FlowCAM image acquisition protocol
FlowCAM primer
Natalie Mladenov - 27 May 2011
Materials:
· Clean 100 mL beaker
· Focusing solution (some kind of solution with particles in it that lets you focus the sample so that you don’t waste real sample).
· Unfiltered sample
Procedure:
Preparation
1. Have at least 10 mL of an unfiltered sample ready. If the sample has a lot of visible, large detritus it will need to be pre-filtered.
2. Check that the FlowCAM is set up with a 10x objective and accompanying flow cell. If it’s not, you will need to do this. Watch the video or ask Natalie Mladenov or Sarah Spaulding for help.
3. Check the size of the tubing.
4. Look at the tables to see what speed the dial should be set to so that multi-imaging does not occur.
Multi-imaging = when the camera picks up the same particle in more than one frame, thereby counting it twice (or more).
5. Find the tubing that is out of position and replace it.
6. Make sure one end of tubing is discharging into a waste beaker and the other has a clean pipette tip attached.
7. Turn on the FlowCAM.
8. Turn on the FlowCAM software by double clicking the icon that corresponds to the 10x objective.
Clean the tubing
1. Add ethanol to the pipette tip.
2. Flip the left switch to “forward”. And flip the right switch to “Prime”
3. Prime the ethanol all the way through the tubing for about 5 minutes to clean the tubing.
4. Turn off the pump with the left switch.
5. Flip the right switch to “Slow” or “Fast” depending on the settings you determined from the tables. Turn the dial to the number that corresponds to this setting.
6. Add focusing solution to the pipette tip and flip the pump on again with the left switch.
Focus the microscope
1. In the software click on “Setup” in the top menu. Under setup, click “Setup and Focus”. A gray screen will appear.
2. With your hand slide the flow cell translator to the right or left. You may also have to turn the x-y knob until you see the white line. This is the side of the flow cell. Once you spot the white line, tighten down the flow cell translator.
3. Adjust the fine focus until particles are in focus.
Imaging particles
1. Exit the “Setup and Focus” window.
2. Allow all the focusing solution to be pumped out of the tubing.
3. Switch out the waste beaker for the clean 100 mL beaker – you will be collecting your unfiltered sample in this beaker.
4. Make sure the pump is on and that the dial and right switch are set to the appropriate settings.
5. Add your unfiltered sample to the pipette tip and wait for it to flow past the flow cell.
6. Click “Analyze” in the top menu. Under analyze, click “Autoimage mode”.
7. The software will prompt you to save the images that you are about to collect. Choose an appropriate folder.
8. Once you hit “save” the FlowCAM will start imaging the particles it sees.
9. Allow the FlowCAM to collect images for exactly 10 minutes. This is equal to ____________ mL. You may need to set a timer.
10. Click on the “x” in the top right corner when it’s time to stop collection.
11. Repeat #6-10 above two more times so that you have imaged the sample three times in total. You may have to reuse sample from the 100 mL beaker. If there are any problems with the images collected, you can always collect additional image files. You may need to refocus if the images begin to look blurry.
12. Run the remaining sample through the tubing until the tubing is empty. Turn off the pump.
13. Save the sample that’s in the beaker.
14. Label the bottle in some way to indicate that the sample has already been run.
Analyzing the data
· This depends on what particles you are looking for. You can build a library and do a lot with particle recognition. Watch the videos.
Closing down
· Place the end of the tubing back into the waste beaker and flip the switches to “forward” and “prime”.
· Run ethanol through the flow cell for 15 minutes.
· Make sure the tubing is empty and turn off the pump.
· Turn off the instrument. Unhitch the tubing so that it is loose while not in use.
Natalie Mladenov - 27 May 2011
Materials:
· Clean 100 mL beaker
· Focusing solution (some kind of solution with particles in it that lets you focus the sample so that you don’t waste real sample).
· Unfiltered sample
Procedure:
Preparation
1. Have at least 10 mL of an unfiltered sample ready. If the sample has a lot of visible, large detritus it will need to be pre-filtered.
2. Check that the FlowCAM is set up with a 10x objective and accompanying flow cell. If it’s not, you will need to do this. Watch the video or ask Natalie Mladenov or Sarah Spaulding for help.
3. Check the size of the tubing.
4. Look at the tables to see what speed the dial should be set to so that multi-imaging does not occur.
Multi-imaging = when the camera picks up the same particle in more than one frame, thereby counting it twice (or more).
5. Find the tubing that is out of position and replace it.
6. Make sure one end of tubing is discharging into a waste beaker and the other has a clean pipette tip attached.
7. Turn on the FlowCAM.
8. Turn on the FlowCAM software by double clicking the icon that corresponds to the 10x objective.
Clean the tubing
1. Add ethanol to the pipette tip.
2. Flip the left switch to “forward”. And flip the right switch to “Prime”
3. Prime the ethanol all the way through the tubing for about 5 minutes to clean the tubing.
4. Turn off the pump with the left switch.
5. Flip the right switch to “Slow” or “Fast” depending on the settings you determined from the tables. Turn the dial to the number that corresponds to this setting.
6. Add focusing solution to the pipette tip and flip the pump on again with the left switch.
Focus the microscope
1. In the software click on “Setup” in the top menu. Under setup, click “Setup and Focus”. A gray screen will appear.
2. With your hand slide the flow cell translator to the right or left. You may also have to turn the x-y knob until you see the white line. This is the side of the flow cell. Once you spot the white line, tighten down the flow cell translator.
3. Adjust the fine focus until particles are in focus.
Imaging particles
1. Exit the “Setup and Focus” window.
2. Allow all the focusing solution to be pumped out of the tubing.
3. Switch out the waste beaker for the clean 100 mL beaker – you will be collecting your unfiltered sample in this beaker.
4. Make sure the pump is on and that the dial and right switch are set to the appropriate settings.
5. Add your unfiltered sample to the pipette tip and wait for it to flow past the flow cell.
6. Click “Analyze” in the top menu. Under analyze, click “Autoimage mode”.
7. The software will prompt you to save the images that you are about to collect. Choose an appropriate folder.
8. Once you hit “save” the FlowCAM will start imaging the particles it sees.
9. Allow the FlowCAM to collect images for exactly 10 minutes. This is equal to ____________ mL. You may need to set a timer.
10. Click on the “x” in the top right corner when it’s time to stop collection.
11. Repeat #6-10 above two more times so that you have imaged the sample three times in total. You may have to reuse sample from the 100 mL beaker. If there are any problems with the images collected, you can always collect additional image files. You may need to refocus if the images begin to look blurry.
12. Run the remaining sample through the tubing until the tubing is empty. Turn off the pump.
13. Save the sample that’s in the beaker.
14. Label the bottle in some way to indicate that the sample has already been run.
Analyzing the data
· This depends on what particles you are looking for. You can build a library and do a lot with particle recognition. Watch the videos.
Closing down
· Place the end of the tubing back into the waste beaker and flip the switches to “forward” and “prime”.
· Run ethanol through the flow cell for 15 minutes.
· Make sure the tubing is empty and turn off the pump.
· Turn off the instrument. Unhitch the tubing so that it is loose while not in use.
