Interferometric Scattering (iSCAT) microscopy is a sophisticated optical method that allows researchers to observe objects far smaller than the wavelength of light. This technique offers a powerful way to visualize nanoscale entities, such as individual proteins and viruses, without the need for fluorescent tags. By detecting the faint light that samples naturally scatter, iSCAT provides insight into the physical world at the molecular level, enabling scientists to study biological processes with clarity and speed.
The Physics Behind Interferometric Scattering
The underlying principle of iSCAT is to transform the extremely weak light scattered by a nanoscale object into a much stronger, detectable signal using an optical phenomenon called interference. When a laser beam illuminates a sample, any particle within the light path will scatter a tiny amount of light in all directions, a process known as Rayleigh scattering. This scattered light field is far too weak to be detected directly against the bright background of the illuminating beam.
To overcome this limitation, iSCAT combines the scattered light with a strong, controlled “reference” beam, often light reflected directly from the glass coverslip beneath the sample. When these two light waves are superimposed, they interfere with one another. This interference can be either constructive, where the waves reinforce each other, or destructive, where they partially cancel each other out.
This superposition mechanism is highly sensitive because the resulting image intensity is linearly proportional to the product of the two light fields. The large amplitude of the reference light essentially acts as an amplifier for the minuscule signal from the scattered light. The contrast generated in the final image is based on the difference in the refractive index between the particle and the surrounding medium, a mechanism often described as extinction. This physical interaction allows the microscope to generate an image where the presence of a subwavelength object is revealed by a localized increase or decrease in light intensity.
Advantages of Label-Free Imaging
The ability of iSCAT to visualize samples without chemical labels provides several advantages for studying dynamic biological events. Traditional fluorescence methods require attaching a bulky dye molecule to the target, which can unintentionally alter the molecule’s natural behavior or function. Label-free imaging ensures that the observed motion and interactions of molecules are native, providing a more accurate representation of biological reality.
Another benefit is the elimination of photobleaching and photoblinking, which are inherent drawbacks of fluorescent tags. Photobleaching occurs when a fluorophore permanently loses its ability to emit light after repeated exposure, limiting observation time. Since iSCAT relies on light scattering, a physical property that is not destroyed by light exposure, researchers can track objects for extended periods, enabling the study of long-term molecular processes.
The high signal-to-noise ratio achieved through interferometric amplification also permits high temporal resolution. Because the system does not have to wait for the relatively slow process of fluorophore excitation and emission, imaging speeds are limited only by the camera technology. This enables frame rates up to hundreds of thousands per second, allowing scientists to resolve fleeting, ultrafast molecular movements with nanometric precision. This combination of speed, stability, and minimal perturbation is useful for observing the rapid diffusion and conformational changes of single molecules in real-time.
Current Applications in Biological Research
iSCAT microscopy is used for addressing fundamental questions in cell and molecular biology that require high-speed tracking of tiny objects.
Molecular Motors
One prominent application is the study of molecular motors, such as myosin and kinesin, which are responsible for movement within cells. The high temporal resolution of iSCAT allows scientists to directly observe the individual, nanometer-scale steps these motors take along cellular tracks, revealing the mechanics of their stepping cycles.
Viruses and Proteins
The technique is also widely used for tracking the dynamics of viruses and proteins. Researchers can monitor how single viral particles bind to cell surfaces or how they move across a membrane before infecting a cell. Furthermore, iSCAT can be used in mass photometry, where the intensity of the scattered light is measured to determine the mass of individual, unlabeled protein molecules in solution.
Membrane Dynamics
Observing membrane dynamics in living cells is another area where iSCAT excels. It provides insight into how proteins and lipids diffuse within the complex and organized environment of the cell membrane. This allows for the visualization of processes like the formation of lipid rafts or the clustering of membrane receptors, which are crucial for cellular communication and signaling.