54  V
22 4194304 2.38  H
24 16777216 596 AD
2 8 = 256
2 10 = 1024
.
.
.
Full scale measurement range = 0 to 10 volts
ADC resolution is 12 bits: 212 = 4096 quantization levels
ADC voltage resolution is: (10-0)/4096 = 0.00244 volts = 2.44 mV
K. Souček Bi9393 Analytical cytometry
Logarithmic gain & dynamic range
adapted from JPRobinson
tench
logo
Data analysis
◼ View data
– histogram
– dot plot
– isometric display
– contour plot
– chromatic (color) plots
– 3D projection
◼ Gating
Ways to display data
K. Souček Bi9393 Analytical cytometry
Summary
◼ Fluid systems
◼ Sorting
◼ Signal, data – basic principle
At the end of today's lecture you should :
1. Know the basic principles of light scattering
2. and fluorescence;
3. to know what light sources are used in flow cytometry ;
4. and how it is detected;
5. know the basic principles of fluid systems and laminar flow.
6. Know the basic principle of data processing and visualization
At the end of today's lecture you should :
1. Know the basic principles of light scattering
2. and fluorescence;
3. to know what light sources are used in flow cytometry ;
4. and how it is detected;
5. know the basic principles of fluid systems and laminar flow.
6. Know the basic principle of data processing and visualization
K. Souček Bi9393 Analytical cytometry