1. Home
  2. >
  3. Applications & Technologies
  4. >
  5. Attune® Cytometer
  6. >
  7. Marine Sample Analysis With The Attune® Flow Cytometer

Marine Sample Analysis With The Attune® Flow Cytometer

Cell Cycle Analysis With The Attune® Flow Cytometer

Picophytoplankton Analysis of Marine Samples
Flow cytometry is a powerful tool for studying the biology, ecology, and biogeochemistry of marine photosynthetic picoplankton. Populations of photosynthetic picoplankton are intrinsically fluorescent due to their photopigment content, and differences in photopigment compositions are used to distinguish the various groups.

Download the marine sample analysis application note [PDF]


Photosynthetic Picoplankton


Prochlorococcus spp. and Synechococcus spp. are the two major groups of prokaryotes that comprise photosynthetic picoplankton and have been extensively studied for their principal role in primary production. Prochlorococcus spp. are the smallest and most abundant photosynthetic organisms known, and, along with Synechococcus spp., have a large impact on the global carbon cycle. Prochlorococcus spp. are approximately 0.6 μm in size and contain the red-fluorescent molecules divinyl-chlorophylls a and b. At 1 μm, cells of Synechococcus spp. are larger and contain the orange-fluorescent phycoerythrin in addition to red-fluorescent chlorophyll. These differences allow for the discrimination of natural populations of Prochlorococcus spp. and Synechococcus spp. in environmental samples.1-5


Typical Photosynthetic Picoplankton Population Analysis


Analysis of marine photosynthetic picoplankton is routinely performed using flow cytometry, although this
testing has presented some challenges. The excitation of the intrinsically fluorescent photosynthetic picoplankton has conventionally been performed using a 488 nm laser, a wavelength not optimal for the divinyl-chlorophyll containing–Prochlorococcus spp. Instruments that utilize high velocity or high volumetric sheath fluid to focus cells (hydrodynamic focusing) before laser interrogation have been used. Most flow cytometers are pressure driven, and direct cell counting of discrete populations is not possible without weighing of the sample pre- and post-analysis or adding counting beads to the sample.

In addition, the inclusion of 488 nm–excitable nucleic acid binding dyes for determining cell counts of the heterotrophic population (Bacteria and Archaea) obscures the intrinsic fluorescence of the picophytoplankton populations, requiring multiple samples to assess the entire microbial population.

(back to top)

Simplified Photosynthetic Picoplankton Population Detection


Conventional cytometers employ large sheath-to-sample flow rates to hydrodynamically focus particles. In contrast, the Attune® Acoustic Focusing Cytometer uses sound energy to focus particles and requires significantly lower sheath fluid flow rates. The Sensitive mode on the Attune® further reduces the instrument sheath flow rate, thereby slowing the particle velocity. By slowing the particle velocity, the researcher can increase the laser interrogation and photon collection times for dim, low-background populations (e.g., the inherently dimly fluorescent Prochlorococcus spp. from oligotrophic surface water samples).


The 405 nm laser enables better excitation of divinyl-chlorophylls from Prochlorococcus spp. and enhances separation of distinct picophytoplankton populations from background signal (Figure 1). Syringe-driven sample fluidics permits the direct counting of cells in a given population. Combining syringe-driven sample handling with excitation of divinyl-chlorophylls with the 405 nm laser allows for direct enumeration of Prochlorococcus spp. in SYBR® Green I–stained samples. Figure 2 demonstrates the utility of combining a slow particle flow rate and excitation of divinyl-chlorophylls with the 405 nm laser.


  Figure 1. Absorption (solid line) and fluorescence emission spectra (dotted line) of divinyl-chlorophyll a.



Figure 2. Oligotrophic Station ALOHA surface water sample analyzed  with Sensitive and Standard transit times (particle flow rates).
click to enlarge

Figure 2. Oligotrophic Station ALOHA surface water sample analyzed with Sensitive and Standard transit times (particle flow rates). Sensitive 25 μL /min (A) allows for better separation of the inherently dim red-fluorescent (y-axis) Prochlorococcus spp. (Pro) populations from the remaining SYBR® Green I–stained cells as compared Standard 25 μL/min (B). Slowing the particle flow rate can increase the laser interrogation and photon collection times from dim, low background populations.

