What are the differences between forward and side scatter in flow cytometry?

What are the differences between forward and side scatter in flow cytometry?

In flow cytometry, forward scatter (FSC) and side scatter (SSC) are two complementary light-scattering signals used to infer cell properties as cells pass through a laser beam.

Key differences:

• What they measure
  • Forward Scatter (FSC): Correlates with cell size (more precisely, the cross-sectional area of the cell as it diffracts light). Larger cells tend to scatter more light in the forward direction.
  • Side Scatter (SSC): Correlates with internal complexity or granularity (internal organelles, vesicles, granules, nuclei). More complex or granular cells scatter more light to the side.
• Detection geometry
  • FSC: Collected by detectors placed in the forward direction, typically around 0–15 degrees relative to the laser axis.
  • SSC: Collected by detectors placed at a 35–70 degree angle to the laser axis, perpendicular to the forward path.
• Informational content
  • FSC provides information about cell size, which can help distinguish cell populations by size (e.g., lymphocytes vs. monocytes, platelets vs. red blood cells are often gated by FSC).
  • SSC provides information about internal structure and granularity, helping to distinguish cell types with different internal complexity (e.g., granulocytes have higher SSC than lymphocytes; dead/damaged cells may have altered SSC).
• Sensitivity and limitations
  • FSC can be influenced by cell shape, refractive index, and the instrument’s optical alignment. It is less reliable for very small particles (e.g., platelets or beads near instrument noise) where size differences are minimal.
  • SSC is influenced by granularity and internal composition, which can also be affected by fixation, staining, or cell state (e.g., activation, apoptosis). It is often more variable between samples and vendors due to differences in detector geometry and laser configuration.
• Practical use in experiments
  • Gating strategy: FSC vs. SSC plots (e.g., FSC-A vs. SSC-A) are commonly used to gate cell populations by size and granularity before applying fluorescent antibody gates.
  • Paired analysis: Combining FSC and SSC with fluorescence channels allows better separation of cell types (e.g., lymphocytes typically appear low FSC and low-to-moderate SSC, granulocytes appear higher SSC, monocytes intermediate FSC/SSC).
• Example interpretation
  • Lymphocytes: relatively small with low internal complexity ⇒ low FSC, low SSC.
  • Monocytes: larger than lymphocytes and more granulated ⇒ higher FSC and higher SSC.
  • Granulocytes (neutrophils, eosinophils): intermediate to high FSC and high SSC due to multi-lobed nuclei and granules.
  • Platelets: very small, very low FSC and SSC, often distinguished by specific platelet markers rather than FSC/SSC alone.
• Technical tips
  • Proper gating: Use a clean population (e.g., singlets via FSC-A vs. FSC-H or SSC-A vs. SSC-H) to exclude doublets.
  • Instrument calibration: Regularly calibrate FSC and SSC using beads of known size and refractive index, and be aware that different cytometers (different lasers, objectives) will have different FSC/SSC scales.
  • Sample state: Fixation, permeabilization, or cell activation can shift FSC/SSC distributions; include appropriate controls.