How does mass photometry work?

Mass photometry is a bioanalytical technology that measures the mass of individual biomolecules or particles in solution by quantifying light scattering. 

What is the principle behind mass photometry?

The principle behind mass photometry is simple: A single molecule on a measurement surface (e.g. a glass coverslip) exposed to a beam of light produces a small but measurable light scattering signal, which is directly proportional to the molecule’s mass. The greater the mass of the molecule, the more intense the signal (Fig. 1). 

Figure 1 Mass photometry concept. A biomolecule (light blue) that is exposed to light (yellow) and placed in a mass photometer generates a light-scattering signal (grey circles beneath the biomolecules). The signal’s intensity is correlated with the molecule’s mass. 

In a mass photometry measurement, a sample of proteins (or other biomolecules or particles) is illuminated with a beam of light. Some of that light is reflected by the measurement surface and some is scattered by molecules on the measurement surface (Fig. 2).   

Mass photometry measures the interference between the light scattered by the molecule and the light reflected by the measurement surface. The signal measured is called the contrast (or interferometric contrast) and is directly correlated with molecular mass, as demonstrated by the seminal paper introducing mass photometry (Young, et al. 2018). 

Figure 2 The principle of mass photometry. The light scattered by a molecule in contact with a measurement surface interferes with light reflected by that surface. The interference signal scales linearly with the molecule’s mass. 

Why mass photometry is useful

Several valuable benefits set mass photometry apart from other bioanalytical techniques: 

1. It measures true molecular mass. Mass photometry does not infer the mass indirectly from a different physical parameter. This is different from other techniques, such as dynamic light scattering (DLS) and size-exclusion chromatography (SEC), which infer molecular size from the hydrodynamic radius. Instead, the mass photometry signal measured is directly correlated with the true molecular mass, enabling measurement of the mass of molecules in the range 30 kDa to 6 MDa. 
2. It shows sample heterogeneity. Mass photometry measures the mass of each molecule or particle on the measurement surface. These single-molecule measurements make it possible to detect subpopulations of species or detail the heterogeneity – aspects of a sample that are invisible to analytical methods that use bulk measurement (such as DLS, SDS-PAGE, analytical ultracentrifugation (AUC) and SEC). 
3. It works in solution. Mass photometry measurements are performed in solution, and mass photometry is compatible with water as well as a wide range of buffers. Measuring biomolecules in an environment that mimics the intracellular aqueous environment allows their true native behavior to be studied. 
4.  It uses very little sample. Volumes as little as 10 µL can be enough for a single mass photometry measurement, and the recommended concentration range is 100 pM – 100 nM. The low concentration range means that biomolecules can be studied at concentrations that are physiologically relevant – again helping to mimic the intracellular environment and observe native behavior. A rapid-dilution microfluidics device enables measurement of samples at higher concentrations – up to the tens of micromolar. 
5. It requires no modification of the sample. Mass photometry does not require any labelling of the molecules or particles under investigation. This simplifies sample preparation and eliminates the risk of labels interfering with native behavior. 
6.  It provides results rapidly. Mass photometry workflows are very quick, with both sample preparation and measurement together usually taking just minutes. 
7.  It is easy to use. It takes less than half a day to learn how to do a mass photometry measurement and the data is intuitive to interpret. The instruments also fit conveniently on a lab benchtop. 

What types of molecules and particles can you measure with mass photometry? 

Mass photometry is a bioanalytical method suitable for measuring the mass of particles in the 30 kDa – 6 MDa mass range, depending on the mass photometer (30 kDa – 5 MDa with the TwoMP, 500 kDa – 6 MDa with the Samux).  

Most published studies have applied mass photometry to proteins – to study protein-protein interactions (Higuchi, et al. 2021), protein oligomerization mechanisms (Naftaly, et al. 2021) and characterize protein sample stability (Nuber, et al.  2021).  

Mass photometry has also been used successfully to study heteromolecular interactions such as DNA-protein interactions (Hickman, et al. 2020).  

Mass photometry can be applied to nucleic acids, enabling the detection and quantification of nucleic acids at sub-picomolar concentrations (Li, et al. 2020) as well as the measurement of the length and integrity of RNA samples (Camperi et al. 2024, Schmudlach et al. 2020).  

Mass photometry has also been applied to vesicles and micelles (Lebedeva, et al.  2020), and polysarcosine star polymers for drug delivery (England, et al. 2020).  

The linear correlation between the contrast (the signal measured in mass photometry) and molecular mass holds for a wide variety of biomolecules  (Young, et al. 2018) – making mass photometry a universal tool for biomolecules and particles in solution. The precise linear relationship between the contrast and mass may differ for each class of molecule, however, requiring calibration with an appropriate standard (e.g. a protein calibrant for measuring proteins, a DNA calibrant for measuring DNA, etc.).  

How is mass photometry used?

With mass photometry, you can measure the molecular mass of single biomolecules, oligomers, polymers, macromolecular assemblies, nanostructures and small viral capsids (of e.g. adeno-associated viruses, AAVs). 

You can also quantify the oligomerization and aggregation of biomolecules (Naftaly, et al. 2021, Balakrishnan et al. 2024), characterize sample heterogeneity (Olerinyova, et al. 2021, Sonn Segev, et al. 2020), monitor the stability of sample components (Nuber, et al. 2021), and study the effects of molecular or experimental modifications on sample integrity (Bertosin, et al. 2021). 

Mass photometry is especially valuable for studying biomolecular interactions – including protein-protein interactions (Higuchi, et al. 2021; Soltermann, et al. 2020) and protein-nucleic acid interactions (Hickman, et al. 2020; Acharya, et al. 2021). Mass photometry makes it possible to determine stoichiometries in biochemical reactions (Xu et al., 2024) , and quantify affinities and rate constants in molecular interactions (Wu and Piszczek 2020; Soltermann, et al. 2020). 

Thanks to this versatility, mass photometry is seeing use across a wide range of different applications, such as assessing antibody affinity and aggregation (den Boer et al. 2021, Cramer et al. 2023), characterizing membrane proteins (Olerinyova, et al. 2021, Dodge et al. 2024) and supporting structural studies (Vasquez et al. 2023, Crowe et al. 2024). 

Mass photometry is also widely used to characterize AAV samples (Wu, et al. 2021, Wagner et al. 2023, Wagner et al. 2024, Ebberink et al. 2024). Macro mass photometry, a newer technology that builds on the principles of mass photometry, characterizes samples of larger viral vectors (e.g. adenovirus), virus-like particles (VLPs) and lipid nanoparticles (). Wu et al. 2025). 

Although mass photometry was introduced relatively recently (in 2018), its range of applications has expanded rapidly as users discover the technology and its versatility. For an up-to-date list of publications citing mass photometry, visit our publications page.