Download COAS Flow Cytometer

The COAS Flow Cytometry Facility, Burt Hall 378
Common use (fee charges!),
acquired by an NSF Major Research Equipment Grant, Fall 2000
Graduate student Mike Wetz enumerating bacterial abundance
Flow cytometry
- Flow cytometers analyze characteristics of particles, including fluorescence
intensity at various wavelengths and light-scattering properties.
- Individual particles pass through a laser beam in a laminar flow stream, and
fluorescence intensity, plus forward and side light scatter, are recorded for each
‘event’. Light scatter is a function of particle shape, texture, internal structure,
and size.
- FCM software (e.g. BD “Cell Quest”) allows for the plotting of fluorescence
intensity vs light scatter, or for plotting different fluorescence intensities against
each other, yielding event ‘clouds’ in dot plots, that define discrete classes of
particles present in the sample. Histograms, 3D- and Contour plots can also be
viewed; dot plots and histograms in real-time and the others in ‘Analysis’ mode.
- The number of events in each ‘event cloud’, together with volume of sample
processed, yields estimates of abundances of discrete classes of particles. In
addition, raw and statistical data are available on a per cell basis for export into
other software for further analysis.
Advantages of flow cytometry
compared to microscopy
1) SPEED & ACCURACY: Decreased sample variance due to
larger number of cells counted per sample, more samples processed per
unit time, so allows higher resolution data sets. Small sample volumes, ~
0.5 ml/sample.
2) BEST METHOD FOR SOME TYPES OF CELLS: e.g, Preferred
method for enumeration of Prochlorococcus
3) CELL-SPECIFIC INFORMATION: High-speed multi-parametric
data acquisition on cell-by-cell basis, allows multivariate data analysis
and separation of distinct populations of cells.
4) SEGREGATING CELLS BY SORTING: Can sort out particular
cells of interest in a sample, and then further test those cells, e.g.
phylogenetic identification
COAS Common Use Instrument:
Becton Dickinson FACSCalibur, 4-color, with cell sorter unit
Salient features:
- Can handle particle sizes of up to 180 um, counts cells at
abundances of > 1000 per ml very accurately
- We typically process ~ 30 – 300 ul of sample in ~ 3 minutes for
bacterial and phytoplankton abundance data
- Cytometer is controlled, and data collected, using B-D software,
data can be exported to spreadsheets
FACSCALIBUR detection system:
Note: every particle analyzed by the FCM is
characterized by a number of different variables
I - PARTICLE SIZE/SHAPE
• FALS: Forward Angle Light Scatter, detected by a
photodiode, influenced by particle size and shape
• SSC: Side Scatter (light scatter), detected by a
photomultiplier tube (PMT), influenced by particle size
and shape, refractive index, and internal cell structure
II - PARTICLE FLUORESCENCE
Blue light laser (488 nm)
• FL1: green light fluorescence (515 – 545 nm), detected by
a PMT
• FL2: orange fluorescence (564 – 606 nm), detected by a
PMT
• FL3: long band-pass red fluorescence (> 670 nm), detected
by a PMT
Red light laser (635 nm)
• FL4: narrow band-pass fluorescence (653 – 669 nm),
detected by a PMT
SIP
On/off and flow
rate controls
PMTs
Fluidics
system
Lasers
SIP
Sorting
system
PMTs
Flow cell
Blue laser, 488 nm
Red laser, 635 nm
FACSCalibur fluorescence detection
• FL1 – GREEN: Molecular Probes nuclear stains, e.g. Syto & Sybr Green
• FL2 – ORANGE: Phycoerythrine of cyanobacteria and cryptomonad algae, CTC
• FL3 – LONG-PASS RED: Chlorophyll-a, propidium iodide, Texas Red
• FL4 – NARROW RED: Phycocyanine of cyanobacteria and cryptomonad algae
Phytoplankton via photopigment fluorescence
Coccoid cyanobacteria
Eukaryotic picophytos
July 2001, NH-line, station 85 depth profile
Relative fluorescence per cell, or cell abundance per ml
0
2000
4000
6000
-5
Abundance/ml
Depth, m
-25
-45
-65
coccoid cyanobacteria,
per ml x 10
-85
eukaryotic phytoplankton,
per ml
-105
Fluorescence/cell
High nucleic acid, high scatter
High nucleic acid,
low scatter
Low nucleic acid,
low scatter
Typical dot plot of heterotrophic bacterioplankton in Oregon coastal
waters, showing three groups of bacteria based on amount of SYBR
(nucleic acid) staining and side scatter (~cell size)
100 m sample depth
3.2 x 10^5 cells/ml
10 m sample depth
2.8 x 10^6 cells/ml
Heterotrophic bacterioplankton data presented as contour plots of green
fluorescence [ SYBR (nucleic acid) staining] versus side scatter for two sample
depths. Note differences in distribution of High-DNA bacteria in these two
samples.
Comparison of 0-50 m integrated eukaryotic phytoplankton and bacterioplankton with
surface fluorescence off the Oregon coast, September 2001
A. Color = eukaryotic phytoplankton
contours = surface fluorescence
2.0
1.5
1.0
0.5
B. Color = bacterioplankton
contours = surface fluorescence
0.0
14
44.5
44.5
44.0
44.0
43.5
43.5
43.0
43.0
42.5
42.5
42.0
42.0
-125.5
-125.0
-124.5
-124.0
12
-125.5
10
-125.0
8
6
-124.5
4
2
-124.0
SEGREGATING CELLS BY SORTING:
The cell sorting capacity of the FCM allows us to separate out
particular cells of interest in a sample, for further testing of
those cells, e.g. phylogenetic identification, or cell-specific rates
of uptake of radiolabeled substrate.
The FACSCalibur sorter is mechanical and slow, can sort a
maximum of 300 target particles per second.
But, the sort system is sealed, so we can sort samples labeled
with radioisotopes.
FACSCalibur
Sorting System
Three sort modes:
Graduate student Krista Longnecker preparing to sort CTC+ labeled bacteria from a
seawater sample for phylogenetic analysis
Flow cytometer sort window defined as a region on the dot plot of red fluorescence
(FL3) vs orange fluorescence (FL2) for samples containing CTC+ bacteria. Note
that coccoid cyanobacteria (Synechococcus), which also fluoresce orange, appear in
a different region on the dot plot.