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Cytometry

Small Particle Analysis

Overview of Small Particle Analysis

  • Definition: Small particle analysis refers to the use of flow cytometry to characterize particles that are smaller than typical cells, ranging in size from approximately 50 nm to 1 μm
  • Challenges:
    • Size Limitations: Conventional flow cytometers are not optimized for detecting particles below 1 μm
    • Low Signal Intensity: Small particles scatter less light and emit less fluorescence than larger cells, resulting in weak signals
    • High Background Noise: High background noise can obscure the signal from small particles
    • Resolution: Resolving different populations of small particles can be challenging
  • Importance:
    • Extracellular Vesicle (EV) Research: To study the role of EVs in cell-cell communication and disease
    • Virus Detection: To detect and quantify viruses
    • Nanoparticle Characterization: To characterize the size, concentration, and surface properties of nanoparticles
  • Applications:
    • Extracellular Vesicles (EVs)
    • Viruses
    • Nanoparticles

Extracellular Vesicles (EVs)

  • Definition: Membrane-bound vesicles that are released by cells and can transport proteins, nucleic acids, and other molecules to other cells
  • Types:
    • Exosomes: Small vesicles (30-150 nm) that are formed inside cells and released upon fusion of multivesicular bodies with the plasma membrane
    • Microvesicles: Larger vesicles (100-1000 nm) that are shed directly from the plasma membrane
    • Apoptotic Bodies: Vesicles that are released during apoptosis
  • Clinical Significance:
    • Cell-Cell Communication: EVs play a role in cell-cell communication
    • Disease Biomarkers: EVs can serve as biomarkers for various diseases
    • Therapeutic Delivery: EVs can be used as vehicles for delivering drugs or other therapeutic agents
  • Methods for EV Analysis:
    • Sample Preparation:
      • Remove Cells and Debris: Centrifuge or filter the sample to remove cells and debris
      • Concentrate EVs: Use ultracentrifugation, ultrafiltration, or other methods to concentrate the EVs
    • Staining:
      • Label EVs with fluorescent antibodies against surface markers
      • Use appropriate controls to account for non-specific binding
    • Flow Cytometry:
      • Use a flow cytometer that is optimized for small particle detection
      • Use appropriate gating strategies to identify EVs

Virus Detection

  • Definition: Identifying and quantifying viruses using flow cytometry
  • Methods:
    • Direct Detection:
      • Principle: Uses fluorescent antibodies that bind directly to viral antigens
      • Advantages: Can detect viruses without amplification
      • Disadvantages: Requires high-affinity antibodies
    • Indirect Detection:
      • Principle: Uses fluorescent antibodies that bind to cells that are infected with the virus
      • Advantages: Can detect viruses even if antibodies against viral antigens are not available
      • Disadvantages: Requires cell culture
  • Considerations:
    • Virus-Specific Antibodies: You must have antibodies that bind to the virus of interest
    • Techniques: Due to the small size of viruses, they can be difficult to detect by flow cytometry
  • Applications:
    • Tracking a viral infection
    • Assessing the efficacy of anti-viral compounds

Nanoparticle Characterization

  • Definition: Characterizing the size, concentration, and surface properties of nanoparticles using flow cytometry
  • Methods:
    • Size Measurement: Use calibrated beads to create a standard curve for size measurement
    • Concentration Measurement: Add a known concentration of fluorescent beads to the sample as an internal standard
    • Surface Characterization: Use fluorescent antibodies or dyes to label the surface of the nanoparticles
  • Considerations:
    • Nanoparticle Aggregation: Nanoparticles can aggregate, which can affect the accuracy of the measurements
    • Nanoparticle Stability: Nanoparticles can degrade over time, which can affect the results
  • Applications:
    • Confirm the delivery of a drug
    • Determine the purity of the sample

Instrument Considerations for Small Particle Analysis

  • High-Sensitivity Flow Cytometer:
    • Blue laser for optimal light scatter properties
    • Flow cytometers with optimized optics can detect smaller particles
  • Triggering:
    • Use a trigger parameter that is sensitive to small particles (e.g., side scatter or fluorescence)
    • Adjust the trigger threshold to minimize background noise
  • Sheath Fluid:
    • Use particle-free sheath fluid to minimize background noise
    • Filter the sheath fluid through a 0.1 μm filter
  • Cleaning Protocols:
    • Use rigorous cleaning protocols to remove contaminants from the flow cytometer
    • Flush the flow cytometer with cleaning solutions before and after each experiment

Sample Preparation Considerations for Small Particle Analysis

  • Sample Filtration:
    • Filter samples through a 0.2 μm or 0.1 μm filter to remove large particles and debris
  • Sample Concentration:
    • Concentrate samples to increase the number of small particles
    • Use ultracentrifugation, ultrafiltration, or other methods to concentrate the samples
  • Blocking Reagents:
    • Use blocking reagents to reduce non-specific binding
    • Use appropriate controls to validate the blocking protocol

Gating Strategies for Small Particle Analysis

  • Size-Based Gating:
    • Use forward scatter (FSC) to gate on particles of a specific size range
    • Use calibrated beads to create a standard curve for size measurement
  • Fluorescence-Based Gating:
    • Use fluorescent antibodies or dyes to identify particles that express specific markers
    • Use appropriate controls to define gating boundaries and account for background noise
  • Sequential Gating:
    • Use a combination of size-based and fluorescence-based gating to identify specific populations of small particles

Controls for Small Particle Analysis

  • Blank Controls:
    • Samples that contain no particles, used to measure background noise
  • Bead Controls:
    • Samples that contain calibrated beads of known size and fluorescence intensity, used to calibrate the flow cytometer
  • Isotype Controls:
    • Antibodies that are the same isotype as the primary antibody but do not bind to the target antigen, used to assess non-specific antibody binding
  • Buffer Controls:
    • Samples that contain only the buffer used for staining, without any particles or antibodies, used to measure background fluorescence

Troubleshooting Small Particle Analysis

  • No Events Detected:
    • Possible Causes:
      • Low concentration of small particles
      • Instrument settings
      • Sample loss
    • Troubleshooting Steps:
      • Increase concentration by adjusting sample
      • Inspect equipment for malfunctions
  • High Background:
    • Possible Causes:
      • Instrument Noise, non-specific binding
    • Troubleshooting Steps:
      • Make sure proper control are in place and reagents/settings are optimal
  • Aggregation:
    • Possible Causes:
      • Inadequate mixing or cell stress
    • Troubleshooting Steps:
      • Adjust preparation methods