Scattering-based Light Microscopy: From Metal Nanoparticles to Single Proteins

Abstract

Our ability to detect, image, and quantify nanoscopic objects and molecules with visible light has undergone dramatic improvements over the past few decades. While fluorescence has historically been the go-to contrast mechanism for ultrasensitive light microscopy due to its superior background suppression and specificity, recent developments based on light scattering have reached single-molecule sensitivity. They also have the advantages of universal applicability and the ability to obtain information about the species of interest beyond its presence and location. Many of the recent advances are driven by novel approaches to illumination, detection, and background suppression, all aimed at isolating and maximizing the signal of interest. Here, we review these developments grouped according to the basic principles used, namely darkfield imaging, interferometric detection, and surface plasmon resonance microscopy.

Figure 1

Commonly employed approaches to scattering-based total internal reflection microscopy. (A) Prism-type total internal reflection microscopy. (B) Objective-type total internal reflection microscopy. P, prism; OBJ, objective; L, tube lens; M, mirror; BB, beam block; C, camera. (C) Image of a 20 nm GNP on a glass coverslip taken with an optimized objective-type TIRS microscope, scale bar = 0.5 μm. (D) Schematic illustrating the fields involved with TIRS microscopy, E_i incident field, E_s scattered field, E_e evanescent field, θ incident angle of illumination, which is greater than the critical angle, θ_c. (C) adapted with permission from ref (58). Copyright 2021 Philipp Kukura.

Figure 2

Perforated mirror TIR darkfield microscopy. (A) Schematic demonstrating the separation of scattered light from the reflected illumination beam. OBJ, objective lens; PM, perforated mirror. (B) View from the back aperture of the objective looking down the optical axis. (C) Image of 40 nm GNP in water immobilized on a glass slide. (D) Rotational motion of 40 nm gold-nanoparticle-labeled F₁-ATPase at 2 mM ATP, with the inset displaying an example trace from which the data is obtained. (C,D) Adapted with permission from ref (59). Copyright 2010 Elsevier.

Figure 3

ROCS microscopy principle and representative results. (A) Schematic of ROCS microscopy. OBJ, objective; BS, beamsplitter. (B) Translation of the illumination beam in a ring pattern around the back aperture of the objective lens. (C) Schematic of interference between adjacent scatterers <λ/2 apart. Under the condition of normal incidence, the scattered waves constructively interfere at the detector, whereas for oblique illumination, a phase delay of Δφ = π is introduced between the two scattered waves, leading to destructive interference at the detector and enabling the scatterers to be resolved. (D) TIR-darkfield ROCS image of 200 nm polystyrene beads illumination from a single azimuthal angle. (E) An image from the same sample once the beam is scanned in the pattern from B during a single detector exposure. (F) and (G) are the TIR-brightfield equivalents to D and E, respectively. (C) adapted with permission from ref (85). Copyright 2016 Springer Nature under Creative Commons Attribution 4.0 International License https://creativecommons.org/licenses/by/4.0/. (D–G) adapted with permission from ref (91). Copyright 2018 Optical Society of America.

Figure 4

Schematic and data from an objective coupling mirror darkfield microscope. (A) Schematic of the experimental setup. OBJ, objective lens; RM, rod mirror. (B) View from the back aperture of the objective looking down the optical axis toward the rod mirror. (C) Image of a 100 nm GNP attached to a flagellar motor on top of an Escherichia coli cell, scale bar = 1 μm. (D) Traces of the displacement of a 100 nm GNP attached to a bacterial flagellar motor, the black and gray lines correspond to X and Y displacement, respectively. (C,D) Adapted with permission from ref (95). Copyright 2010 AIP Publishing.

Figure 5

Darkfield stop schematics and performance. (A) Schematic illustrating the separation of illumination and scattered light. OBJ, objective; DS, darkfield stop. (B) View from the back aperture of the objective lens looking along the optical axis toward a 4 mm darkfield stop. (C) Image of 20 nm GNPs in water with 1 ms integration time. (D) Fluorescence image of ATTO 610 molecules without the use of an optical filter with 100 ms integration time. (E) Traces of the fluorescence emission of the molecules labeled in (D). (C–E) adapted with permission from ref (42). Copyright 2014 American Chemical Society.

Figure 6

Darkfield imaging in optical waveguides. (A–C) Evanescent scattering using waveguide illumination. (A) Schematic of the experimental setup. OBJ, objective; SMF, single mode fiber; WC, waveguide core. (B) Image of 150 nm fluorescently labeled lipid vesicles, scale bar = 2 μm. (C) Scattering intensity trace of 18 nm GNPs binding to a 100 nm lipid vesicle. (D–F) Darkfield imaging in a nanofluidic optical fiber. (D) Schematic of experimental design. OBJ, objective; WC, waveguide core; NC, nanochannel. (E) Example traces of CCMV virions diffusing in the optical fiber. (F) Image of a mixture of 19–51 nm polystyrene beads. (B,C) adapted with permission from ref (106). Copyright 2015 American Chemical Society. (E,F) adapted with permission from ref (112). Copyright 2015 American Chemical Society.

