Label-Free Optical Imaging of Nanoscale Single Entities

Abstract

The advancement of optical microscopy technologies has achieved imaging of nanoscale objects, including nanomaterials, virions, organelles, and biological molecules, at the single entity level. Recently developed plasmonic and scattering based optical microscopy technologies have enabled label-free imaging of single entities with high spatial and temporal resolutions. These label-free methods eliminate the complexity of sample labeling and minimize the perturbation of the analyte native state. Additionally, these imaging-based methods can noninvasively probe the dynamics and functions of single entities with sufficient throughput for heterogeneity analysis. This perspective will review label-free single entity imaging technologies and discuss their principles, applications, and key challenges.

Keywords: evanescent scattering; interferometric scattering; label-free optical microscopy; nanofluidic scattering; nanoscale single entities; plasmonic scattering; single molecules; surface plasmon resonance.

Figure 1.

SPRM for single entities imaging in biology. a). Schematic of SPRM. b). Images of nanoparticles with different sizes and H1N1 influenza A virus. (a, b) Reprinted/Adapted with permission from ref , Copyright 2010, National Academy of Sciences. c). Stretched λ-DNA and zoomed in image (Up right and left) and corresponding fluorescent images (bottom left and right). Reprinted/Adapted with permission from ref , Copyright 2014, American Chemical Society. d) k-space of a SPRM image. e). SPRM image of a 100-nm silica nanoparticle (left) and after image reconstruction (right). f). TEM and SPRM (bottom, iPM-interferometric plasmonic microscopy) images. (d-f) Reprinted/Adapted with permission from ref , Copyright 2018, National Academy of Sciences. g). Sandwich scheme for ultrasensitive analyte detection through micromanipulation. (g) Reprinted/Adapted with permission from ref , Copyright 2022, National Academy of Sciences. h). Schematic of bacterial cell interacts with the surface and its SPRM signal. (h) Reprinted/Adapted with permission from ref , Copyright 2020, National Academy of Sciences.

Figure 2.

SPRM for measuring single entity electrochemical properties. a). Schematic of SPRM electrochemical current imaging. Reprinted/Adapted with permission from ref , Copyright 2010, Science. b). Current density of a single platinum nanoparticle at different potentials, scale bar, 3μm. Reprinted/Adapted with permission from ref , Copyright 2012, Nature Publishing Group .c). Continues recording of one Ag nanoparticle land on and oxidate on the surface, blue curve is the image intensity. d). Ag particle size versus peak potential in the oxidation. Reprinted/Adapted with permission from ref , Copyright 2014, American Chemical Society.

Figure 3.

ISCAT and its single entity imaging applications. a). Schematic of iSCAT setup. Reprinted/Adapted with permission from ref , Copyright 2017, American Chemical Society. b). Image of 5nm AuNP. Two line-profiles are shown at bottom. Reprinted/Adapted with permission from ref , Copyright 2004, American Physical Society. c). An EGFR protein is labeled with AuNP through EGF EFGR interaction on the cell membrane. d). A track obtained using method in c). (c, d) Reprinted/Adapted with permission from ref , Copyright 2019, Nature Publishing Group. e). One shot tracks of virion (blue) and QD (red), the corresponding three-dimensional motion is in the right. Reprinted/Adapted with permission from ref , Copyright 2009, Nature Publishing Group. f). ISCAT contrast versus mass. Reprinted/Adapted with permission from ref , Copyright 2018, Science. g). Example of protein association on the SLBs. Reprinted/Adapted with permission from ref , Copyright 2021, Nature Publishing Group.

Figure 4.

PSM image principle and applications. a). PSM setup with a second top-mounted objective. b). Digital counting of IgA for binding kinetics measurement with anti-IgA modified on the surface. Images details are shown in c). (a-c) Reprinted/Adapted with permission from ref , Copyright 2020, Nature Publishing Group. d). Schematic of Single protein pulldown of cells. Reprinted/Adapted with permission from ref , Copyright 2022, American Chemical Society. e). PSM imaging of a cell, box indicate focal adhesions and arrows points to their moving directions and displacement after activation. f). Track details of box 1 in b). (e, f) Reprinted/Adapted with permission from ref , Copyright 2021, American Chemical Society.

Figure 5.

Evanescent scattering microscopy applications. a). BSA images under different potential using evanescent scattering. Reprinted/Adapted with permission from ref , Copyright 2020, Nature Publishing Group. b). AuNP z axis displacement before (up) and after (bottom) miRNA binding measured under ESM. Reprinted/Adapted with permission from ref , Copyright 2022, Nature Publishing Group.

Figure 6.

Nanofluidic scattering. a). Schematic of fiber nanofluidic scattering imaging. Reprinted/Adapted with permission from ref , Copyright 2015, American Chemical Society. b). Nanofluidic chip for scattering imaging, objective is not shown. c). Scatter plot of optical contrast and mass. R_s (left Y axis) and D (right Y axis) are related with Stokes-Einstein equation. d). Kymography of lipoproteins (red arrows) and an extracellular vesicle (blue arrow). (b-d) Reprinted/Adapted with permission from ref , Copyright 2022, Nature Publishing Group.