Interferometric Scattering Microscopy: Seeing Single Nanoparticles and Molecules via Rayleigh Scattering

Abstract

Fluorescence microscopy has been the workhorse for investigating optical phenomena at the nanometer scale but this approach confronts several fundamental limits. As a result, there have been a growing number of activities toward the development of fluorescent-free imaging methods. In this Mini Review, we demonstrate that elastic scattering, the most ubiquitous and oldest optical contrast mechanism, offers excellent opportunities for sensitive detection and imaging of nanoparticles and molecules at very high spatiotemporal resolution. We present interferometric scattering (iSCAT) microscopy as the method of choice, explain its theoretical foundation, discuss its experimental nuances, elaborate on its deep connection to bright-field imaging and other established microscopies, and discuss its promise as well as challenges. A showcase of numerous applications and avenues made possible by iSCAT demonstrates its rapidly growing impact on various disciplines concerned with nanoscopic phenomena.

Nanostructured Alkali-Metal Vapor Cells

“…4) has been the most widely adopted detection technique for studying thermal vapors due to the simplicity of implementation. However, when the optical depth of the system is decreased, as in the case of a nanothickness vapor, it becomes more challenging to detect the extinction signal due to a reduced signal-to-noise ratio [69]. Our geometry allows us to instead perform fluorescence measurements efficiently, employing a high-NA objective lens to collect atomic fluorescence through the thin cover glass.…”

“…The architecture of our nanocell makes it well adapted to the application of microscopy and spectroscopy techniques that are routine in nano-optics. As well as TIRF and TPFM, further potential examples include stimulated emission depletion fluorescence microscopy [79]; fluorescence correlation spectroscopy [80]; and interferometric scattering-a scheme that has previously been used to detect single nanoparticles and even charge transport via Rayleigh scattering [69,81]. These detection schemes, along with the versatility of the cell design and manufacturing process, offer access to a multitude of investigative routes.…”

Differential diffusional properties in loose and tight docking prior to membrane fusion

“…Detection of the LUV scattering signal was greatly improved with a background-correction procedure (compare upper and lower image in Figure 1b) described in more detail in Methods. The iSCAT contrast of a nano-object is directly proportional to its polarizability, which is in turn linked to the particle size (23). The size of the LUVs was estimated from the observed iSCAT contrast to be about 90 nm (see Figure 1c).…”

“…We employed interferometric scattering (iSCAT) microscopy to record three-dimensional (3D) trajectories of particle diffusion on the surface of GUVs. This technique has been shown to reach microsecond temporal resolution and nm precision in detecting and tracking individual nanoparticles via the interference of their scattered light with a reference beam (22,23, and see Methods). Using iSCAT, we could investigate the diffusion of unlabeled vesicles arrested at a loosely or tightly docked intermediate state using synaptobrevin point mutants as previously described (17).…”

Detection of cellular micromotion by advanced signal processing

“…This promises to reveal signals on a millisecond timescale, which would be the timescale of electric activity and some previously reported micromotion 20,21,41 . Illumination by coherent light could further enhance small motions 42 and thus reduce photon load of the cells, and more advanced microscopy schemes such as phase imaging or differential interference contrast could be employed to enhance the signal. The neural network architecture could be improved as well.…”