Questions for Resolution Optics

What is maximum recommended particle concentration? User manual says "In order to achieve accurate images, inline holography requires that a reasonable amount of the reference wave (unscattered light) reaches the camera sensor. Too many objects between the PS and the camera can reduce the amount of reference wave reaching the camera and will therefore result in poor reconstructions." RO looked at 5-10 micron plankton at 5x106 cells/liter with no trouble.

What exactly is output of realtime processing? Predetermined stack of images? Configurable? Can select single image depth for realtime processing. Can save other depths for later offline processing.

Automated image depth determination? This capability will be provided by RO's "Stingray" software, now in development

Realtime particle classification?  Realtime particle size distribution, other statistics in one image plane provided by "Swordfish" software

How does spatial resolution vary with image plane depth? Highest resolution (1 micron) when target is against source window. Lowest resolution (4 microns) when target against camera window

How to discrimate between "old" and "new" particles in each image? (not pumped?)
If not a fresh volume every time, how to discriminate shift direction, etc? How to get adequate volumetric statistics?

How to integrate instrument with mobile platform, e.g. flow cell with pump, vs "passive" flow based on vehicle motion? Turbulent vs laminar flow, etc?

Instrument fouling issues? What if particle stuck to window? (e.g. can processing eliminate specific image planes, like against the window?) Smooth sapphire windows may discourage biofouling, but likely to be an issue. Particles stuck to window can be eliminated through software.

What about embedded processing system? RO is working on an embedded battery-powered version; maybe available September 2014?

Julio's questions:
- instrument flow rate
- resolution range (size range of organisms the instrument can image)
- percentage of flow-through volume imaged by the instrument's "eye"
- power consumption 5 Watts
- image storage (memory) capacity and total number of images possible Depends on laptop drive capacity
- options for variable sampling — burst mode versus continuous sampling over a deployment.  Possible for the AUV to trigger sampling in an adaptive manner?

ain outcome - MBARI can borrow a Submersible Microscope (older version) for 1.5 months maximum, starting "in April". Sergey or John might be able to visit MBARI to help us get started. Instrument is rated to 2000 meters, but currently has a 10 meter gigabit ethernet cable (R.O. might have a longer cable to loan us). Sample spacer is adjustable on this model.

Notes from the telecon

Here are my actual notes of the meeting - please feel free to comment or correct.

Attendees:
Stephen Jones, Sergey Missan, George McMurtry, John Sampson
John Ryan, Brett Hobson, Francisco Chavez, Steve Haddock, Julio Harvey, Danelle Cline, Hans Thomas, Thom Maughan, Tom O'Reilly

Loaner SM instrument has same optical characteristics as newest version.

Spatial resolution varies with source-to-target distance. Best resolution is about 1 micron when close to source, 4 microns when close to camera.

Imaged volume is about 12 microliters: 2 x 2 mm FOV, 3 mm depth
Sampling rate is 16 fps, 190 microliters/sec
Single plane image transformation at 16 fps, i.e. can keep up in real time. Other planes can be processed later.

Check out the SM gallery

Software packages

Octopus: "manual" processing (hologram-to-image transform, measure size, etc)

Swordfish: High-speed real-time particle characterization (16 fps), historgrams, statistics

Stingray (in development): Automatic target tracking across image planes, morphological features, classifier. Problematic to track when targets overlap.

Particle concentration: if too many particles, there's not enough light for good reconstruction. But RO looked at 5-10 micron plankton at 5x106 cells/liter with no trouble.

Sample spacer - adjustable in older model (the model we'd borrow), fixed at 8 mm in latest model.

Instrument has been deployed for up to 1 day at least.
Sapphire windows are smooth, but biofouling likely an issue for longer deployments.

To process, need reference holograms(s) - to options:
1. Collect clear-water reference at start of deployment, or
2. Alternate between reference and sample images during deployment (not sure how this works operationally - Tom)

Processing requires Windows laptop with Invidia gpu; raw data and images stored to laptop drive.

Instrument requires 12 V power, gigabit ethernet connection to laptop.

R.O. is working on an embedded, battery-powered data logger - maybe available in August or September 2014?

Camera: 2048 x 2048 pixel, 8 bits monochrome, 16 fps, up to 50 fps in binned mode
Holograms and images stored as png, tiff (other formats?)

Data can be analyzed with LabView, other software thanks to standard image formats.

Loaner instrument is rated to 2000 meters, but has 10 meter cable (R.O. might be able to loan us a longer one).

Loaner sample spacer can be adjusted.
Sergey or John Sampson may be able to come to MBARI to help set up and use the instrument.

Sergey will send document that describes method limitations.

Sergey has not seen any processing bottlenecks running at 16 fps for 24 hours.