Spinning disk interferometric scattering confocal microscopy captures millisecond timescale dynamics of living cells

Abstract

Interferometric scattering (iSCAT) microscopy is a highly sensitive imaging technique that uses common-path interferometry to detect the linear scattering fields associated with samples. However, when measuring a complex sample, such as a biological cell, the superposition of the scattering signals from various sources, particularly those along the optical axis of the microscope objective, considerably complicates the data interpretation. Herein, we demonstrate high-speed, wide-field iSCAT microscopy in conjunction with confocal optical sectioning. Utilizing the multibeam scanning strategy of spinning disk confocal microscopy, our iSCAT confocal microscope acquires images at a rate of 1,000 frames per second (fps). The configurations of the spinning disk and the background correction procedures are described. The iSCAT confocal microscope is highly sensitive—individual 10 nm gold nanoparticles are successfully detected. Using high-speed iSCAT confocal imaging, we captured the rapid movements of single nanoparticles on the model membrane and single native vesicles in the living cells. Label-free iSCAT confocal imaging enables the detailed visualization of nanoscopic cell dynamics in their most native forms. This holds promise to unveil cell activities that are previously undescribed by fluorescence-based microscopy.

A paintbrush for delivery of nanoparticles and molecules to live cells with precise spatiotemporal control

“…Figure 3j shows the localized delivery of 200 nm carboxylate-coated fluorescent beads, which readily adhere to the cell membrane. The simultaneously recorded fluorescence and confocal iSCAT 41 , 42 images of a COS-7 cell are overlaid. The close-up of the region marked in the left panel of Fig.…”

Real-Time Monitoring and Control of Nanoparticle Formation

“…Although iSCAT is capable of subnanometer precision measurements of object location, high densities are still challenging. Super-resolved variants of iSCAT microscopy are in their infancy, , and to the best of our knowledge, they only offer spatial resolutions at length scales of approximately 100 nm, larger than might be useful for close-packed plasmonic NPs, for example.…”

Scattered‐light‐sheet microscopy with sub‐cellular resolving power

“…SPIM and LSI also represent exceptional platforms for elastic scattering-based imaging [16][17][18][19]. In these imaging modalities, an object of certain refractive index contrast (Δn) will exhibit a nonzero probability for an incident photon to elastically scatter into a unit solid cone oriented at an angle relative to the photon's original trajectory [11].…”