What is light scatter?
Light scatter is one of the basic measurements performed by a flow cytometer. Scatter from any of the lasers on the system can be utilised, but traditionally the 488 nm laser is used. Additionally, scatter from more than one laser can be used; this will be further explored later in this CytoEssential.
How is light scatter measured in flow cytometry?
Scattered light is a complex mixture of refracted, diffracted, and reflected light. The intensity of each depends on the cell shape, surface, and composition. To try and simplify measurement, scattered light is normally detected at 2 different angles from the incident laser beam as these may yield a contrast in cellular information. As we will see, these are typically split into Forward Scatter (a detector assembly placed in line with the path of the laser) and Side Scatter (a detector assembly placed at roughly 90 degrees to the laser path) measurements.
Scattered light is mostly detected through low sensitivity detectors (as compared to fluorescence). However, as events become smaller, a more sensitive detector may be required. Conversely, the detection of bigger cells (or higher intensity illumination sources) may require the reduction of light entering the detector, by the use of a neutral density filter.
It is essential that stray scattered light from the sheath stream or flow cell is excluded from the detector, as scattered light although much higher intensity that fluorescent light, is much less intense compared to the incident laser light, which if allowed to enter the detector may disable or damage it.
Example light scatter collection assemblies
This illustration shows the moment at which the cell has entered the laser beam and how the light is delivered to different detectors.
Light is scattered all around the cell. However, much of the laser light passes straight through the system. This light is normally blocked by a blocker bar placed in the laser’s path after it has exited the intersection area. On “sense in air” cell sorters, an extra blocker bar is required to block stray light from the laser/stream intersection. This is placed in front of the Side Scatter collection lens.
Lens and pinhole arrangements are commonly used to further eliminate stray light to maximise sensitivity.
Forward Scatter vs Side Scatter
Forward Scatter: Light is refracted and diffracted at a low angle by the cell as it passes through the laser beam. This combination of scattered light is a comparatively high intensity signal, thus on many systems requires the use of a comparative low sensitivity photodiode. On some instruments the refracted and diffracted light is often focused through a pinhole onto a detector as mentioned before to reduce the interference of stray light.
There are various factors which contribute to the forward scatter from different cells. Cell size has a significant effect on forward scatter. Other important factors include: wavelength of the laser, the relative refractive index of the cell, and its nuclear to cytoplasmic ratio. It is therefore reasonable to expect that particles of equal size may have very different forward scatter values. Conversely, particles of the same forward scatter may have a different size if their composition is different.
Side Scatter: Light is reflected from the cell; this is a comparatively low intensity signal compared to forward scatter and so usually requires the use of a PMT (albeit a low sensitivity one) or an Avalanche Photodiode (APD).
It is commonly collected through the same lens as is used for fluorescence collection which is normally placed perpendicular to the laser beam. Often collected in the same area as fluorescence signals, a dichroic mirror is used to deflect the scattered laser light to a detector.
Typically termed “Granularity” side scatter is mostly generated by internal structures and folds of the cell membrane. Particles which are empty and smooth surfaced generate very different side scatter values than ruffled/granular particles.
Why is the collection angle of light scatter important?
The selection of the forward scatter angle can affect cell measurement. It has been shown that instruments with different FS angles are sensitive to changes in size in different ranges. Instruments utilising narrow angles of FS show good sensitivity size changes in the 5-to-10 micron range. Whereas instruments with a wider angle of collection show good sensitivity to size changes in the 10-to-20 micron range1.
Adjusting the angle of light collected can improve discrimination between cells and debris, especially if cells have been pretreated with RBC lysis buffers of different types. This can be seen by the recommendation of different lysis buffers for different instrument types, as different manufacturers use different geometry to collect scatter so, samples may not look the same.
Counter-intuitive to this, the collection of wide-angle forward/side scatter has been shown to enhance the detection of sub-micron particles, such as platelets, bacteria, viruses, and extracellular vesicle. Mie scattering is strong in particles above 1um, anything below that is Rayleigh scatter. We use Mie scatter for large particles but the smaller they become, wide angle scatter is better than narrow angle FSC.
The wavelength of illuminating light can also create an effect, particularly on the resolution of small particles from noise wavelengths, such as Violet (405nm) and UV (365nm, 355nm, 325nm) have been used. Generally, instruments either offer scatter detectors utilising either 488nm or 405nm lasers.
Scatter calibration
Calibration of scatter to estimate the size of particles on flow cytometers not specifically designed for this purpose can be quite difficult. As discussed above the scatter detectors on the system may have been optimised for different purposes, other than specifically for cell sizing. The use of size calibration particles is useful to estimate the detection limits of systems to those particles, but not to compare the sizes of calibration particles to other particles.
Different examples of light scatter
Cells with very specific cell morphology form clusters on scatter plots, such as those from whole blood show below. Cells which have been grown in culture or may be derived from adherent cells have much more amorphous clusters on scatter plots, such as the Chinese Hamster Ovary Cells (CHO) show below.
Examples where native cell features can be combined with light scatter measurements to aid specific gating.
We hope this short article will help you understand how we measure light scatter in flow cytometry, the differences between forward scatter and side scatter and the importance of each.
- Ormerod, M.G. et al. (1995) ‘Discrimination of apoptotic thymocytes by forward light scatter’, Cytometry, 21(3), pp. 300–304. doi:10.1002/cyto.990210311. ↩︎
- Kiehl, T.R. et al. (2011) ‘Observations of cell size dynamics under osmotic stress’, Cytometry Part A, 79A(7), pp. 560–569. doi:10.1002/cyto.a.21076. ↩︎
- O’Donahue, M. (2016) Lysing methods and reagents for flow …, International Clinical Cytometry Society. Available at: https://www.cytometry.org/web/modules/module1.pdf (Accessed: 13 March 2025). ↩︎
- Scientific, T. (2021) Bigfoot Spectral Cell Sorter, ThermoFisher Scientific. Available at: https://assets.thermofisher.com/TFS-Assets/BID/Reference-Materials/bigfoot-spectral-cell-sorter-light-scatter-white-paper.pdf (Accessed: 13 March 2025) ↩︎
