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Enhanced axial resolution in three-dimensional structured illumination microscopy using CellBrite® Fix Membrane Dyes

Structured illumination is a computational imaging system that involves the projection of a known pattern of light onto a sample; the way the pattern deforms when the projected light strikes different components in the sample allows imaging systems to calculate additional information about the sample. This technique is used in several technologies, including a popular forensic imaging device, the 3D scanner. In three-dimensional structured illumination microscopy (3D SIM), a fluorescently labeled microscopic sample is excited with structured light, providing information outside the diffraction limit. Computational interpretation of this information yields a super-resolution image with double the resolution of standard light microscopy. Though these gains are modest among super-resolution methods, this technique offers advantages in improved optical sectioning, lower required illumination dose and acquisition speed, and has provided numerous biological insights. One limitation of 3D SIM is its axial resolution, which is normally limited to ∼300-nm, versus ∼120-nm lateral resolution, leading to distortion of fine features along the axial dimension.

Li et al. describe methods of overcoming this issue with 3D SIM axial resolution in their recent Nature Biotechnology article, which includes several striking images. They present two discrete, complementary methods to improve axial resolution, which require little or no alteration to existing optical systems. They first showed that placing a mirror on the opposite side of the sample and back-reflecting the central beam enables four-beam interference offering near-isotropic imaging. They also developed a multi-step deep learning pipeline that produced reconstructions with isotropic spatial resolution. They then use a panel of dyes, including Biotium’s CellBrite® Fix 488 and CellBrite® Fix 555 fixable membrane stains, to demonstrate the potential of their novel methods by imaging a variety of prokaryotic and eukaryotic cellular samples and delineating the nanoscale distribution of subcellular elements. In bacteria, they were able to resolve the structure of membranes, cell division proteins, and core components of the spore coat (Figure 1 a-d; Video 1). In eukaryotic cells, they achieved nanoscale resolution of membrane-encased actin filaments and pores that traversed thin membrane extensions, discrete cytoskeletal elements, and caveolar coat proteins. They also performed time-lapse imaging of organellar and cytoskeletal dynamics (Figure 1 h-n; Video 2). Together these results demonstrate the utility of these two approaches for improving 3D SIM. Such advances are important, as SIM microscopy offers a potential replacement for electron microscopy for some medical diagnoses, including those related to kidney disorders, kidney cancer, and blood diseases.

Figure 1. Biotium CellBrite® Fix dye proves useful for four beam 3D SIM in both prokaryotic and eukaryotic cells. a, Maximum intensity projection of live B. subtilis stained with Biotium’s CellBrite® Fix 488 membrane stain, imaged in four-beam SIM. bc, Axial views along the yellow (b) and orange (c) dashed lines in image a, comparing wide-field microscopy (top), 3D SIM (middle) and four-beam SIM (bottom). Yellow arrowheads highlight upper and lower cell membranes and red arrowheads highlight membrane invaginations. d, Line profiles corresponding to vertical orange line in image c. h, Maximum intensity projection image of fixed mouse liver sinusoidal endothelial cells (LSECs) stained with Biotium’s CellBrite® Fix 488 membrane stain (cyan), and fluorescent phalloidin, marking actin filaments (magenta). i, Axial view corresponding to the dashed line in h, highlighting membrane signal encapsulating actin. j, Higher magnification view of the dashed rectangular region in h with membrane pores emphasized (white arrows). k, l, Analogous axial views to j, again highlighting pores (white arrows). Red arrows: actin encapsulated within membrane; blue arrows: void areas enclosed by membrane; yellow arrows: non-specific labeling of coverslip. m, Higher magnification view of the dashed rectangular region in h, with accompanying cross-sectional view (n) corresponding to white dashed line in m, emphasizing membrane-bound organelles (white arrows). Scale bars, 2 µm (a,f); 1 µm (b,i–n); 10 µm (c,h). Credit: X. Li, et al. reproduced under the CC BY-NC-ND 4.0.


Video 1. 3D projections of live vegetative B. subtilis stained with CellBrite® Fix 488, marking membranes. Wide-field (top), 3D SIM (middle) and four-beam SIM reconstructions (bottom) are compared. See also Fig. 2b. Credit: X. Li, et al. reproduced under the CC BY-NC-ND 4.0.
Video 2. Four-beam SIM imaging of fixed mouse LSECs with CellBrite® Fix 488 label, marking membrane (cyan) and Alexa Fluor® 568 phalloidin, marking actin filaments (magenta). See also Fig. 3h. Credit: X. Li, et al. reproduced under the CC BY-NC-ND 4.0.

Learn more about Biotium’s broad selection of membrane dyes and high-performance CF® Dyes with multi-color flexibility, along with other reagents for immunofluorescence microscopy. We also offer several product lines that have been validated for STORM and other super-resolution microscopy techniques.

Full Citation

Li, X., Wu, Y., Su, Y., Rey-Suarez, I., Matthaeus, C., Updegrove, T. B., … & Shroff, H. (2023). Three-dimensional structured illumination microscopy with enhanced axial resolutionNature Biotechnology, 1-13.

Other References

Pullman, J. M., Nylk, J., Campbell, E. C., Gunn-Moore, F. J., Prystowsky, M. B., & Dholakia, K. (2016). Visualization of podocyte substructure with structured illumination microscopy (SIM): a new approach to nephrotic disease. Biomedical Optics Express, 7(2), 302-311.  doi:10.1364/BOE.7.000302

Liu, J., Wang, M., Tulman, D., Mandava, S. H., Elfer, K. N., Gabrielson, A., … & Lee, B. R. (2016). Nondestructive diagnosis of kidney cancer on 18-gauge core needle renal biopsy using dual-color fluorescence structured illumination microscopyUrology98, 195-199. doi:10.1016/j.urology.2016.08.036

Westmoreland, D., Shaw, M., Grimes, W., Metcalf, D. J., Burden, J. J., Gomez, K., … & Cutler, D. F. (2016). Super‐resolution microscopy as a potential approach to diagnosis of platelet granule disordersJournal of Thrombosis and Haemostasis14(4), 839-849. doi:10.1111/jth.13269