New and Notable

Angling for A Better View Christopher M. Yip1,* 1

Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada

Viewing long-standing questions and challenges through a different lens can often provide unique insights and perspectives. This has been the key to many of the innovations emerging in the field of optical microscopy. Whether one is devising novel superresolution localization microscopies (1), new imaging reagents (2,3), or exploiting new imaging modalities to study challenging cellular structures and processes (4,5), the key driver remains acquiring quantitative insights into structure, dynamics, and ultimately functionality. Our understanding of how cells interact with surfaces has been greatly facilitated by innovative imaging strategies. What is certainly clear is the spatial-temporal complexity of the cell-substrate region and the need for quantitative approaches that can robustly probe changes in local topography, ideally in real-time. The challenge in examining these changes is that the vertical scale of interest is small, approximately nanometers in z, for regions such as focal adhesions, which may extend over several hundreds of square nanometers. Moreover, it is important to characterize the spatial organization and dynamics of the cell membrane (and its constituent elements) both for regions that are in

Submitted July 20, 2016, and accepted for publication July 20, 2016. *Correspondence: [email protected] Editor: David Piston.

immediate contact with the surface and for those that are not. To accomplish this, the tool itself must be able to quantitatively determine the vertical position of the cell membrane with nanometer-scale precision, in a loose molecular analogy of a civil engineer’s theodolite. This latter point is particularly important because interaction forces between a cell and a surface necessarily arise from both specific and nonspecific interaction forces (the latter of which have a clear distance dependence). Optical microscopy is perhaps the best approach for interrogating and mapping this interfacial region; however, there are certainly clear caveats. These could include, but not be limited to, the mechanism of image contrast, imaging objective specifications (i.e., numerical aperture), and the internal optics of the microscope itself (i.e., point spread function). While conventional tools such as confocal or deconvolution microscopy could be applied in this context, their vertical resolution is relatively poor compared with interference-based strategies such as reflectance interference contrast microscopy, or fluorescence interference contrast microscopy (6), which can afford nanometer-scale resolution. While there has been renewed interest in reflectance interference contrast microscopy (as it is a label-free approach relying on differences in refractive index (7)), there remain clear challenges in its use for live cell imaging. These largely arise because the cytoplasm is

a complex heterogeneous environment that cannot realistically be modeled as having a uniform, well-behaved, and known refractive index (n) (8). In the context of mapping the nearsurface region of a cell, total internal reflection fluorescence microscopy (TIRF) has become the tool of choice (9). Originally a prism-based technique, objective-based TIRF has gained popularity with the introduction of high numerical-aperture oil-immersion objectives (10). While the general model of the evanescent decay of the excitation beam has certainly been well established, there continue to be exciting developments in how one can exploit this behavior and interpret the resulting images. For example, recent work has described strategies for addressing the nonuniformity of the excitation field, and compensating for both under- and supercritical angle contributions to yield quantitative TIRF images (11–14). It has long been recognized that by acquiring TIRF images at various incident angles—so-called variable-angle TIRF (vaTIRF)—one can then optically section through a volume of interest. There are many challenges in vaTIRF, especially when practiced in an objective-based versus prism-based configuration, not the least of which is ensuring that, as the incident angle is varied, that the illuminated sample region remains the same. In the work by Cardoso Dos Santos et al. (15) published in this issue of the Biophysical Journal, the concept of vaTIRF

http://dx.doi.org/10.1016/j.bpj.2016.07.034 Ó 2016 Biophysical Society.

Biophysical Journal 111, 1141–1142, September 20, 2016 1141

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imaging is revisited and the development of a robust and simple means of performing objective-based vaTIRF imaging is presented (15). The approach that the authors chose is one that is simple and easy to implement. Their design involves placing a rotating mirror at the rear focal plane of the focusing lens, which allows them to tune the incident angle and minimize any lateral displacement of the beam during a vaTIRF experiment. In doing so, and thus controlling the penetration depth of the TIRF field, the authors contend that it is now feasible to locate structures with nanometer precision. The authors then go on to present a compelling and innovative theoretical framework that addresses the complexity of how the evanescent wave propagates through multiple dielectric media (glass, solution, cell cytoplasm). Their model also bundles difficult-to-quantify parameters, such as the point-spread function, local fluorophore concentration, and orientation into a single angle-invariant term, with the proviso that it only works with s-polarized illumination. The work by Cardoso Dos Santos et al. (15) is timely and the formalism that they have developed is quite accessible. While their work suggests that collecting a vaTIRF dataset and fitting the resulting pixelwise intensity data as a function of angle will allow one to easily map the cell surface topography, cell-substrate separation distances, and effective local indices of refraction (neff), due to imaging arti-