(back to top)

Protocol: Direct Picophytoplankton Cell Counting-Intrinsic Fluorescence


Sample Preparation for Unstained Samples

Marine water samples can be analyzed with or without fixation (final concentration of 1% paraformaldehyde or 0.1% gluteraldehyde, 20 min). If an internal reference is desired, dilute Fluospheres® 1.0 µm yellow-green fluorescent microspheres (Invitrogen Cat. No. F13081) to 105 beads/mL in distilled water and add approximately 3x10^5 beads/mL of the diluted bead suspension to 1 mL of sample.


Instrument Setup for Unstained Samples

1. Replace the standard 603/48 BP filter in Violet Laser channel 3 (VL3) with a 640 LP filter. Note: The detection of Prochlorococcus spp. requires red-sensitive photomultiplier tubes in VL3 and BL3 detection channels.


  Figure 3. VL3 histogram of unstained Station ALOHA surface water sample with the threshold set near the point where background noise is beginning to enter the data (arrow).


2. Set collection rate to Sensitive 25 µL/min and acquisition volume to 50 µL. The acquisition volume can be changed before recording data to adjust for the number of events, time, or volume desired as the stop criteria for the analysis.

3. Select the View tab and select Instrument Settings. Select the Threshold tab and change the Boolean value under FSC to Ignore. Change the Boolean on VL3 to Or and set the value to 2.

4. With Instrument Settings still open, select the Voltages tab and use the following voltages as a starting point for your experiment (further adjustment will be necessary):


  • FSC: 1600
  • SSC: 2100
  • BL2: 2500
  • BL3: 2500
  • VL3: 2500

5. Select the Insert tab to view the available plots. Create the following dot plots for your data, making sure that SSC-H is set to logarithmic scaling:


  • BL2-H vs SSC-H (Synechococcus spp. population)
  • BL3-H vs SSC-H (Prochlorococcus spp. and picoeukaryotes populations from 488 nm excitation)
  • VL3-H vs SSC-H (Prochlorococcus spp. and picoeukaryotes populations from 405 nm excitation)
  • VL3-H vs BL3-H (All populations will be visible on this plot)

6. Begin collecting data using a sample that will contain the dimmest red population (e.g., oligotrophic surface water). Adjust the VL3 threshold and VL3 voltage to position the Prochlorococcus spp. on scale while eliminating background noise from the data. This is easiest to gauge on a VL3-H vs. SSC-H dot plot or VL3 histrogram (Figure 3). Voltage and threshold settings are sample dependent and will vary. Optimization of these settings should be performed for each experiment.


Acquisition and Analysis for Unstained Samples

With the collection rate set to Sensitive 25 µL/min, change the acquisition volume to 150 µL. Acquisition volumes will depend on the stop criteria used for the analysis. For the data presented here, the stop criteria were set to analyze a volume 125 µL (the equivalent of a 5 min run at a low flow rate on a conventional cytometer). To set the stop criteria, uncheck the boxes next to the event and time stop criteria in the collection panel and check the volume stop criteria, entering 125 µL as the volume to be analyzed. Press the Run button to begin the acquisition and the Record button as events begin to show in the displayed plots to begin the recorded acquisition of data. The acquisition will stop when 125 µL of sample has been analyzed (according to the set stop criteria). To assure correct cell counts, lower the sample tube lifter in between analyses of samples with dim fluorescence.


Select the Insert tab to view the Gating Tools. Gates are applied by selecting a plot on the workspace, selecting one of the gates from the Gating Tools bar and placing it around the population of interest. After gating the populations of interest, insert a Statistics box by selecting the gated plot and clicking the Statistics box. This creates a Statistics box specific to the gated plot. Right click on the Statistics box and scroll to Customize. Select the Concentration radio button to display population counts in events/µL. The samples in this experiment were not diluted; to obtain population counts in cells/mL, simply multiply the displayed number under Concentration by 1,000.


Results from unstained samples

Excitation of the endogenous divinyl-chlorophyll with the 405 nm laser combined with the increased interrogation time and photon collection from acoustic focusing cytometry, allowed for detailed separation and direct cell counting of Prochlorococcus spp. from the very oligotrophic Station ALOHA surface water sample where these marine picophytoplanktons contain low amounts of divinyl-chlorophyll (Figures 4 and 5).



