Fluorescence Microscopy Journal Club

Rapid 3D-STORM Imaging of Diverse Molecular Targets in Tissue

by Nicholas Albrecht, Danye Jiang, Robert Hobson, Colenso Speer, Melanie Samuel

Key Points

  • A set of imaging parameters relating to tissue preparation, fixation, quenching, labeling, and imaging conditions is empirically linked to quantifiable improvements in the quality of 3D SMLM images of tissues;
  • The optimized protocol identified here is applicable to a broad range of sample tissue types, molecular targets, and biological structures — including clinically derived human tissue;
  • The RAIN-STORM method provides a detailed approach to optimizing sample preparation, and should serve as a guide for those interested in pursuing SMLM studies in tissues; and
  • This approach, when combined with appropriate instrumentation, makes SMLM imaging of tissues in 3D much more accessible.



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This review appeared in the March 2022 edition of the Fluorescence Microscopy Journal Club — a monthly email brief highlighting leading-edge research and the latest discoveries supported by Bruker fluorescence microscopes.

Cell Reports Methods, Dec. 2021
DOI: 10.2139/ssrn.3985168

Single-molecule localization microscopy (SMLM) is a powerful tool for visualizing and quantifying nanoscale organization across a variety of biological systems. However, the ability to image tissue samples in 3D and at high resolutions presents a challenge for many SMLM systems. This is partially due to limitations of instrumentation along with complex sample preparation requirement; standard total internal reflection (TIRF) systems restrict imaging to the coverslip surface and preparing thin tissue slices can be a daunting task.

In this paper from Albrecht et al., researchers conducted a comprehensive experiment to optimize sample preparation conditions for 10µm-thick retinal tissue sections. Using Bruker’s Vutara 352 microscope and SRX software to execute a new super-resolution imaging method — RAIN-STORM (Rapid Imaging of Tissues at the Nanoscale Stochastic Optical Reconstruction Microscopy) — the authors:

  • Collected and analyzed a database of ~375 3D STORM images;
  • Identified and systematically tested 125 parameters relating to tissue preparation, fixation, quenching, labeling, and imaging conditions; and
  • Defined a set of imaging parameters based on quantified improvements in resolution, antibody labeling density, and reduction in background signal.

In the case of these 10µm sections, the Vutara microscope is the ideal system for 3D imaging due to its epi-illumination and biplane detection means of obtaining 3D data with every acquisition without the need for an astigmatic lens. As a result, they were able to image the entirety of the 10µm sections by scanning in Z with more accuracy at larger depths than would be obtainable with astigmatic TIRF-based 3D imaging. For quantifying each image, researchers assessed the total number of localizations, sample detail preservation, background signal, and planar and axial resolutions. OPTICS (Ordering Points to Identify the Clustering Structure) and Fourier Ring Correlation analysis, which are available directly in the SRX statistical analysis suite, were used to identify associated contiguous structure from unstructured data, and to assess planar and axial resolution of all images.

The optimized conditions were tested on and are applicable to a wide range of tissue types and many molecular targets and biological structures. Furthermore, the optimized protocol can be successfully applied to clinically derived human tissue.


      KEY TERMS:

  • 3D Nanoscopic Imaging; Human Samples; Molecular Features in Intact Tissue; Nanoscopic Tissue Imaging; Sample Preparation Protocols