facts the range of usable angles is small (~63.6–~67.5 ). While it is easy to see how this modification can be implemented on any TIRF system with easy access to the optical train, the ease of data analysis, the value-add of this implementation and the fact that many commercial TIRF platforms already incorporate some form of angle control does argue quite persuasively for facile commercial integration. What remains to be seen, however, is how effective this approach is for multicolor TIRF imaging. Because penetration depth is wavelength-dependent, this certainly presents a more challenging integration scheme, particularly from the optical alignment perspective. The mathematical processing approach that has been developed should have straightforward extensions that would allow for a robust postacquisition interpretation and analysis of multicolor vaTIRF datasets. REFERENCES 1. Betzig, E. 2015. Single molecules, cells, and super-resolution optics (Nobel lecture). Angew. Chem. Int. Ed. Engl. 54:8034–8053. 2. Baddeley, D., I. D. Jayasinghe, ., C. Soeller. 2009. Light-induced dark states of organic fluochromes enable 30 nm resolution imaging in standard media. Biophys. J. 96:L22–L24. 3. Enterina, J. R., L. Wu, and R. E. Campbell. 2015. Emerging fluorescent protein technologies. Curr. Opin. Chem. Biol. 27:10–17. 4. Weber, M., M. Mickoleit, and J. Huisken. 2014. Light sheet microscopy. Methods Cell Biol. 123:193–215.

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5. Chung, K., J. Wallace, ., K. Deisseroth. 2013. Structural and molecular interrogation of intact biological systems. Nature. 497:332–337. 6. Parthasarathy, R., and J. T. Groves. 2004. Optical techniques for imaging membrane topography. Cell Biochem. Biophys. 41:391–414. 7. Klein, K., C. E. Rommel, ., J. P. Spatz. 2013. Cell membrane topology analysis by RICM enables marker-free adhesion strength quantification. Biointerphases. 8:28. 8. Limozin, L., and K. Sengupta. 2009. Quantitative reflection interference contrast microscopy (RICM) in soft matter and cell adhesion. ChemPhysChem. 10:2752–2768. 9. Axelrod, D. 2008. Chapter 7: total internal reflection fluorescence microscopy. Methods Cell Biol. 89:169–221. 10. Axelrod, D. 2001. Selective imaging of surface fluorescence with very high aperture microscope objectives. J. Biomed. Opt. 6:6–13. 11. Winterflood, C. M., T. Ruckstuhl, ., S. Seeger. 2010. Nanometer axial resolution by three-dimensional supercritical angle fluorescence microscopy. Phys. Rev. Lett. 105:108103. 12. Ruckstuhl, T., D. Verdes, ., S. Seeger. 2011. Simultaneous near-field and far-field fluorescence microscopy of single molecules. Opt. Express. 19:6836–6844. 13. Brunstein, M., M. Teremetz, ., M. Oheim. 2014. Eliminating unwanted far-field excitation in objective-type TIRF. Part I. Identifying sources of nonevanescent excitation light. Biophys. J. 106:1020–1032. 14. Brunstein, M., K. He´rault, and M. Oheim. 2014. Eliminating unwanted far-field excitation in objective-type TIRF. Part II. Combined evanescent-wave excitation and supercritical-angle fluorescence detection improves optical sectioning. Biophys. J. 106:1044–1056. 15. Cardoso Dos Santos, M., R. De´turche, ., R. Jaffiol. 2016. Nanoscale topography of cells in adhesion revealed by variable-angle total internal reflection fluorescence microscopy. Biophys. J. 111:1316–1327.

Angling for A Better View.

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