Figure 4. Divinyl-chlorophyll fluorescence from 488 nm excitation (x-axis) versus divinyl-chlorophyll fluorescence from 405 nm excitation (y-axis) showing separation of populations (A) and direct cell counts (B) of picophytoplankton from unstained Station ALOHA site surface water sample.
click to enlarge

Figure 4. Divinyl-chlorophyll fluorescence from 488 nm excitation (x-axis) versus divinyl-chlorophyll fluorescence from 405 nm excitation (y-axis) showing separation of populations (A) and direct cell counts (B) of picophytoplankton from unstained Station ALOHA site surface water sample. The analyzed sample is from an oligotrophic area of the ocean. Few commercial cytometers are capable of resolving the Prochlorococcus spp. population from this very oligotrophic part of the ocean, and fewer will resolve this picophytoplankton population and provide a direct cell count. The concentration for Prochlorococcus (Pro) (27,000 events) observed from the oligotrophic Station ALOHA surface water sample was 216,000 cells/mL. The Synechococcus (Syn) cell count from this sample was 2,100 cells/mL. Picoeukaryote (Euk) cell count for this sample was 1,100 cells/mL. Fluospheres® 1.0 µm yellow-green fluorescent microspheres were added as an internal reference.

Results from unstained samples

Excitation of the endogenous divinyl-chlorophyll with the 405 nm laser combined with the increased interrogation time and photon collection from acoustic focusing cytometry, allowed for detailed separation and direct cell counting of Prochlorococcus spp. from the very oligotrophic Station ALOHA surface water sample where these marine picophytoplanktons contain low amounts of divinyl-chlorophyll (Figures 4 and 5).



Figure 5. Divinyl-chlorophyll fluorescence from 488 nm excitation (x-axis) versus divinyl-chlorophyll fluorescence from 405 nm excitation (y-axis) showing separation of populations (A) and direct cell counts (B) of picophytoplankton from unstained Station ALOHA site Deep Chlorophyll Maximum (DCM) water sample.
click to enlarge

Figure 5. Divinyl-chlorophyll fluorescence from 488 nm excitation (x-axis) versus divinyl-chlorophyll fluorescence from 405 nm excitation (y-axis) showing separation of populations (A) and direct cell counts (B) of picophytoplankton from unstained Station ALOHA site Deep Chlorophyll Maximum (DCM) water sample. The direct cell count for Prochlorococcus observed for the Station ALOHA site DCM sample was 113,000 cells/mL. The Synechococcus cell count calculated from the DCM sample was 300 cells/mL. Picoeukaryote (Euk) cell count for this sample was 2,400 cells/mL. Fluospheres® 1.0 µm yellow-green fluorescent microspheres were added as an internal reference.
(back to top)

Protocol: Bacterioplankton & Prochlorococcus Spp. Detection --SYBR® Green & Intrinsic Fluorescence


Sample preparation for SYBR® Green I–stained samples

Marine water samples may be analyzed with fixation (final concentration of 1% paraformaldehyde or 0.1% gluteraldehyde, 20 min). The fixation step ensures that all cells within the sample are stained with SYBR® Green I.


For DNA staining, prepare a 1:10 dilution of SYBR® Green I (Invitrogen Cat. No. S7563, supplied as a 10,000X stock solution) in distilled water. Add 1 µL of 1:10 dilution of SYBR® Green I dye per milliliter of sample and incubate in the dark for 30 min. The sample should not be washed before analysis.


Instrument setup for SYBR® Green I–stained samples

1. Replace the standard 603/48 BP filter in Violet Laser channel 3 (VL3) with a 640 LP filter. Note: The detection of Prochlorococcus spp. requires red-sensitive photomultiplier tubes in VL3 and BL3 detection channels.


2. Set collection rate to Sensitive 25 µL/min and acquisition volume to 50 µL. The acquisition volume can be changed before recording data to adjust for the number of events, time, or volume desired as the stop criteria for the analysis.


3. Select the View tab and select Instrument Settings. Select the Threshold tab and change the Boolean value under FSC to Ignore. Change the Boolean on BL1 to Or and the value to 10. Direct cell counts of bacterioplankton and Prochlorococcus spp. based on SYBR® Green I DNA staining and intrinsic fluorescence with the Attune® cytometer


4. With Instrument Settings still open, select the Voltages tab and use the following voltages as a starting point for your experiment:


  • FSC: 1600
  • SSC: 2100
  • BL2: 2500
  • BL3: 2500
  • VL3: 2500

5. Create the following dot plots for your data, make sure that SSC-H is set to logarithmic scaling. Click the Insert tab to view the available plots.


