Flow Cytometry Methods for Small Particle and Exosome Analysis

Author: Mike Blundell | Reviewer: Chloe Fenton

What Are Small Particles in Flow Cytometry?

Small particles in flow cytometry refer to submicron-sized particles such as exosomes, extracellular vesicles (EVs), and nanoparticles. Detecting these particles is challenging due to their low light scatter signals, requiring sensitive instrumentation and fluorescence-based detection strategies.

Which Small Particles Can Be Detected Using Flow Cytometry?

The type of small particles now being detected and evaluated by flow cytometry is increasing. Small particles can include platelets, which are typically 2–3 µm in diameter, bacteria, which can range from 0.3–5 µm, and cellular extravesicles, which can be further split into apoptotic bodies, microvesicles, and exosomes, with the smallest being exosomes, which are as little as 50 nm in diameter. Exosomes are mostly composed of cytoskeletal proteins, mRNA, microRNA, and actin receptors. They are thought to be important in cross-talk and regulation of cells by transferring proteins and RNA between cells. The particles can be identified by forward and side scatter but the scale is logarithmic to allow identification and better separation.

Detection of these particles can be problematic as light scatter depends on the particle diameter, the wavelength of the interrogation light (it is difficult to detect particles smaller than the wavelength), the particle refractive index, the angle of collection, and the intensity of the laser. To account for this, cytometers have been developed that have extra PMT detectors in the forward scatter (e.g., the ZE5 Cell Analyzer from Bio-Rad) and the trigger for data collection changed to a fluorescent signal or multiple fluorescent markers.

How Are Small Particles Detected by Flow Cytometry?

Typical workflow for detecting small particles:

1. Prepare samples (e.g., isolate extracellular vesicles or exosomes).
2. Label particles with fluorescent antibodies or dyes.
3. Optimize instrument sensitivity and detection thresholds.
4. Use appropriate controls and calibration beads.
5. Acquire and analyze data using careful gating strategies.

Which Methods Can Be Used to Analyze Small Particles?

The advantages and limitations of different small particle detection methods.

How Do You Choose the Right Method for Small Particle Analysis?

• Use flow cytometry—for high-throughput phenotyping of extracellular vesicles and particles
• Use NTA—for accurate sizing and concentration measurements of nanoparticles
• Use electron microscopy—for structural validation and visual confirmation

Alternative Methods

Alternative methods for enhanced detection include using shorter light wavelengths as this generally results in increased scatter and using antibody coated beads to increase the size of the particle being detected. Care should be taken to reduce the noise by filtering the sheath fluid and carefully setting the threshold level. As with all cytometry experiments, sample preparation can be crucial to obtaining good results. It should be noted however that as the particles decrease in size, the available antigen will also reduce, leading to decreased sensitivity or resolution.