Measuring molecular mass with light

Interferometric analysis of the weak light scattered from proteins makes it possible to determine their mass.

Mass is one of the fundamental properties of matter. For example, it plays a central role in two of the most famous equations in physics formulated by Isaac Newton and Albert Einstein for describing force and energy, namely F = ma and E = mc2. As atoms and molecules have their own characteristic mass, measurements of the mass of the constituents within a sample can readily be used as a means to identify them even in a complex mixture of different species. This is the principle of mass spectroscopy and over the last three decades, advances in the field have been remarkable, enabling the accurate analysis of atoms and molecules, including biological macromolecules such as DNA and proteins. Now, writing in Science, Young et al. report an optical image-based technology that uses the signature of light scattering to accurately determine the mass of proteins and protein complexes in aqueous solution down to the level of single molecules1.

How is the mass of biomolecules usually measured? The answer is that conventional mass spectrometry records the time-of-flight of ionized molecules under an electric field. As the mass-to-charge ratio of the molecule is known to be proportional to the square of the time-of-flight, this allows the mass to be determined (Fig. 1a). The approach works well and is trusted, but it requires a considerable amount of sample (typically more than 10 μg) and the biomolecular complex may undergo a change during the process of ionization and separation. Recently, nanomechanical oscillators were shown to be capable of measuring the mass of single proteins by detecting a subtle change in the resonant frequency of the oscillator on molecular adsorption in vacuum and at low temperature2,3. While this approach is highly sensitive, it is not accurate as the frequency shift per molecule is affected by the adsorption location and the amount of accumulated molecules over time.

Fig. 1: Imaging mass of single biomolecules.

iSCAMS uses the interference between the scattered light from biomolecules and the reflected light from the surface. The interference signal is linearly proportional to the molecular mass, and gives spatial and temporal information. In conventional mass spectrometry, a molecule’s mass-to-charge ratio is determined from the time-of-flight measured. V+ is the voltage relative to the group indicated by 0.

To overcome these problems, Young and colleagues have now developed an optical technique called interferometric scattering mass spectrometry (iSCAMS) (Fig. 1b) that uses light to perform quantitative mass analysis with single-protein sensitivity1. Light scattering is a universal process and is routinely used as a prominent contrast mechanism for the optical detection of particles. However, due to the sixth-power dependence of the strength of the scattering on the object size, it is difficult to detect the scattering signal from nanometre-scale objects as the signal is so small. However, by averaging the signal from thousands of particles diffusing through a focused laser beam, dynamic light scattering can characterize the diffusion of a nanoscale object such as the bovine serum albumin (BSA) protein that has a molecular mass of 67 kDa (1 kDa = 103 atomic mass unit) and infer its size.

Young et al. adapted a scheme called interferometric scattering microscopy (iSCAT) that they had previously developed to study gold nanoparticles4 and the dynamics of virus particles with a 45-nm diameter5. In iSCAT, laser light is scanned over a glass coverslip that is covered with an aqueous solution of biomolecules; the concentration of biomolecules is kept very low to ensure single-molecule detection. The reflected and scattered light signals are collected by a high-numerical-aperture objective and imaged using a complementary metal-oxide–semiconductor (CMOS) camera. Three components contribute to image formation: the light reflected from the glass/water interface, the light scattered by biomolecules, and the interference between the scattered and reflected signals. Since the scattered signal is much smaller than the other terms, the signal contrast is determined by an amplitude ratio between the scattered light and the reflected light. After differential image processing, the image of a single molecule appears as a destructive interference spot.

iSCAT shares a similarity with interference reflection microscopy that has been used for imaging cell adhesion6, but the main differences are that iSCAT uses a coherent light source to obtain a high-contrast image, uses beam scanning or narrow-field illumination to minimize speckle patterns, and achieves single-molecule-level detection. Recently, the image contrast of iSCAT has been improved from 10–4 to 10–2 by attenuating the reflected light amplitude using a partial reflector that preferentially transmits light from a dipole on the surface radiating with a near critical angle7,8.

Now, importantly, Young et al. have taken another step forward and discovered that the interferometric signal is actually linearly proportional to the mass of biomolecules, as illustrated with tests with eight different proteins with different molecular masses. They showed that the signals of monomer, dimer and trimer of BSA are equally spaced, and precisely determined the relative abundance of different oligomeric species. Even a very rare species of BSA tetramer was detectable (0.25% abundance).

Structural differences did not affect the scattering contrast. iSCAMS was also applicable to other biomolecules such as lipids and carbohydrate-coated proteins. Because the experiment was limited by shot noise that is in turn determined by the photon flux, the mass resolution depends on the molecular mass and can be expressed as a fraction of the molecular mass. Remarkably, it was even possible to detect the binding of small ligands to a protein due to the impressive mass precision of 1.8%.

Additionally, iSCAMS can monitor the mass change of a single biomolecular complex in real time, which is impossible in conventional mass spectrometry. The researchers analysed the assembly process of several proteins, for example, alpha-synuclein, a key player in Parkinson’s disease, and actin, a major component of the cytoskeleton. Such capabilities will potentially provide insight into mechanisms of neurodegenerative diseases and other cellular processes.

This work by Young et al. represents the first optical mass imaging technique that can quantitatively measure the mass of single biological macromolecules in ambient conditions. Because this study used non-specific binding of molecules to a clean coverslip, it does not provide molecular specificity. It will be challenging to analyse complex samples such as cell lysates because other biomolecules will adhere to the surface. However, these limitations might be overcome by passivating the surface and functionalizing it with antibodies to capture specific molecules of interest9. By combining iSCAMS with sensitive fluorescence detection, it should be possible to investigate the conformational changes of molecular machinery during self-assembly. Sub-shot-noise measurements using correlated light10 may be able to further improve the precision of the mass measurement to below a few kDa so that a drug binding to biomolecules or small chemical modifications can be identified.


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Correspondence to Kyu Young Han or Taekjip Ha.

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Han, K.Y., Ha, T. Measuring molecular mass with light. Nature Photon 12, 380–381 (2018).

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