Despite recent advances in super-resolution methodologies, real-time imaging with nano-scale precision remains challenging, especially along the optical axis. Current techniques (reviewed in1
) have revealed the architecture of molecular complexes such as focal adhesions with unprecedented detail in fixed cells, yet the dynamic behavior of these structures remains largely unknown2
. A need still exists for imaging methodologies capable of recording nano-scale structural details and movements in living cells as occurs during processes such as motility, where nano-scale dynamics of molecules are presumably coordinated across large spatial regions of the cell3-5
The leading approach for measuring the topography of lipid membranes with nanometer-scale z-resolution is fluorescence interference contrast microscopy (FLIC)6
. In FLIC, axially varying structured illumination is used to determine the vertical position of nanometer-sized objects. To eliminate periodic replication artifacts and obtain unambiguous positions, the pattern of structured illumination is manipulated by varying the angle of incidence of excitation light to provide additional spatial information7
. Although FLIC has the potential to visualize the rapid movements and spatial organization of proteins, interference contrast methodologies have not been widely adopted in biology, likely due to the technical challenges of implementation, including the assembly of custom microscopy suites and/or the generation of micro-fabricated substrates, and the assumptions that must be made regarding fluorophore molecular orientation.
Here we modify FLIC to develop a new interference contrast method with nanometer precision, which we term scanning angle interference microscopy. This approach can be implemented on commercial total internal reflection (TIRF) microscopes with no hardware modifications (Supplementary Note
), and it overcomes many of the limitations of FLIC. We determine unambiguous positions of fluorescent structures over a wide axial range (approaching a micron or more) by actively scanning the incidence angle of the excitation. Furthermore, by manipulating the polarization state of light, we have eliminated the requirement for knowledge of the orientation of fluorescence dipoles in the labeled structure. This approach enables imaging with several nanometer axial precision and temporal sampling rates on the order of one second.
Samples for scanning angle interference microscopy are prepared on reflective silicon wafers with a thin layer of silicon oxide that functions as a sample substrate ()
. Axially varying patterns of excitation light intensity are generated through the interference of incident and reflected light, and the structure of the pattern is manipulated by controlling the incidence angle of the excitation light. To quantify performance, we adsorbed nano-sized fluorescent beads on silicon substrates with oxide spacer layers of varying thickness and scanned them with excitation light of varying incidence angle. The recorded fluorescence intensity profiles across varying incidence angles were then fit to an optical model to obtain the axial position of the bead center from the silicon surface with nanometer-scale precision. The use of excitation light linearly polarized perpendicular to the plane of incidence (s-polarized) improved interference intensity contrast by as much as five-fold over non-polarized or circularly polarized light (Supplementary Fig. 1
), eliminated the dependence of the optical model on fluorophore dipole orientation (See Optical theory
, Online Methods
), and significantly improved the accuracy of object localization (Supplementary Fig. 1
Scanning Angle Interference Microscopy
Measured bead heights above the silicon were on average within a few nanometers of expected heights based on thin film measurements of the silicon oxide ( and Supplementary Fig. 2
) with the standard deviation ranging from 3.6 to 5.1 nm (). Beads varying in axial position by as little as 13 nm had distinct interference contrast profiles, allowing their height differences to be resolved (). Similarly, analysis of mCherry and mEmerald conjugates of the focal adhesion protein paxillin in cells positioned at four heights above the silicon substrate revealed a standard deviation of just 1.8 nm from the mean height across eight independent measurements (Supplementary Fig. 3
). We found that a signal to noise ratio of approximately two or better yielded the most accurate results in cellular samples (Supplementary Fig. 4
). The error of measurement in our approach due to natural refractive index variations in cells was theoretically predicted to be ~3.8 nm for structures positioned 0 to 350 nm above the substrate (Supplementary Fig. 5
). These predictions were experimentally borne out with a nearly identical mean height and standard deviation obtained for beads absorbed on the silicon substrates when they were imaged through either buffer or cells (Supplementary Fig. 6
). Lateral resolution of the technique in cells was approximately diffraction limited (full width at half maximum ~300 nm; Supplementary Fig. 7
We used scanning angle interference microscopy to image the vertical positions of three types of cellular structures: the plasma membrane (), the microtubule cytoskeletal network (), and molecules within the focal adhesion complex (). Visual reconstructions of dye-labeled, fixed ventral cell membranes revealed fine axial detail, displaying distinct topographical features corresponding to the sites of focal adhesion complexes, the cortical actin network proximal to the plasma membrane, and the variations in glycocalyx thicknesses ranging from 40 to 85 nm ( and Supplementary Fig. 8
). In fixed epithelial cells with fluorescently-labeled microtubules, the scanning angle interference images showed a downward bending of the microtubule network in the lamella ().
