Multimodal illumination platform for 3D single-molecule super-resolution imaging throughout mammalian cells

Abstract

Single-molecule super-resolution imaging is instrumental in investigating cellular architecture and organization at the nanoscale. Achieving precise 3D nanometric localization when imaging structures throughout mammalian cells, which can be multiple microns thick, requires careful selection of the illumination scheme in order to optimize the fluorescence signal to background ratio (SBR). Thus, an optical platform that combines different wide-field illumination schemes for target-specific SBR optimization would facilitate more precise 3D nanoscale studies of a wide range of cellular structures. Here, we demonstrate a versatile multimodal illumination platform that integrates the sectioning and background reduction capabilities of light sheet illumination with homogeneous, flat-field epi- and TIRF illumination. Using primarily commercially available parts, we combine the fast and convenient switching between illumination modalities with point spread function engineering to enable 3D single-molecule super-resolution imaging throughout mammalian cells. For targets directly at the coverslip, the homogenous intensity profile and excellent sectioning of our flat-field TIRF illumination scheme improves single-molecule data quality by providing low fluorescence background and uniform fluorophore blinking kinetics, fluorescence signal, and localization precision across the entire field of view. The increased contrast achieved with LS illumination, when compared with epi-illumination, makes this illumination modality an excellent alternative when imaging targets that extend throughout the cell. We validate our microscopy platform for improved 3D super-resolution imaging by two-color imaging of paxillin - a protein located in the focal adhesion complex - and actin in human osteosarcoma cells.

Fig. 1.

Design and performance of the multimodal illumination platform. (a) Simplified schematic of the microscopy platform (not to scale). Definitions of optical elements abbreviations: cylindrical lens (Cyl.), de-speckler (DS), dichroic mirror (DM), galvanometric mirror (GM), motorized mirror (MM), notch filter (NF), objective lens (Obj), periscope (P), phase mask (PM), removable mirror (RM), translation stage (TS). “f” indicates lenses with the indicated focal lengths in mm. (b) Images of both light sheet (LS) orientations acquired in fluorescent solution at a fixed LS Z-position show the LS thickness (side view, left) and width (top view, right). Scale bars are 10 µm. (c) Graph with line scans of the LS images shown in (b) at the indicated dashed lines used to determine the LS thickness and width. (d) Images of a dense layer of fluorescent beads showing the flat-field (FF, left) and Gaussian (right) TIRF illumination intensity profiles. Scale bars are 10 µm. (e) Graph with line scans of the images shown in (d) at the indicated dashed lines used to quantify the uniformity of the FF intensity profile compared to the Gaussian intensity profile. (f) Images of fluorescent beads at the indicated axial positions demonstrating the double-helix PSFs with 2 µm (left) and 12 µm (right) axial range. Scale bars are 1 µm and 3 µm for left and right panels, respectively. Color bars indicate the linear color scale used. The contrast of each image is normalized independently.

Fig. 2.

Light sheet (LS) and flat-field (FF) TIRF illumination improve the contrast for cellular imaging. (a) Representative images of a U-2 OS cell immunolabeled with CF568 for paxillin and illuminated using Gaussian epi-illumination (top left), Gaussian TIRF illumination (top right), flat-field (FF) epi-illumination (bottom left), and FF TIRF illumination (bottom right). (b) Representative images of a U-2 OS cell where actin is labeled using phalloidin conjugated with AF647 and illuminated using epi-illumination (top) and light sheet (LS) illumination (bottom). (c) Graph showing line scans as indicated by the dashed lines in (a), demonstrating the contrast improvement and the more homogeneous illumination achieved when using FF TIRF illumination compared to the other illumination modalities. (d) Graph showing lines scans as indicated by the dashed lines in (b), demonstrating the significant contrast improvement when using LS illumination compared to epi-illumination. All scale bars are 10 µm. Color bars indicate the linear color scale used. The contrast of each image is normalized independently.

Fig. 3.

Flat-field TIRF illumination provides more uniform performance across the field of view for single-molecule localization microscopy. (a,b) A dense layer of CF568 fluorophores imaged with (a) Gaussian TIRF illumination and (b) FF TIRF illumination showing the field of view split into six annuli of equal area for parameter quantification. Scale bars are 10 µm. (c-e) Graphs showing Gaussian and FF TIRF comparison across annuli (top panel) and overall difference, Δ, in mean values (bottom panel) between annulus 5 and 0 for the (c) localization uncertainty, (d) number of localizations, and (e) the number of photons per localization (intensity) from single-molecule imaging. The error bars show mean ± standard deviation from three measurements for each illumination modality.

Fig. 4.

3D single-molecule super-resolution imaging throughout mammalian cells. (a) 3D super-resolved reconstructions of actin (color coded based on Z position) and paxillin (white) in a U-2 OS cell. (b) 3D super-resolved reconstruction of the actin shown in (a). (c) 3D super-resolved reconstruction of the paxillin shown in (a), here color coded by depth. (d) Zoom in of the paxillin shown in the red dashed box in (c). (e) Zoom in on the actin and paxillin connection shown in the yellow dashed box in (a). (f) Histogram of Z positions of actin and paxillin localizations of a representative example used to determine the separation between the two targets. Scale bars are 10 µm in (a), (b), and (c), and 1 µm in (d) and (e).