Flow Cytometry

Small Particle Detection

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 µm-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™ from Bio-Rad) and the trigger for data collection changed to a fluorescent signal or multiple fluorescent markers.

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.