  • BL1-H vs SSC-H (SYBR® Green I positive population)
  • BL1-H histogram (SYBR® Green I positive population)
  • VL3-H vs BL1-H (Prochlorococcus spp. populations from 405 nm excitation)

6. Begin collecting data using a sample that will contain the dimmest red population (e.g. oligotrophic surface water). Adjust the BL1 threshold and voltage to position the SYBR® Green I positive populations on scale while eliminating background noise from the data. This is easiest to gauge from a BL1-H histogram (Figure 6). Voltage and threshold settings are sample dependent and will vary. Optimization of these settings should be performed for each experiment.


Figure 6. BL1 histogram of SYBR® Green I–stained Station ALOHA surface water sample with the threshold set near the point where background noise is beginning to enter the data.

Figure 6. BL1 histogram of SYBR® Green I–stained Station ALOHA surface water sample with the threshold set near the point where background noise is beginning to enter the data.(A) For analysis of bacterioplankton populations the threshold should be set so as not to include the virus population, which was shown here for clarity. Another strategy is to set a histogram gate for the SYBR® Green I positive populations of interest (B)


Acquisition and analysis for SYBR® Green I–stained samples

With the collection rate set to Sensitive 25 µL/min, change the acquisition volume to 150 µL. Acquisition volumes will depend on the stop criteria used for the analysis. For the data presented here, the stop criteria was set to analyze a volume 125 µL (the equivalent of a 5 min run at a low flow rate on a conventional cytometer). To set the stop criteria, uncheck the boxes next to the event and time stop criteria in the collection panel and check the volume stop criteria, entering 125 µL as the volume to be analyzed. Press the Run button to begin the acquisition and the Record button as events begin show in the displayed plots to begin the recorded acquisition of data. The acquisition will stop when 125 µL of sample has been analyzed (according to the set stop criteria). To assure correct cell counts, lower the sample tube lifter in between analyses of samples with dim fluorescence.


Select the Insert tab to view the Gating Tools. Gates are applied by selecting a plot on the workspace, selecting one of the gates from the Gating Tools bar and placing it around the populations of interest. After gating the populations of interest, insert a Statistics box by selecting the gated plot and clicking the Statistics box. Right click on the Statistics box and scroll to Customize. Select the Concentration radio button to display population counts in events/µL. When utilizing the gating strategy shown in Figure 6 be sure to set the population for each of the plots on the workspace to SYBR® Green I + gate. To do this, right click on the plot, scroll to Set Population, and select SYBR® Green I +. The samples in this experiment were not diluted; to obtain population counts in cells/mL, simply multiply the displayed number under Concentration by 1,000.


Setup a null compensation matrix to correct for the spillover of SYBR® Green I fluorescence into BL3 and VL3 channels by selecting the Home tab and then clicking the Compensation Setup button. Check the Height and all the fluorescent channel radio buttons for Unstained Control, BL1, BL3 and VL3 (Figure 7). Numerical values may be entered to subtract SYBR® Green I spillover or manually adjust the compensation on any bivariant fluorescent plot (e.g. VL3-H vs BL1-H) with the compensation slider bars.



Figure 7. Compensation matrix for SYBR® Green I spillover correction from the BL3 and VL3 channels show –5.00 and –1.30 respectively for this sample.

Figure 7. Compensation matrix for SYBR® Green I spillover correction from the BL3 and VL3 channels show –5.00 and –1.30 respectively for this sample.

Results from SYBR® Green I–stained samples SYBR® Green I staining of environmental marine samples enables the fluorescent detection and cell counting of the total microbial population present. Performing this analysis with the Attune® Acoustic Focusing Cytometer with 405 nm excitation allows direct cell counting of the divinyl-chlorophyll containing Prochlorococcus spp. in SYBR® Green I–stained samples (Figures 8 and 9).



Figure 8. SYBR® Green I fluorescence (x-axis) versus divinyl-chlorophyll fluorescence from 405 nm excitation (y-axis) showing separation (A) and direct cell counts of SYBR® Green I–stained bacterioplankton and Prochlorococcus spp. (Pro) from Station ALOHA surface water sample.
click to enlarge

Figure 8. SYBR® Green I fluorescence (x-axis) versus divinyl-chlorophyll fluorescence from 405 nm excitation (y-axis) showing separation (A) and direct cell counts of SYBR® Green I–stained bacterioplankton and Prochlorococcus spp. (Pro) from Station ALOHA surface water sample.Prochlorococcus spp. population cell count of 216,000 cells/mL strongly agrees with the cell count from the unstained sample. The heterotrophic population cell count calculated from this analysis was 530,000 cells/mL. Fluospheres® 1.0 µm yellow-green fluorescent microspheres were added as an internal reference.

