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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.