Bellingham's notes

Notes from telecon with the FlowCAM folks:

Attendees: Bellingham, Chavez, Klimov, Jac Fought, Harry Nelson (Fluid Imaging)

Francisco:

160-170 codes for organisms quantified in 'standard' water analysis.  larger by microscopy, smaller by flow cytometry, some ~um size in both.

Samples 250 ml in size.

Preserve material with glutaraldehyde, filter 25 ml of fluid.
- microscopy done from filter
- some fluid frozen w liquid N2 for cytometer (sent to U of Hawaii)

Catalog organisms to 200 um in size

Lots of small stuff - use staining to see bacteria, etc.

For the microscopy - use different magnifications and look over different areas

Harry:

We would probably use 10x objective for 100x magnification over 100um couvette w lower limit of about 10um critter.

4x objective has lower effective size resolution of 40um.

Can use 20x objective w 50um couvette but 'takes a while'

Harry will send us all some sample images of organisms at the various resolutions.

User builds their own library, does not come with library.  Library is used for classification.  Have taxonomic photos, but not a library.

How many entries does it need?  Depends.

Three step process - build a library, build a filter, build a template from the filters.  Is dynamic - can add and subtract from them.  Use the filter iteratively (first pass for organism 1, then for organism 2, etc.).

Could build some libraries ahead of time.

Chavez:

Can we also get size distribution of what is in sample?

Harry:

Yes, that just comes from pixels.  Easily done.  Can also use fluorescence, but if the sample is really concentrated then several organisms might be in field of view.

1/4 ml/min volumetric flow rate for 100 um couvette.

Outcome of  the meeting is that Francisco sent them some preserved material.  They are running it this week.  We are going to get a copy of their software and work on developing a library for the resulting images, and use it to develop a classifier for the remainder of the data....

Additional notes from Klimov:

10x objective, 100x magnification, 10-20um resolved microorganisms, 100um flow cell. This is a most common configuration.
4x objective, 40x magnification, 25-30um resolved microorganisms, 200um flow cell.
25x objective, 250x magnification, 50um flow cell. For highest resolution but also slowest pumping rate.

It was not clear what is the actual optical resolution of the system with say 10x objective. It should be higher then specified resolved microorganism size, perhaps 1-2 um.
Harry said "10 um" is "many pixels", as can be seen in "sample pictures".