Flow cytometry is a tecnique that measures and then analyzes multiple physical characteristics of single particles, such as cells, as they flow in a fluid stream through a beam of light, the laser. Any sustended particle or cell from 0.2 -150 μm in size is suitable for analysis. The properties measured include a particle’s :
- relative size (Forward Scatter);
- relative granularity or internal complexity ( Side Scatter);
- relative fluorescence intensity ( Fluorescent Channels).
Systems:
For a more detailed view of the flow cytometry the components are the fluid sheath, laser, focusing optics lents, photomultiplier tubes (PMTs), analogue-to-digital converter and analysis work station.
Fig: www.semrock.com/flow-cytometry.aspx
Reference: Tavares, S. and Tavares A. (2009) .Principios gerais de Cultura de Células e Citometria de Fluxo para avaliação dos efeitos da radiação ionizante.Relatório Interno. FEUP
Silva, T., Reis, A., Hewitt, C., Roseiro, J. (2004) Citometria de Fluxo- Funcionalidade celular on-line em bioprocessos. Boletim de Tecnologia pp 32-40
- The fluidics: transports particles in a stream to the laser beam;
- The optics: lasers to illuminate the particles in the sample stream;
- The electronics: converts the detected light signals into electronic signals that can be processed by the computer. For some instruments equipped with a sorting feature, this system is also capable of initiating sorting decisions to charge and deflect particles (voltage).
For a more detailed view of the flow cytometry the components are the fluid sheath, laser, focusing optics lents, photomultiplier tubes (PMTs), analogue-to-digital converter and analysis work station.
Fig: www.semrock.com/flow-cytometry.aspx
Reference: Tavares, S. and Tavares A. (2009) .Principios gerais de Cultura de Células e Citometria de Fluxo para avaliação dos efeitos da radiação ionizante.Relatório Interno. FEUP
Silva, T., Reis, A., Hewitt, C., Roseiro, J. (2004) Citometria de Fluxo- Funcionalidade celular on-line em bioprocessos. Boletim de Tecnologia pp 32-40
Fluids System
- Interrogation point is the point at which particles pass one at a time through the laser light beam.
- Event is the light scatter registered for each particule or cell.
- The cell enters the laser beam and the voltage pulse is created.
There can be two flow rates from witch the sample is analysed in the flow chamber:
It´s crucial for a good analysis for the fluidics system to be free of air bubbles and debris and properly pressurized at all times, in order for the particles intersect the laser beam correctly.
Fig: Leach, M., Drummond, M., and Doig, A. (2013) Pratical Flow Cytometry in Haematology Diagnosis. John Wiley & Sons, Ltd.
Reference: Leach, M., Drummond, M., and Doig, A. (2013) Pratical Flow Cytometry in Haematology Diagnosis. John Wiley & Sons, Ltd.
http://probes.invitrogen.com/resources/education/tutorials/4Intro_Flow/player.html
- Slow flow rate = low sample pressure = narrow sample stream = cells pass beam in single file.Ex: DNA analysis
- High flow rate = high sample pressure = wider sample stream = more than one cell passes through the beam at a time. Ex: Immunophenotyping
It´s crucial for a good analysis for the fluidics system to be free of air bubbles and debris and properly pressurized at all times, in order for the particles intersect the laser beam correctly.
Fig: Leach, M., Drummond, M., and Doig, A. (2013) Pratical Flow Cytometry in Haematology Diagnosis. John Wiley & Sons, Ltd.
Reference: Leach, M., Drummond, M., and Doig, A. (2013) Pratical Flow Cytometry in Haematology Diagnosis. John Wiley & Sons, Ltd.
http://probes.invitrogen.com/resources/education/tutorials/4Intro_Flow/player.html
Optical System
As a cell passes through the interrogation point, light from the laser beam is scattered:
The FSC and SSC signals give us information on the physical properties of the cells allowing differentiation within a fluid sample. The light that has a wavelength different from that emitted, which can be adjusted to a specific wavelength, depending on different fluorophores, is detected by the Side FLuorescence detector (SFL).
Reference: Leach, M., Drummond, M., and Doig, A. (2013) Pratical Flow Cytometry in Haematology Diagnosis. John Wiley & Sons, Ltd.
As a cell passes through the interrogation point, light from the laser beam is scattered:
- Forward scattered light (FSC): Light diffracted at forward angle to the laser beam. The amount of FSC is proportional to the surface area or size of the cell. FSC is collected by a detector placed in line with the laser beam on the opposite side of the sample stream.
- Side scattered light (SSC): the light at 90º angles will pass through the cell membrane and is refracted and reflected by cytoplasmic organelles or nucleus of the cell. This light is collected by a photodiode positioned at approximately 90º to the laser beam. SSC is proportional to the granularity or internal complexity of the cell.
The FSC and SSC signals give us information on the physical properties of the cells allowing differentiation within a fluid sample. The light that has a wavelength different from that emitted, which can be adjusted to a specific wavelength, depending on different fluorophores, is detected by the Side FLuorescence detector (SFL).
Reference: Leach, M., Drummond, M., and Doig, A. (2013) Pratical Flow Cytometry in Haematology Diagnosis. John Wiley & Sons, Ltd.