: Non-specific binding is prevented using anti-Fc antibodies. Cells are then stained with fluorophore-conjugated antibodies, viability dyes, or nuclear dyes like DAPI or Hoechst to provide spatial landmarks.
IFC provides a "visual verification" for every data point, allowing researchers to see exactly what their gated populations look like.
: Systems like the ImageStream use Charge-Coupled Device (CCD) cameras with Time Delay Integration (TDI) to track cells in flow, preventing motion blur and enabling the detection of dim markers. Imaging Flow Cytometry: Methods and Protocols
: If internal markers (e.g., nuclear proteins) are the target, cells are fixed with formaldehyde and permeabilized with detergents like Triton X-100.
: Beyond fluorescence, IFC provides data on cell size, shape, circularity, and internal complexity. : Non-specific binding is prevented using anti-Fc antibodies
While IFC shares many preparatory steps with conventional flow cytometry, it requires specific optimizations to ensure high-quality imagery.
: Researchers must set specific software parameters, such as magnification and laser power, to maximize the resolution of the captured images. Common Applications : Systems like the ImageStream use Charge-Coupled Device
: Cells must be in a single-cell suspension to avoid clogging the thin fluidic channels used for imaging.