Hydrodaynamic Focusing

Core Concept: Hydrodynamic Focusing

• Definition: Hydrodynamic focusing is a process that uses a sheath fluid to narrow the stream of sample fluid, forcing cells or particles to pass through an interrogation point in a single file
• Purpose:
  • Ensures that only one cell/particle at a time passes through the laser beam, preventing coincidence (multiple events being detected as one).
  • Optimizes light scatter and fluorescence signal detection by controlling cell position
  • Increases sample throughput
• Mechanism:
  • Sheath Fluid: A clean, particle-free fluid that surrounds the sample core
  • Pressure Differential: The sheath fluid is introduced at a higher pressure than the sample fluid
  • Nozzle/Flow Cell Design: Specific geometry of the flow cell directs and constricts the sample stream
  • Laminar Flow: The fluid dynamics are designed to create laminar flow (smooth, layered flow), preventing turbulence and mixing
  • Focused Core Stream: The higher pressure of the sheath fluid compresses the sample stream into a narrow core, typically a few micrometers in diameter

Key Factors Affecting Hydrodynamic Focusing

• Sheath Fluid Pressure:
  • Increased pressure: Narrower core stream, higher cell velocity, increased sample throughput, lower resolution
  • Decreased pressure: Wider core stream, lower cell velocity, decreased sample throughput, better resolution
• Sample Fluid Pressure:
  • Increased pressure: Wider core stream
  • Decreased pressure: Narrower core stream
• Flow Cell Geometry:
  • The design of the nozzle or flow cell (e.g., size, shape, angle) significantly impacts the focusing efficiency and core stream dimensions
• Fluid Viscosity:
  • Higher viscosity: affects the flow rate
• Fluid Temperature:
  • Temperature changes: can affect the fluidic properties
• Coaxial Alignment:
  • Misalignment: affects the flow profile

Sheath Fluids: Properties and Considerations

• Composition: Typically a balanced salt solution (e.g., PBS) or deionized water with added components
• Key Properties:
  • Purity: Must be free of particles (bacteria, debris) that could interfere with cell detection or clog the system
  • Sterility: Prevents microbial growth within the instrument’s fluidic system
  • Isotonicity: Should be isotonic to cells to prevent osmotic stress (swelling or shrinking)
  • pH: Maintained at a physiological pH (usually around 7.4) to preserve cell viability and antibody binding
  • Viscosity: Affects the flow rate and focusing efficiency; usually optimized for the specific instrument
  • Refractive Index: Can affect light scatter measurements
  • Electrical Conductivity: Important for instruments that use impedance-based cell counting
  • Compatibility: Must be compatible with the dyes and reagents used in the assay
• Common Additives:
  • Antibiotics: To inhibit bacterial growth
  • EDTA: As a metal chelator, to prevent aggregation of cells
  • Protein (e.g., BSA): To block non-specific binding of antibodies to the flow cell
  • Surfactants: To reduce surface tension and prevent bubble formation
  • Stabilizers: To prevent the degradation of components in the sheath fluid
• Preparation and Storage:
  • Prepared using high-quality reagents and sterile techniques
  • Filtered through a 0.2 μm filter to remove particles
  • Stored properly to prevent contamination and degradation
• Maintenance:
  • Regularly replaced to ensure purity and prevent clogging
  • Fluid filters are replaced
  • The fluidic system is cleaned with detergent solution

Troubleshooting Fluidic Issues

• Clogging:
  • Symptoms: Erratic flow rates, increased pressure, poor resolution, or complete blockage
  • Causes: Particulate matter in the sheath fluid or sample, cell aggregates, precipitation of reagents
  • Solutions: Filter sheath fluid and samples, use cell preparation techniques to minimize aggregates, flush the system with cleaning solutions, and replace clogged filters
• Bubble Formation:
  • Symptoms: Erratic flow rates, unstable readings, or signal fluctuations
  • Causes: Air leaks in the fluidic system, improper degassing of sheath fluid, or surfactants in the sample
  • Solutions: Check for leaks, degas sheath fluid, adjust surfactant concentrations, and ensure proper fluid levels in reservoirs
• Contamination:
  • Symptoms: High background noise, unexpected cell populations, or microbial growth.
  • Causes: Non-sterile sheath fluid, improper handling of samples, or contamination of the flow cell
  • Solutions: Use sterile techniques, replace contaminated sheath fluid, decontaminate the flow cell appropriate controls.
• Pressure Issues:
  • Symptoms: Inconsistent flow rates, unstable focusing, or failure to aspirate samples
  • Causes: Blocked lines, pump malfunction, or improper pressure settings
  • Solutions: Check for blockages, inspect pump function, verify pressure settings, and calibrate the instrument
• Carryover:
  • Symptoms: False positive results
  • Causes: Insufficient washing between samples
  • Solutions: Increase the wash volume, reduce the sample concentration, or use a carryover reduction solution