Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
[Preprint]. 2023 May 26:2023.05.26.542523.
doi: 10.1101/2023.05.26.542523.

Super-resolution imaging of neuronal structure with structured illumination microscopy

Tristan C Paul  1 , Karl A Johnson  1 , Guy M Hagen  1
Affiliations

Super-resolution imaging of neuronal structure with structured illumination microscopy

Tristan C Paul et al. bioRxiv. .

Update in

Abstract

Super-resolution structured illumination microscopy (SR-SIM) is a method in optical fluorescence microscopy which is suitable for imaging a wide variety of cells and tissues in biological and biomedical research. Typically, SIM methods use high spatial frequency illumination patterns generated by laser interference. This approach provides high resolution but is limited to thin samples such as cultured cells. Using a different strategy for processing the raw data and coarser illumination patterns, we imaged through a 150 µm thick coronal section of a mouse brain expressing GFP in a subset of neurons. The resolution reached 144 nm, an improvement of 1.7 fold beyond conventional widefield imaging.

PubMed Disclaimer

Conflict of interest statement

Competing interests

The authors declare that they have no competing interests.

Figures

Figure 1:
Figure 1:
(left) simplified optical diagram; (right) connection diagram. Shown is the connection setup for two-wavelength acquisition, in this study only 470 nm illumination was used.
Figure 2:
Figure 2:
(a), Overview SIM image. The yellow box indicates the temporal association area where neurons were imaged with super-resolution MAP-SIM. (b), Allen brain atlas slice 92 of 132.
Figure 3:
Figure 3:
(a), neuron imaged at a depth of 41 μm to 66 μm using a 100x / 1.4 NA oil immersion objective. (b-c) show zoomed in views of the selected areas indicated in a by yellow boxes. The width of the spine neck, selected in (d) was fit to a Gaussian function, FWHM 164.0 ± 4.9 nm.
Figure 4:
Figure 4:
(a), TeA neuron shown in widefield, basic SIM, and MAP-SIM. (b), MAP-SIM image color coded by depth.
Figure 5:
Figure 5:
Neuron imaged using a high frequency (1 pixel on, 5 off) pattern (a) and a low frequency (2 pixels on, 10 off) pattern (b) as well as graphs of their circularly averaged power spectral density (c,d). Insets in images (a,b) are the corresponding FFTs with yellow dotted circles indicating the region of support.
See this image and copyright information in PMC

References

    1. Hell S. W. and Wichmann J., “Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy,” Opt Lett 19, 780 (1994). - PubMed
    1. Rust M. J., Bates M., and Zhuang X., “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat Methods 3, 793–795 (2006). - PMC - PubMed
    1. Betzig E., Patterson G. H., Sougrat R., Lindwasser O. W., Olenych S., Bonifacino J. S., Davidson M. W., Lippincott-Schwartz J., and Hess H. F., “Imaging intracellular fluorescent proteins at nanometer resolution,” Science (1979) 313, 1642–1645 (2006). - PubMed
    1. Dertinger T., Xu J., Naini O., Vogel R., and Weiss S., “SOFI-based 3D superresolution sectioning with a widefield microscope,” Opt Nanoscopy 1, 2 (2012). - PMC - PubMed
    1. Gustafsson M. G. L., “Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy,” J Microsc 198, 82–87 (2000). - PubMed

Publication types