Figure 7

Interferometric scattering microscopy principles and example experimental arrangements. (A) Schematic of widefield illumination iSCAT. (B) Schematic of rapid beam scanning illumination iSCAT. OBJ, objective; BS, beamsplitter (nonpolarizing); QWP, quarter-waveplate; PBS, polarizing beamsplitter; L1, illumination lens; L2, imaging lens; C, camera. (C) Confocal detection iSCAT image of 20 nm GNPs on a glass coverslip in water. (D) Principle of interferometric scattering microscopy, E_i incident field, E_s scattered field, E_r reflected reference field, Δφ phase difference between the scattered and reflected fields. (C) Adapted with permission from ref (120). Copyright 2006 Optical Society of America.

Figure 8

Combination of interferometric scattering detection and single particle traps. (A–C) Anti-Brownian electrokinetic trap (ISABEL). (A) Illustration of the scanning pattern of the illumination beam. (B) Schematic of the trapping algorithm. (C) Trace of iSCAT contrast with time of a trapped 40 nm GNP. (D–G) Nanofluidic rocking Brownian motor capable of sorting GNPs based on their size. (D) Topography of the sorting device. (E) Schematic of the method of separation of 60 and 100 nm GNPs. (F) iSCAT images displaying the separation of 60 and 100 nm GNPs. (G) Plot of position vs median relative brightness of the particles in the device before and after sorting. (A–C) Adapted with permission from ref (143). Copyright 2019 American Chemical Society. (D–G) Adapted with permission from ref (148). Copyright 2018 The American Association for the Advancement of Science.

Figure 9

Interferometric scattering microscopy of microtubules and type IV pili. (A) Images of a microtubule acquired with light polarized parallel (left) and perpendicular (right) to the microtubule. Scale bar: 1 μm. (B) Composite kymograph of isotropic (red) and anisotropic (green) scattering combined. Scale bars: 1 μm (vertical) and 1 s (horizontal). (C) The profile of the mean contrast (black) and anisotropic contrast (red) averaged from disassembling microtubules. (D) Cross sections of the iSCAT (top left) and super-resolved (top right) microtubule images inset. (E–H) iSCAT images of different steps in the type IV pili cycle of a Pseudomonas aeruginosa cell, scale bar = 2 μm. (E), (F), (G), and (H) correspond to extension, attachment, tension, and detachment, respectively. (A–D) Adapted with permission from ref (153). Copyright 2021 John Wiley and Sons. (E–H) Adapted with permission from ref (154). Copyright 2019 Springer Nature.

Figure 10

Numerical aperture filtered iSCAT. (A) Schematic of the dipole emission pattern for a point scatterer at a refractive index interface; n₀ and n₁ represent the refractive indices of the media (n₁>n₀). (B) Emission pattern at the back aperture of a high-NA (1.42) microscope objective for a nanoscopic scatterer at a glass–water interface illuminated with circularly polarized light. The inner outlined circle indicates the position of the partial reflector (top panel). Normalized emission density as a function of numerical aperture. Gray shaded region corresponds to the position of the partial reflector (bottom panel). (C) Raw iSCAT image of a glass coverslip covered by water. (D) Equivalent image with the addition of a T = 1% partial reflector in the optical setup. Scale bar: 1 μm. (E) Illustration of a photothermal spatial light modulator (PT-SLM). (F) XY cross-section of the temperature profile across the PT-SLM (top left panel), equivalent XZ cross-section (top right panel). Scale bar: 100 μm. The two white dashed lines indicate the boundaries of the thermo-optic material. XY cross-section of the resulting refractive index variation (bottom left panel) and equivalent XZ cross-section (bottom right panel). (G) Phase-shift profile of a plane wave propagating through the structure in z at the position of the red dashed line in (I). (H,I) Normalized iSCAT images of 30 nm single gold nanospheres at total heating powers incident on the modulator structure of 0 mW (L) and 110 mW (M). Scale bar: 500 nm. (B–D) Adapted with permission from ref (43). Copyright 2017 American Chemical Society. (E–I) Adapted with permission from ref (168). Copyright 2021 Springer Nature under Creative Commons Attribution 4.0 International License https://creativecommons.org/licenses/by/4.0/.

Figure 11

Mass photometry principle and applications. (A) Scattering contrast histogram acquired from binding events of 12 nM BSA. (B) MP images of a BSA monomer, dimer, trimer, and tetramer, scale bars = 200 nm. (C) Contrast to mass relationship for a range of proteins. (D,E) Relationship between contrast and number of base pairs in double-stranded (D) and single-stranded (E) DNA. (F) Mass histogram of Escherichia coli bo₃ oxidase isolated from lauryl maltose-neo-pentyl glycol detergent micelles. (G) Mass histograms of different ratios of Escherichia coli outer membrane protein trimers solubilized in amphipols. (A–C) Adapted with permission from ref (34). Copyright 2018 American Association for the Advancement of Science. (D,E) Adapted with permission from ref (175). Copyright 2020 Oxford University Press under Creative Commons Attribution 4.0 International License https://creativecommons.org/licenses/by/4.0/. (F,G) Adapted with permission from ref (184). Copyright 2020 Philipp Kukura.

