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This page last changed on Jul 11, 2013 by klimov.
Categories, methods
| Method |
Notes |
Applicability
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Adaptability
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Flowcytometry in a flow with optical detection
- Optical Forward Scatter or (FSC). Seems to be most efficient for optical power utilization.
- Optical Side Scatter or (SSC)
- Optical Absorption
- Fluorescence
- Multispectral with single detector: color-space-time coding (UCSD)
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FSC seems to be most efficient for optical power utilization and most common "signature" parameter, proxy to the organism biomass
SSC is quite dependent on the size and configuration of the scattering organism and if collected at one angle only, can be hard to interpret.
Fluorescence is common for taxonomy; signal levels are much lower, and usually require calibration with fluorescent bits.
"Traditional" flowcytometry and most common for bench top instruments.
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Pros:
- established method
- seems to be best for taxonomy (with fluorescence)
- resolution and ID size range down to sub um
Cons:
- size range is relatively narrow
- method of counting cells is optimized for relatively narrow range of target organism sizes
- hard to interpret data for larger cells (> 50um) with complex morphology
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Pros:
- probably can be adapted in a reasonable size / power configuration (miniaturization of a Seaflow)
- CytoSub can propable be integrated to Dorado
Cons:
- high cost
- usually bulky and complex instruments
- requiring multiple detectors and alignment
- miniaturized Seaflow must compromise on power and sensitivity, and would have even narrower dynamic range
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Imaging flow cytometry
- Configuration of the channel: round or flat
- Primary imaging system, vs. "image on trigger".
- Combination with adherent cytometry: Celico (Cyntelect, purchased by Brooks Automation) |
Commercial imaging flow cytometers seems to be oriented for oceanographic/ environmental markets.
"Image on trigger" helps to identify larger cells with complex morphology as add-on to "traditional" cytometer
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Pros:
- wider dynamic range
- better resolves larger cells with complex morphology
Cons:
- fundamental limit on low side of dynamic range; optical resolution > 1-2 um, ID limit is > 5-10 um
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Pros:
- relatively wide detection size range
- relatively simple fluid handling/ maintenance
Cons:
- require processing power;
- training/ image database;
- harder to implement fluorescence
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Holographic Imaging
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SHM, Submersible Holographic Microscope (Resolution Optics)
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Pros: ???
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Pros:
- LISST-HOLO adapted to Dorado
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Bulk optical properties
- Size-dependent diffraction
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Pros:
- optically, this is fast acquisition method
Cons:
- can be computationally intensive
- bulk optical properties measured as oppose to individual cells
- set of assumption on optical properties/ shapes of cells
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Pros:
- and LISST-100 adapted to Dorado
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Mass-spectrometry cytometry
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| Microfluidic cytometry |
Traditional flow cytometry with cell counting/, with the exception that sample handling is performed in a microfluidic structure usually made of silicone rubber.
Emerging technology; attempt to reduce size, cost of traditional flow cytometer; and also to simplify maintenance or confine the fluid handle for toxic/ biohazardous specimens
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Pros:
- confined fluid path
- simplified integration of solid-state detectors and light sources
Cons:
- how to maintain/replace microfluidic component
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Adherent cell cytometry.
- Proxy to membrane properties, mobility, cells size
- Resistance in a micro channel. Coulter counter, and proxy to cell volume |
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Trends:
Continue to decrease in size and energy consumption.
Use of smaller laser diodes/ solid state lasers.
Increase in detection and precision measurements
Lower cost especially for medical and bio sciences
Challenges
Limited dynamic range vs. unpredictable wide range population composition
Trade-off between size range, concentration range, and throughput is really optimized for *narrow range* of those parameters
If detection volume is set to be able to resolve pulses from small particles, then statistics on larger cells with low concentration is becoming unacceptably slow
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