Achieving Perfect Flow

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Achieving Perfect Flow
Joyce Carafa Coordinator -
CIC Flow Cytometry Core
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Flow Cytometry is a Mongrel Science

• Cells, in suspension, flow single-file
• through a focused light beam
• which they scatter according to their size and
  density
• and from which they absorb and re-emit
  fluorescence energy
• these signals are collected, filtered and
  amplified
• then they are converted to digital values that
  are stored on a computer

Fluidics
Optics
Fluorescent Staining
Electronics
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Fluidics

• Need to have the cells flow, in suspension, in
  single file, through an illuminated volume
• In most instruments this accomplished by
  injecting the sample into a sheath fluid as it
  passes through a small (50-300 µm ) orifice
• When the conditions are right, the sample fluid
  flows in a central core which does not mix with
  the sheath fluid - this is termed Laminar Flow.
• This introduction of a large volume into a
  smaller one in such a way that it becomes focused
  along an axis is called Hydrodynamic Focusing.

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Flow Cell Fluidics
Sheath Fluid
Hydrodynamic Focusing
Sample Fluid
Laminar Flow
Light Source
Illuminated Volume
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Fluidics - Flow Chambers

• Four basic flow chamber types
• Closed cross flow
• best optical properties, cant sort
• Open flow across surface
• best optical properties, cant sort
• Jet-in-air
• best for sorting, inferior optical properties
• Flow-through cuvette
• excellent optical properties, can be used for
  sorting

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Flow Channel Optics
PMT 4
Beam-splitters or Dichroic Filters
PMT
3
Flow cell
PMT
2
PMT
Bandpass Filters
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Laser
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Excitation Optics

• Usually consists of
• A Laser
• can provide a single wavelength of light (a laser
  line) or (more rarely) a mixture of wavelengths
• can provide from milliwatts to watts of light
• can be inexpensive, low power, air-cooled units
• or expensive, high power, water-cooled units
• provide coherent light

Lenses - to shape and focus the laser beam
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Collection Optics

• Usually consists of
• A collection lens to collect light emitted from
  the particle-laser beam interaction
• A system of optical mirrors and filters to route
  specified wavelengths of collected light from the
  illuminated volume down a specific optical
  channel with minimal signal loss
• Optical elements provide separation of channels
  and wavelength selection
• A designated optical detector

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Filter Layout

• To simultaneously measure more than one scatter
  or fluorescence from each cell, we typically use
  multiple channels (multiple detectors)
• Design of multiple channel layout must consider
• spectral properties of fluorochromes being used
• proper order of beam-splitters, filters and
  mirrors

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Flow Channel Optics
PMT 4
Beam-splitters or Dichroic Filters
PMT
3
Flow cell
PMT
2
PMT
Bandpass Filters
1
Laser
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Dichroic Filter or Mirror

• When a filter is placed at a 45o angle to a light
  source, light which would have been transmitted
  by that filter is still transmitted but light
  that would have been blocked is reflected (at a
  90o angle). This results in much less signal loss
  than when using a beam-splitter

Dichroic Filter at 45 deg
Transmitted Light
Light Source
Reflected light
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Filter Properties

• When using Laser light sources, filters must have
  very sharp cut-ons and cut-offs since there will
  be many orders of magnitude more scattered laser
  light than fluorescence
• To collect specific wavelengths a filter must
  reject to certain tolerance
• e.g. reject 488 nm light at 10-6 level
• only 0.0001 of incident light at 488 nm gets
  through

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Filter Properties
Long Pass Filters

• Long pass filters transmit only signal above a
  cut-on wavelength

520 nm Long Pass Filter
Light Source
Transmitted Light
gt520 nm
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Filter Properties
Short Pass Filters
Short pass filters transmit only signal
below a cut-off wavelength
575 nm Short Pass Filter
Light Source
Transmitted Light
lt575 nm Light
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Filter Properties
Band Pass Filters
Band pass filters transmit only signal in a
narrow range around a specified wavelength
630/20 nm BandPass Filter
Transmitted Light
Light Source
620 -640 nm Light
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Optical Detectors

• Two common detector types
• Photodiode
• used for strong signals when saturation is a
  potential problem (e.g., forward scatter
  detector)
• Photomultiplier tube (PMT)
• more sensitive than photodiode but can be
  destroyed by exposure to too much light

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Fluorescence

• The fluorescence emitted by each fluorochrome is
  usually detected in a unique fluorescence channel
• The specificity of detection is controlled by the
  wavelength selectivity of optical filters and
  mirrors

