Tutorials, the basics spelled out for you.
Flow Cell - Illustrates the fluorescence signal produced when a cell or particle labeled with a fluorochrome passes through a laser excitation beam. Note that a signal is produced only when there is cell or particle passing through the laser beam. Also note that the flow stream is in laminar flow with a concentric sheath stream traveling at a higher rate surrounding it to keep the cell or particles centered while they pass through the laser beam.
Spectra - Illustrates the intensity (excitation efficiency) of the emission signal as a function of the excitation wavelength. Note that the position and shape of the emission spectrum does not change as a function of the excitation wavelength. Only the intensity of the emission changes with excitation wavelength and it is a maximum at the peak of the excitation spectrum.
Filters - These animations illustrate the various types of barrier filters and mirrors used in a flow cytometer to direct and limit the optical path of the excitation and emission energies.
• Short Pass Filter - This filter will only pass light below 500nm.
• Long Pass Filter - This filter will only pass light above 650nm.
• Band Pass Filter - This filter will only pass light between 560 and 590nm.
• Short Pass Dichroic Mirror - This filter will reflect light greater than 500nm and pass light less than 500nm.
• Long Pass Dichroic Mirror - This filter will reflect light less than 650nm and pass light greater than 650nm.
Log Sample Space - This animation illustrates fluorescence intensity Log Sample Space and the relationship between non-specifically and specifically labeled cells, as well as spectrally matched standards. The relative positions of each population will remain fixed and independent of the instrument measuring them. Note that the fluorescence intensities cover only a small portion of the intensity scale.
pH vs. Intensity - This animation illustrates the affect of pH on the fluorescence intensity of fluorescein (FITC)-labeled standards (B) and cells (C). Note that the fluorescence intensity of standards (A), which are not labeled with fluorescein, is not affected by change in pH. Note that cells (C) and standards (B) labeled with phycroerythrin (PE) are not responsive to pH until the pH is above 8.0. This indicates that environmentally-responsive standards (having the same fluorochrome in the same environment) should be used as reference standards.
• pH vs. FITC
• pH vs. PE
PMT - This animation illustrates the affect of the position of the Window of Analysis as a function of the photomultiplier (PMT) setting. Note that position of the sample does not move in Log Sample Space, rather only the Window of Analysis shifts as a function of the PMT setting. Also note that the Window of Analysis does not change size or shape with changing PMT settings.
Log Amplification - This animation illustrates the portion of Sample Space that an instrument can analyze as a function of the log amplification. Note as the log decades decrease, the range of Sample Space viewed by the instrument also decreases with each of the populations appearing to occupy a larger portion of the analysis range.
Filter vs. Target - This animation illustrates the relative positions between a reference standard and a labeled cell as a function of the analysis barrier filters. Note when the spectra of the standard and sample match (B) and (C), they move proportionally when different barrier filter wavelengths are used. However, when non-matching spectral standards are used, the relative positions between the sample and standard vary as a function of the filter.
Furthermore, the animation illustrates that matching spectra allow a fixed Target Channel to be assigned, which keeps the reference standard and sample in the same position of the Window of Analysis.
Antibody Binding - These animations illustrate how antibodies bind specifically to cell surfaces and through the Fc binding site to microbead standards. These microbeads have polyclonal anti-Mouse antibodies covalently bound to their surface, which acts as the receptor.
• Specific Fab Antibody Binding
• Fc Antibody Binding to Microbead Standards
• Fab Antibody Binding to Cells
I. Determination of Antibody Binding using Quantum™ MESF Standards and Simply Cellular® Microspheres
A) Determination of the Effective F/P Ratio
1. Saturate a population of Simply Cellular® microspheres with the antibody used to label the cell sample.
2. Calibrate the flow cytometer in MESF units of the conjugated fluorochrome.
3. Determine the MESF of the labeled Simply Cellular® microspheres.
4. Calculate the Effective F/P ratio of the antibody by dividing the MESF value by the assigned number of binding sites on Simply Cellular® microspheres.
B) Determination of Antibody Binding Capacity (ABC) of Cell Sample
1. Saturate a cell sample with the antibody used to label the Simply Cellular® microspheres.
2. Determine the MESF value of the cell sample using the MESF calibration plot.
3. Divide the MESF value of the cell sample by the effective F/P ratio.
II. Determination of Antibody Binding using Quantum™ Simply Cellular® Standards.
1. Saturate Quantum™ Simply Cellular® with antibody used to label cell sample.
2. Saturate cell sample with antibody used to label the Quantum™ Simply Cellular® microspheres.
3. Calibrate the flow cytometer in ABC units of the antibody.
4. Determine the antibody binding of the cell sample with the ABC calibration plot.