Figure 12

Single-particle interferometric reflectance imaging sensor (SP-IRIS). (A) Schematic illustrating the experimental configuration: OBJ, objective; BS, beamsplitter. (B) Image of H1N1 virus particles immobilized on an SP-IRIS chip. (C) Histogram of the measured H1N1 virus diameters. (D) Composite image of an SP-IRIS microarray. Inset: normalized image intensity image of 25 nm × 71 nm GNRs bound to the surface of a microarray spot. (E) Plot of GNR counts against the concentration of HBsAg. (B,C) Adapted with permission from ref (200). Copyright 2010 American Chemical Society. (D,E) Adapted with permission from ref (202). Copyright 2018 American Chemical Society.

Figure 13

Coherent brightfield microscopy. (A) Schematic illustrating the experimental configuration. OBJ1, water-dipping objective; OBJ2, oil-immersion objective. (B) Image of vaccinia virus particles immobilized on a glass coverslip. (C) Image of a vaccinia virus particle as a function of axial sample position. (D) 3D localization of a tracked vaccinia virus particle immobilized on a glass coverslip at a frame rate of 5 kHz. (E) 3D trace of a virus particle landing and diffusing on a cell plasma membrane at an acquisition rate of 5 kHz. (B–E) Adapted with permission from ref (218). Copyright 2017 American Chemical Society.

Figure 14

Surface plasmon resonance microscopy. (A) Schematic of a typical SPRM optical setup. OBJ, objective; BS, beamsplitter; L, tube lens; C, camera. (B) An example of a SPRM PSF with the arrow indicating the direction of surface plasmon propagation. (C) Near-field light–matter interaction of SPRM illumination. E_i incident field, E_s scattered field, E_r reflected field, E_sp evanescent field, θ_R resonant angle. (B) Adapted with permission from ref (258). Copyright 2018 American Chemical Society.

Figure 15

SPRM detection of single virus particles. (A) SPRM images of H1N1 influenza A virus and three different-sized silica nanoparticles in PBS buffer. (B) Histograms of SPR intensities of silica nanoparticles of different diameters and of influenza A viral particles. (C) Calibration curve of SPR intensity vs particle volume. (A–C) Adapted with permission from ref (276). Copyright 2010 National Academy of Sciences.

Figure 16

SPRM imaging of protein size, charge, and mobility. (A) Schematic showing a single protein tethered to an ITO surface by a 63 nm long polyethylene glycol (PEG) linker. (B) FFT image contrast as a function of potential amplitude and a schematic displaying this relation to PEG tether extension; scale bar = 3 μm. The blue dashed line indicates linear and plateau regimes of tether extension. (C) Size determination of protein–PEG complexes (D_H) as a function of FFT image contrast change (ΔC). (D) 2D plot of mobility (μ) vs size (D_H) of single proteins and protein–ligand complexes. (A–D) Adapted with permission from ref (294). Copyright 2020 Springer Nature under Creative Commons Attribution 4.0 International License https://creativecommons.org/licenses/by/4.0.

Figure 17

SPRM-ARI imaging and Bloch surface wave microscopy. (A) Schematic illustrating the superposition of the planar illumination wave, incident at differing azimuthal angles, and a circular scattered wave from a nanosphere. The bottom image shows the cumulative propagation direction of the planar wave for azimuthal illumination angles 0–360°, similar to SPRM-ARI. (B) SPRM image of a 50 nm nanosphere on a Ag film (left) and a corresponding image acquired with SPRM-ARI (right). (C) Images acquired analogously to those in B but of a winding polymer nanowire. (D) Schematic displaying the dielectric multilayer substrate used for BSWM. (E) Angle-dependent excitation spectrum where n_e is the refractive index of the environment. (A–E) Adapted with permission from ref (298). Copyright 2019 American Association for the Advancement of Science under Creative Commons Attribution-NonCommercial 4.0 International license https://creativecommons.org/licenses/by-nc/4.0/.

Figure 18

Quantitative detection of single molecules by plasmonic scattering microscopy. (A) Schematic illustrating the detection method employed in PSM, in which plasmonic waves scattered by a particle (E_s) and any inhomogeneities on the gold surface (E_b) are gathered from the top and interfere to produce an image at the detector. (B) Background and drift corrected PSM image of immunoglobulin M (IgM) molecules. Scale bar: 5 μm. (C) PSM image intensity vs particle diameter. (D) Histograms displaying intensity changes associated with binding and unbinding of individual IgA molecules. (E) Example of the binding behavior of a single IgA molecule. (B–E) Adapted with permission from ref (305). Copyright 2020 Springer Nature.