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Fluorescence Staining
Emitted Fluorescence Intensity µ Binding Sites
FITC
FITC
FITC
FITC
FITC
Number of Events
FITC
Fluorescent Intensity
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Electronics Creating a Voltage Pulse

• Converts optical signals to proportional
  electronic signals (voltage pulses)
• Analyzes voltage pulse height, area, and width
• Interfaces with the computer for data transfer

Voltage
Laser
Time
Voltage
Laser
Time
Laser
Voltage
Time
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Forward Scatter
LASER
DETECTOR

• Low angle light scatter is proportional to cell
  size
• tends to be more sensitive to surface properties
  of particles (e.g., cell ruffling)
• can be used to distinguish live from dead cells
• But has limitations
• Presumes that cells are spherical
• Only an estimate of cell size

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Forward Scatter Relative Cell Size
SMALL
MEDIUM
LARGE
VERY LARGECELLS
Blasts
Monocytes
Neutrophils
Lymphocytes
RBC Precursors
Non-HP Cells
Plasma Cells
Hematogones
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• is collected at a 90 angle from illumination
  source
• is more sensitive to cellular inclusions and can
  distinguish granulated cells from non-granulated
  cells
• its intensity is proportional to the size,
  shape, reflectivity of internal cell components
  and optical homogeneity of cells
• it has limitations
• what about components that dont reflect?

Side Scatter
LASER
DETECTOR
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SSC Relative Granularity
VERY HIGH
Neutrophils
MED
Non-HP Cells
Monocytes
Increasing Complexity
Plasma Cells
Blasts
RBC Precursors
Lymphocytes
Hematogones
LOW
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FSC/SSC Patterns
Neutrophils
Debris
Side Scatter
Monocytes
RBC Precursors
Lymphocytes
Forward Scatter
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Target Identification Morphologic Correlation

• Learn to recognize the normal pattern of Forward
  Scatter vs. Side Scatter of your target cells
• Identify aberrant populations
• Correlation with microscopic morphology - it is
  the foundation of hematology cytology
• A better understanding of the morphology allows a
  better understanding of the flow cytometric data
  and the changes it depicts
• Simple morphologic findings often clarify
  difficult flow problems and vice versa
• Consider the use of certain monoclonals like
  CD45 or CD14 to identify target population

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Degrees of positivityof CD 45
Dim
Negative
Moderate
Bright
Neutrophils
Monocytes
NonHP Cells
Blasts
Plasma Cells
RBC Precursors
Hematogones
Lymphocytes
CD 45
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CD45/SSCPatterns
Eosinophils
Neutrophils
Plasma Cells
Monocytes
Side Scatter
Basophils
Non-HP Cells
Lympho -cytes
Hematogones
CD 45
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Flow Cytometry Sample Description
FSC/SSC SSC/CD45
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FSC/SSC SSC/CD45
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Fluorescein (FITC)
300 nm 400 nm 500 nm
600 nm 700 nm
FL1
FL2
FL3
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Phycoerytherin (PE)
300 nm 400 nm 500 nm
600 nm 700 nm
FL1
FL2
FL3
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Propidium Iodide
300 nm 400 nm 500 nm
600 nm 700 nm
FL1
FL2
FL3
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Allophycocyanin (APC)
300 nm 400 nm 500 nm
600 nm 700 nm
FL3
FL2
FL1
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Basics of Flow Sorting

• Droplet formation
• Timing / Drop Delay Calculation for Charge
  Application
• Modes - Yield, Purity and Count

As liquid is ejected into air, it will form
droplets. By vibrating the nozzle at a defined
frequency, the size of these droplets and the
position along the stream where they form can be
controlled with great precision.
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Fluorescence Activated
Cell Sorting
FSC Detector
488 Laser
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Fluorescence detector
Charged Plates
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Single cells sorted into test tubes
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Acknowledgments Sources

• Dennis P. OMalley, M.D Hematopathology Fellow
  FLOW CYTOMETRY A Second Look at the First Look
• Bill Gunderman, BD Biosciences
• Robert F. Murphy, October 1996 Lecture Notes for
  Fluorescence Spectroscopy in Biological
  Research
• Flow Cytometry and Sorting, 2nd ed. (M.R.
  Melamed, T. Lindmo, M.L. Mendelsohn, eds.),
  Wiley-Liss, New York, 1990
• Flow Cytometry Instrumentation and Data Analysis
  (M.A. Van Dilla, P.N. Dean, O.D. Laerum, M.R.
  Melamed, eds.), Academic Press, London, 1985

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Acknowledgments Sources (cont)
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Cell Sorting
FSC Detector
488 Laser
Charged Plates
488 Laser
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Single cells sorted into chosen test tubes
Fluorescence detector