Basic Principles in Flow Cytometry - PowerPoint PPT Presentation

1 / 21
About This Presentation
Title:

Basic Principles in Flow Cytometry

Description:

Flow Cytometry is the technological process that allows for the individual ... Biological Oceanography. Parasitology. Bioterrorism ... – PowerPoint PPT presentation

Number of Views:393
Avg rating:3.0/5.0
Slides: 22
Provided by: flowcytome
Category:

less

Transcript and Presenter's Notes

Title: Basic Principles in Flow Cytometry


1
Basic Principles in Flow Cytometry
  • Prepared by Hector Nolla
  • Manager CRL Flow Cytometry Lab
  • University of California, Berkeley

2
Flow Cytometry
  • Flow Cytometry is the technological process that
    allows for the individual measurements of cell
    fluorescence and light scattering. This process
    is performed at rates of thousands of cells per
    second.
  • This information can be used to individually sort
    or separate subpopulations of cells.

3
History
  • Flow cytometry developed from microscopy. Thus
    Leeuwenhoek is often cited in any discussion
    regarding its history.
  • F.T. Gucker (1947)build the first apparatus for
    detecting bacteria in a LAMINAR SHEATH stream of
    air.
  • L. Kamentsky (IBM Labs), and M. Fulwyler (Los
    Alamos Nat. Lab.) experimented with fluidic
    switching and electrostatic cell sorters
    respectively. Both described cell sorters in
    1965.
  • M. Fulwyler utilized Pulse Height Analyzers to
    accumulate distributions from a Coulter counter.
    This feature allowed him to apply statistical
    analysis to samples analyzed by flow.

4
History
  • In 1972 L. Herzenberg (Stanford Univ.), developed
    a cell sorter that separated cells stained with
    fluorescent antibodies.The Herzenberg group
    coined the term Fluorescence Activated Cell
    Sorter (FACS).

5
Fluorescence Activation Process (or
Immunofluorescence)
Antibodies recognize specific molecules in the
surface of some cells
Antibodies are artificially conjugated to
fluorochromes
FITC
Antibodies
When the cells are analyzed by flow cytometry the
cells expressing the marker for which the
antibody is specific will manifest fluorescence.
Cells who lack the marker will not manifest
fluorescence
FITC
But not others
6
Cellular Parameters Measured by Flow
Intrinsic
Extrinsic
  • No reagents or probes required (Structural)
  • Cell size(Forward Light Scatter)
  • Cytoplasmic grabularity(90 degree Light Scatter)
  • Photsynthetic pigments
  • Reagents are required.
  • Structural
  • DNA content
  • DNA base ratios
  • RNA content
  • Functional
  • Surface and intracellular receptors.
  • DNA synthesis
  • DNA degradation (apoptosis)
  • Cytoplasmic Ca
  • Gene expression

7
Flow Cytometry Applications
  • Immunofluorescence
  • Cell Cycle Kinetics
  • Cell Kinetics
  • Genetics
  • Molecular Biology
  • Animal Husbandry (and Human as well)
  • Microbiology
  • Biological Oceanography
  • Parasitology
  • Bioterrorism

8
  • Flow cytometry integrates electronics, fluidics,
    computer, optics, software, and laser
    technologies in a single platform.

9
Cells are presented to the laser using principles
of hydrodynamic focusing
Y
Z
X
Y
Z
X
10
Laminar Fluidic Sheath
Core Sheath
Outer Sheath
11
  • Each cell generates a quanta of fluorescence

Photomultiplier Tubes (PMTs)
PE FL
FITC FL
488nm Sct
Discriminating Filters
Forward Light Scattering Detector
Confocal Lens
Dichroic Lenses
12
Negative cells are also detected
PE FL
FITC FL
488nm Sct
Forward Light Scatter
Dichroic Lenses
Confocal Lens
13
Optical Bench Schematic
14
From Fluorescence to Computer Display
  • Individual cell fluorescence quanta is picked up
    by the various detectors(PMTs).
  • PMTs convert light into electrical pulses.
  • These electrical signals are amplified and
    digitized using Analog to Digital Converters
    (ADCs).
  • Each event is designated a channel number (based
    on the fluorescence intensity as originally
    detected by the PMTs) on a 1 Parameter Histogram
    or 2 Parameter Histogram.
  • All events are individually correlated for all
    the parameters collected.

15
Light Scattering, 2 Parameter Histogram
Bigger
Apoptotic Cells
Bigger Cells
90 degree Light Scatter
Dead Cells
More Granular
Y Axis
X Axis
Live Cells
Forward Light Scatter (FLS)
16
1 Parameter Histogram
Positive
Negative
Brighter
Dimmer
Count
6
4
1
1 2 3 4 6 7
150 160 170 .. 190
Channel Number
Fluorescence picked up from the FITC PMT
17
2 Parameter Histogram
Single Positive PI Population
Double Positive Population
PE FL
Negative Population
Single Positive FITC Population
FITC FL
18
Gating and Statistics
  • Data generated in flow cytometry is displayed
    using Multiparamater Acquisition and Display
    software platforms.
  • Histograms corresponding to each of the
    parameters of interest can be analyzed using
    statistical tools to calculate percentage of
    cells manifesting specific fluorescence, and
    fluorescence intensity.
  • This information can be used to look at
    fluorescence expression within subpopulations of
    cells in a sample (gating).

19
Flow Cytometry Data
Smaller Region, Live cells mostly
Larger Region includes all cells
20
Running Samples
  • Prepare samples.
  • One sample should be completely negative. This
    sample should be analyzed first. This sample is
    used for adjusting the PMTs amplification
    voltage.
  • Adjust the PMT Voltage until you can see a
    population peak in the first decade of your 1
    parameter and or your two parameter plot.These
    samples are used for adjusting Spectral Overlap.
  • Once the instrument settings are optimized, run
    samples and collect data.

21
Flow Cytometry and sorting
Write a Comment
User Comments (0)
About PowerShow.com