Figure 9. SYBR® Green I fluorescence (x-axis) versus divinyl-chlorophyll fluorescence from 405 nm excitation (y-axis) showing separation (A) and direct cell counts of SYBR® Green I stained bacterioplankton and Prochlorococcus (Pro) spp. from Station ALOHA site DCM sample with detection from 405 nm laser excitation.

Figure 9. SYBR® Green I fluorescence (x-axis) versus divinyl-chlorophyll fluorescence from 405 nm excitation (y-axis) showing separation (A) and direct cell counts of SYBR® Green I stained bacterioplankton and Prochlorococcus (Pro) spp. from Station ALOHA site DCM sample with detection from 405 nm laser excitation. Prochlorococcus spp. population cell count of 109,000 cells/mL strongly agrees with the cell count from the unstained sample. The heterotrophic population cell count calculated from this analysis was 358,000 cells/mL. Fluospheres® 1.0 µm yellow-green fluorescent microspheres were added as an internal reference.

(back to top)

Conclusions


Prochlorococcus spp., Synechococcus spp., picoeukaryote and heterotrophic populations are readily detectable and directly enumerated by acoustic focusing cytometry, using the Attune® Acoustic Focusing Cytometer. In addition, Prochlorococcus spp. are detected and directly counted from the very oligotrophic surface waters from Station ALOHA, even when the sample is stained with the DNA stain SYBR® Green I for calculation of the heterotrophic population.

The direct cell counts determined from the samples reported in this brief communication concur with published data from Station ALOHA near Hawaii. For further reading about marine, photosynthetic picoplankton and their global significance, please see the references provided.


(back to top)

Tips & Tricks


1. Use the Sensitive 25 µL/min collection rate to increase the fluorescent signature of divinyl-chlorophyll containing Prochlorococcus spp. in the red detection channels (BL3 and VL3) with 640 LP filters. This is especially important when analyzing very oligotrophic surface water populations of Prochlorococcus spp.


2. Set all parameters for logarithmic scaling. This aids in identification of distinct populations for gating and data analysis.


3. Instrument setup (PMT voltages and fluorescent thresholds) should be performed with the dimmest fluorescent population being analyzed. Ensure the brightest fluorescent population is on scale before data acquisition.


4. Setting proper voltages and thresholds will maintain an event rate =1,000 events/second, minimizes the inclusion of coincidence in the data set and maximizes population resolution.


5. Creating a density plot of BL2 vs. SSC allows viewing the phycoerythrin containing Synechococcus spp. without the Prochlorococcus spp. and picoeukaryotes populations in unstained samples.


6. Lower the sample tube lifter in between analyses of samples with dim fluorescence ensures correct cell counts of populations of interest.


7. Creating a null compensation matrix after data acquisition is an easy way to correct spillover without havingto run single-color compensation controls.


8. Prior to data acquisition by flow cytometry, checking samples by fluorescence microscopy will confirm that the cells are stained.


9. Use LinLog scaling instead of Log scaling with compensated data to improve the resolution of the populations of interest.


(back to top)

Citations


1. Campbell L, Nolla HA, Vaulot D (1994). The importance of Prochlorococcus to community structure in the North Pacific Ocean. Limnol Oceanogr 39:954–961.


2. Marie D, Partensky F, Jacquet S et al. (1997). Enumeration and cell cycle analysis of natural populations of marine picoplankton by flow cytometry using the nucleic acid stain SYBR Green I. Appl Environ Microbiol 63:186–193.


3. Marie D, Partensky F, Vaulot D et al. (1999). Enumeration of phytoplankton, bacteria, and viruses in marine samples. Curr Protocol Cytom 47:11.11.1–11.11.15.


4. Partensky F, Hess WR, Vaulot D (1999). Prochlorococcus, a marine photosynthetic prokaryote of global significance. Microbiol Mol Biol Rev 63:106–127.


5. Veldhuis MJW, Kraay GW (2000). Application of flow cytometry in marine phytoplankton research: current applications and future perspectives. Sci Mar 64:121–134.


(back to top)