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5 views51 pages

flow3 - Tagged

Uploaded by

Diana Carolina Vargas
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Flow Cytometry

An Introduction
Conclusions?
MLS Students
Definitions

• Flow Cytometry
– = Measuring properties of cells in flow
– Flow cytometry is a method to differentiate and
count cells and microparticles
– How many cells analyzed per second?
How Does Flow Cytometer Do It?
• Fluidics System
• Optics System
• Electronics System
Fluidics System
• Responsible for transporting the cells labeled
with fluorescent antibodies to the
interrogation point of the laser
• Hydrodynamic focusing uses differential fluid
flow rates to align cells single file
• Slower sheath fluid stream runs parallel to
sample stream creating laminar flow
Hydrodynamic Focusing
The Fluidics system procures
hydrodynamic focusing
utilizing liquid (Hydro) to force
cells or particles to travel at the
same speed and to form a single
Injector
file. The Sheath Fluid Tip Sheath
(suspension of saline) forces the
fluid
sample into a core stream or
hydrodynamic core and
eventually make their way to
the interrogation point of laser
beam.

Fluorescence
signals

Focused laser
beam
Hydrodynamic Focusing
Flow Rates

• Sample flow rate and sheath fluid flow rate


are different
• A change to pressure regulator can change the
flow rate
• Low sample pressure, 12 µL/min
• High sample pressure, 60 µL/min
– Wider sample core~doublets
Optics System
• One passing thru hydrodynamic focusing, cells
move into the optics system
• A laser serves to scatter light and excite the
fluorochrome-labeled antibodies on cells
• Resultant light is directed to a series of
photomultiplier tubes (PMTs) which digitally
converts light into data points we visualize on
scatter plots or contour plots
Dot Plot vs Contour Plot
Laser
• Light Amplification by Stimulating Emission of
Radiation providing energy that excites the
fluorophore of interest generating a focused
sample of light at one specific wavelength
Contemporary Flow Cytometer
Dichroic Mirrors
• Direct certain wavelengths of light one way,
while reflecting other wavelengths
• Two Types: Long Pass and Short Pass
• Long Pass (LP)
– Allows light equal to or longer than their indicated
wavelength to pass thru to another detector
• Short Pass (SP)
– Allow light equal to or shorter than their indicated
wavelength to pass thru to another detector
Short Pass vs Long Pass
Photomultiplier Tubes (PMT)
• Amplifies signal by generating photons of light
that generate a voltage pulse that is converted
to a digital readout on your computer screen
• 2 parameter dot plot or contour plot
Bandpass Filter
• Allows only a small range of wavelengths to
pass thru to final detector
• Labeled with two numbers
– Example: 550/40 nm would allow light between
530 nm and 570 nm to pass thru but reflect light
below 530 nm and above 570 nm
FITC530 nm
PE580 nm
PE/Cy5-667 nm
PE/Cy7-785 nm PECy7-
785

PECy5 667

PE580

FITC 530
530/10: 525-535
585/42: 564-606
660/20: 650-670
780/60: 750-810
Forward Angle Light Scatter

Laser

FALS Sensor

Purdue University Cytometry Laboratories


90 Degree Light Scatter

Laser

FALS Sensor

90LS Sensor

Purdue University Cytometry Laboratories


Types of Signals

• Forward scatter (FS)


– Approximate cell size
• Side or right angle light scatter (SS or RALS)
– Cell complexity or granularity
• Fluorescent
– Fluorescent labeling is used to investigate
cell markers, structure, and function.
Bandpass filter selection
Electronics System
• Converts emission spectra into digital signals
that can be visualized graphically
• Electronics measure the area, height, and
width parameters
• Flow data plots are the result of signals be
converted into meaningful data
Log Scale vs Linear Scale
• Light Scatter data uses linear scale
– Forward Scatter [indication of cell size]
– Side Scatter [ indication of cell granularity]
• Fluorescence data uses log scale
• DNA Analysis uses linear scale
FSC vs SSC: Linear Scale
Fluorochromes: PE and FITC
Log Scale
DNA Analysis
• Linear Scale

Football goalpost?
Light Scatter Gating
(Contour Plot)
Side Scatter Projection

1000
Neutrophils

Forward Scatter Projection


800
Forward Scatter
600

Monocytes
400

Lymphocytes
200
0

0 200 400 600 800 1000

90 Degree Scatter
The Electronic Gate
Coulter Principle of
Electrical Impedance
Fulwyler Invention
US3380584A
1965 Patent
BD FACScan

Single Laser, blue


488nm Argon;
parameters include
FSC, SSC, FL1, FL2,
FL3
BD FACSort
BD LSR II

Equipped with four


lasers [Violet405,
Blue488, Yellow552,
red637] 16 PMTs,
three flow rates [12,
35, 60 µL/min]
BD LSR Fortessa

Octagon, trigon detector


arrays minimize light loss,
up to 4 lasers [red, blue,
violet, UV], up to 18
colors, 96 well plates
BD FACS Calibur

Blue and Red Lasers,


4 color, with option
for cell sorting;
walkaway and can
accommodate 96 and
384 well plates
BD FACS CANTO II

Equipped with three


lasers: blue 488, red 633,
violet 405
Octagon and trigon
detector arrays, walkway
with sample loader, up to
8 colors
BD Accuri C6

11x14.75x16.5 inch
footprint, 30lbs, blue
and red laser, two
light scatter
detectors, four
fluorescent detectors
CD4 enumeration,
green laser, 3 optical
parameters [SSC,
FL1, FL2]; 20 µL
EDTA WB, 20
samples/hr

Sysmex
CyFlow
Coulter Epics
Becton Dickenson
FACS Calibur
Applications of Flow Cytometry

• Immunophenotyping of cells of interest


• Cell cycle/Apoptosis/DNA studies
• Reticulocyte enumeration
• Functional/metabolic cell studies
• Gene expression/transfection studies
• Chromosome analysis and sorting
• Microbacterial studies
• Cell sorting

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