Notably, the measured microtubule heights ranged from approximately 70 to 350 nanometers, which is beyond the working range of other techniques that offer high axial precision, including variable incidence angle TIRF, interference-based photo-activation localization microscopy, and standard implementations of FLIC.
Scanning angle interference imaging of microtubules
We next applied scanning angle interference microscopy as a molecular ruler in live cells. We generated a construct for expressing talin, a focal adhesion molecule with a rod-like structure 60 nm in length, as a fusion with mCherry at its N-terminus and mEmerald at its C-terminus. We found that the C-terminus was situated on average 37 nm higher than the N-terminus, corresponding to a molecular orientation of 51 degrees relative to the vertical axis. The precision of measurement with our approach was comparable to advanced single molecule localization microscopy methods implemented on two-objective and interference based microscopes ()
, however in contrast to these approaches, our method permits the acquisition of these measurements with 1-10 second sampling rates in living cells8-9
. Scanning angle interference microscopy does not currently incorporate vertical sectioning and is therefore ideally suited for the measurement of discrete structures with nano-scale thickness, < 150 nm, such as plasma membranes and associated proteins, cytoskeletal filaments, microtubules, and vesicles (Supplementary Fig. 9
). For fluorophores distributed vertically in a diffraction-limited spot, it provides an average height of the molecules, as observed with a talin construct double-labeled with mCherry on its C- and N-termini (Supplementary Fig. 9)
The axial precision and ability to image multiple color channels, coupled with the dynamic capability of scanning angle interference microscopy, makes the method useful for exploring the molecular mechanisms and topological arrangements underlying dynamic mechanical processes. We simultaneously measured the heights of paxillin-mEmerald and vinculin-mCherry conjugates, two structural molecules localized to focal adhesions. We observed qualitatively that paxillin increased in height as compared to vinculin in the adhesions that formed adjacent to sites of cell-cell contact following the collision of two motile cells ( and Supplementary Movie). In more quantitative measurements, we observed that paxillin and vinculin were vertically stratified in motile cells; paxillin was at an average height of 61±6.5 nm and vinculin at an average height of 99±7.2 nm (P
< 0.001; n
> 20 cells; Supplementary Fig. 3
). Finally, we imaged paxillin and vinculin during cycles of cell retraction in the maturing adhesions of these motile cells ( and Supplementary Movie). Imaging randomly selected adhesions in these motile cells revealed that the average height of paxillin in maturing adhesions was significantly lower than the average height of paxillin in stable adhesions in non-retracting regions of the cells (), as well as its height in small, newly formed adhesions in protruding regions (n
= 5 cells; P
< 0.001; Supplementary Fig. 11)
. We found that paxillin was typically lowered in maturing adhesions within 5 – 10 minutes of the start of cell retraction cycles. By contrast, axial movement of vinculin in maturing adhesions was more random ( and Supplementary Fig. 10
Nano-scale dynamics of adhesion proteins in migrating cells
Although the mechanism of downward paxillin movement during cell migration remains unclear, one possibility is that these vertical shifts correspond to increased mechanical engagement of the cytoskeleton with the adhesion complex, which is known to stimulate adhesion maturation. Consistent with this notion, we did not observe any downward displacement of paxillin in cells treated with the Rho kinase inhibitor Y-27632 (data not shown). Nevertheless, the findings raise the possibility that adhesion complexes could transduce mechanical information through the spatial reorganization of their molecular components. The experimental data also highlight the ability of this imaging approach to visualize the dynamics of cellular structures in response to force and to provide the experimentalist with quantitative insight. We therefore propose that scanning angle interference microscopy may be a useful new tool for unraveling cellular mechanisms of mechanotransduction.
In summary, we have demonstrated that scanning angle interference microscopy is capable of localizing biological structures with nanometer-scale precision along the optical axis in living cells on time scales suitable for observing dynamic processes. The combination of high axial and temporal resolution, extended depth of field, ease of implementation, and the ability to image live cells in multiple color channels distinguishes this approach from existing super-resolution techniques. Scanning angle interference microscopy will enable the investigation of the many dynamic processes that occur proximal to the cell membrane – for instance, signaling linked to receptor activation and mechanotransduction, endocytosis, exocytosis, and extracellular-matrix remodeling - and should thus be broadly applicable to emerging questions in membrane transport, signal transduction, and mechanobiology.