Keep event rate under 1,000-2,000 events/second. High speed distorts FSC-A due to pulse overlap.
specifically integrates the entire area under the pulse generated as the cell traverses the laser. Imagine a Gaussian curve: as the cell enters the laser, the signal rises; as it passes through the center, the signal peaks; as it exits, the signal falls. The area under this entire curve is the FSC-A value. Part 2: FSC-A vs. FSC-H vs. FSC-W – The Trinity of Pulse Processing Modern digital flow cytometers do not simply record a single number. They record the full pulse shape and derive three parameters: Area (A) , Height (H) , and Width (W) . Understanding the distinction is critical. Keep event rate under 1,000-2,000 events/second
Use a threshold (e.g., FSC-A > 5,000) to exclude electronic noise and debris. Never threshold on a fluorescence channel unless you have a specific reason. Imagine a Gaussian curve: as the cell enters
Plot FSC-A (X-axis) vs. FSC-H (Y-axis). Draw a polygon tightly around the diagonal population. Alternatively, use FSC-W vs. FSC-A. The singlet gate should exclude events with high FSC-W or mismatched A/H ratios. FSC-H vs
Run a mix of small (3µm) and large (6-10µm) beads to check the dynamic range. Adjust FSC voltage so both populations are on scale (usually between 10^2 and 10^5 on a log scale or 100-200K on a linear scale).
refers to light that is scattered by the cell at small angles (typically 0.5 to 10 degrees) relative to the laser axis. This light is collected by a photodiode placed directly in line with the laser beam. The Relationship Between Size and Refractive Index The intensity of forward-scattered light is proportional to the square of the cell diameter (its cross-sectional area). However, it is not solely size-dependent. The cell’s refractive index (RI) – a measure of internal complexity and granularity – also plays a role. A large, pale lymphocyte and a small, granulated neutrophil might produce similar FSC signals, which is why FSC is best described as a measure of optical volume rather than absolute physical size.