Flowcytometrie samenvatting
E-learning
1) Overview
- What is flow cytometry?
- What types of samples are suitable for analysis in a flow cytometer?
- What cell characteristics can a cytometer measure?
- What are the three subsystems in a flow cytometer?
2) Fluidics
- What are the main functions of the fluidics system?
- What is hydrodynamic focusing?
- How does sample pressure affect flow rate and data?
3) Optics
- What are the main functions of the optics system?
- What is the role of lasers in BD cytometers?
- How are longpass, bandpass and shortpass flters used to route specific wavelengths of light
to the appropriate detectors?
4) Electronics
- How does the electronics system convert light into data?
- What is the purpose of the laser time delay electronics?
- What are the functions and applications of threshold?
5) Optical measurement
- What factors affect light scatter?
- How are scattered light and fluorescence used to detect populations?
- Why is it important to know excitation and emission spectra for the fluorochromes in use?
- What is compensation and why is it necessary for multicolor experiments?
6) Data analysis
- How is data saved and displayed?
- What is the purpose of gating?
- What are examples of data analysis methods?
Flowcytometrie = a process of performing measurements on cells or particles that are in liquid
suspension.
- Meet en analyseert tegelijkertijd meerdere eigenschappen van enkele cellen of deeltjes terwijl
ze door een lichtbundel heengaan.
- Met flowcytometrie kan onderzoek gedaan worden en diagnoses gesteld worden.
Samples:
- Verschillende types, zoals cellen, chromosomen, bacteriën en ‘beads’
- Deeltjes moeten in vloeistof zitten
- Deeltjes van 0.5 – 50 μm kunnen met de meeste flowcytrometers gemeten worden.
Metingen:
- Relatieve grootte
- Relatieve interne complexiteit
- Relatieve intensiteit van de fluorescentie
Drie subsystemen:
- Een flowcytrometer bestaat uit drie subsystemen: fluidics, optics en electronics
- Ze werken samen om tegelijkertijd meerdere eigenschappen te meten van deeltjes, terwijl de
deeltjes in een vloeistofstroom door een lichtbundel bewegen.
,Fluidics subsystem
The fluidics subsystem brings particles of interest to the interrogation point where they interact with
the excitation sources.
- Werking:
o The fluidics subsystem transports particles in a fluidic stream to the laser beam, for
interrogation.
o The portion of the fluid stream where particles are located is called the sample core.
o The portion where particles are located is called the sample core.
o The point at which particles meet the laser beam is the interrogation point.
o Incident laser light is scattered and fluorescence is emitted as particles pass through
the interrogation point.
- Belangrijkste functies:
o Transport particles in a fluid stream to the laser beam for interrogation
o Position the sample core in the centre of the laser beam.
- Hydrodynamic focusing:
Used to maintain the sample core in the centre of the sheath fluid
o Sample core: the pressure of the sample fluid can be
adjusted to increase or decrease the diameter of the sample
core.
o Sheath fluid: surrounds the sample core. These fluids do not
mix as they pass through the flow cell.
o Flow cell: the shape of the flow cell enables hydrodynamic
focusing. In some cytometers, the laser beams pass through
the flow cell to interrogate the sample.
, - BD FACSCanto and BD FACSCalibur System Fluidics:
o In these cytometers, the sample stream is pressurized upwards through the flow cell.
o Within the cuvette flow cell, where particles are interrogated by the laser beam, is
called the interrogation point.
- BD FACSAria System Fluidics
o In this cytometer, the sample stream flows downwards through the cuvette flow cell.
o The stream is interrogated by the laser beam in the cuvette, and eventually exits
through the nozzle tip.
E-learning
1) Overview
- What is flow cytometry?
- What types of samples are suitable for analysis in a flow cytometer?
- What cell characteristics can a cytometer measure?
- What are the three subsystems in a flow cytometer?
2) Fluidics
- What are the main functions of the fluidics system?
- What is hydrodynamic focusing?
- How does sample pressure affect flow rate and data?
3) Optics
- What are the main functions of the optics system?
- What is the role of lasers in BD cytometers?
- How are longpass, bandpass and shortpass flters used to route specific wavelengths of light
to the appropriate detectors?
4) Electronics
- How does the electronics system convert light into data?
- What is the purpose of the laser time delay electronics?
- What are the functions and applications of threshold?
5) Optical measurement
- What factors affect light scatter?
- How are scattered light and fluorescence used to detect populations?
- Why is it important to know excitation and emission spectra for the fluorochromes in use?
- What is compensation and why is it necessary for multicolor experiments?
6) Data analysis
- How is data saved and displayed?
- What is the purpose of gating?
- What are examples of data analysis methods?
Flowcytometrie = a process of performing measurements on cells or particles that are in liquid
suspension.
- Meet en analyseert tegelijkertijd meerdere eigenschappen van enkele cellen of deeltjes terwijl
ze door een lichtbundel heengaan.
- Met flowcytometrie kan onderzoek gedaan worden en diagnoses gesteld worden.
Samples:
- Verschillende types, zoals cellen, chromosomen, bacteriën en ‘beads’
- Deeltjes moeten in vloeistof zitten
- Deeltjes van 0.5 – 50 μm kunnen met de meeste flowcytrometers gemeten worden.
Metingen:
- Relatieve grootte
- Relatieve interne complexiteit
- Relatieve intensiteit van de fluorescentie
Drie subsystemen:
- Een flowcytrometer bestaat uit drie subsystemen: fluidics, optics en electronics
- Ze werken samen om tegelijkertijd meerdere eigenschappen te meten van deeltjes, terwijl de
deeltjes in een vloeistofstroom door een lichtbundel bewegen.
,Fluidics subsystem
The fluidics subsystem brings particles of interest to the interrogation point where they interact with
the excitation sources.
- Werking:
o The fluidics subsystem transports particles in a fluidic stream to the laser beam, for
interrogation.
o The portion of the fluid stream where particles are located is called the sample core.
o The portion where particles are located is called the sample core.
o The point at which particles meet the laser beam is the interrogation point.
o Incident laser light is scattered and fluorescence is emitted as particles pass through
the interrogation point.
- Belangrijkste functies:
o Transport particles in a fluid stream to the laser beam for interrogation
o Position the sample core in the centre of the laser beam.
- Hydrodynamic focusing:
Used to maintain the sample core in the centre of the sheath fluid
o Sample core: the pressure of the sample fluid can be
adjusted to increase or decrease the diameter of the sample
core.
o Sheath fluid: surrounds the sample core. These fluids do not
mix as they pass through the flow cell.
o Flow cell: the shape of the flow cell enables hydrodynamic
focusing. In some cytometers, the laser beams pass through
the flow cell to interrogate the sample.
, - BD FACSCanto and BD FACSCalibur System Fluidics:
o In these cytometers, the sample stream is pressurized upwards through the flow cell.
o Within the cuvette flow cell, where particles are interrogated by the laser beam, is
called the interrogation point.
- BD FACSAria System Fluidics
o In this cytometer, the sample stream flows downwards through the cuvette flow cell.
o The stream is interrogated by the laser beam in the cuvette, and eventually exits
through the nozzle tip.
