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High-density three-dimensional localization microscopy across large volumes.

ABSTRACT: Extending three-dimensional (3D) single-molecule localization microscopy away from the coverslip and into thicker specimens will greatly broaden its biological utility. However, because of the limitations of both conventional imaging modalities and conventional labeling techniques, it is a challenge to localize molecules in three dimensions with high precision in such samples while simultaneously achieving the labeling densities required for high resolution of densely crowded structures. Here we combined lattice light-sheet microscopy with newly developed, freely diffusing, cell-permeable chemical probes with targeted affinity for DNA, intracellular membranes or the plasma membrane. We used this combination to perform high-localization precision, ultrahigh-labeling density, multicolor localization microscopy in samples up to 20 ?m thick, including dividing cells and the neuromast organ of a zebrafish embryo. We also demonstrate super-resolution correlative imaging with protein-specific photoactivable fluorophores, providing a mutually compatible, single-platform alternative to correlative light-electron microscopy over large volumes.

SUBMITTER: Legant WR

PROVIDER: S-EPMC4889433 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

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High-density three-dimensional localization microscopy across large volumes.

Legant Wesley R WR Shao Lin L Grimm Jonathan B JB Brown Timothy A TA Milkie Daniel E DE Avants Brian B BB Lavis Luke D LD Betzig Eric E

Nature methods 20160307 4

Extending three-dimensional (3D) single-molecule localization microscopy away from the coverslip and into thicker specimens will greatly broaden its biological utility. However, because of the limitations of both conventional imaging modalities and conventional labeling techniques, it is a challenge to localize molecules in three dimensions with high precision in such samples while simultaneously achieving the labeling densities required for high resolution of densely crowded structures. Here we ...[more]

PMID: 26950745

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Project description:Many biological processes involve the collective generation and transmission of mechanical stresses across cell monolayers. In these processes, the monolayer undergoes lateral deformation and bending because of the tangential and normal components of the cell-generated stresses. Monolayer stress microscopy (MSM) methods have been developed to measure the intracellular stress distribution in cell monolayers. However, current methods assume plane monolayer geometry and neglect the contribution of bending to the intracellular stresses. This work introduces a three-dimensional (3D) MSM method that calculates monolayer stress from measurements of the 3D traction stresses exerted by the cells on a flexible substrate. The calculation is carried out by imposing equilibrium of forces and moments in the monolayer, subject to external loads given by the 3D traction stresses. The equilibrium equations are solved numerically, and the algorithm is validated for synthetic loads with known analytical solutions. We present 3D-MSM measurements of monolayer stress in micropatterned islands of endothelial cells of different sizes and shapes. These data indicate that intracellular stresses caused by lateral deformation emerge collectively over long distances; they increase with the distance from the island edge until they reach a constant value that is independent of island size. On the other hand, bending-induced intracellular stresses are more concentrated spatially and remain confined to within one to two cell lengths of bending sites. The magnitude of these bending stresses is highest at the edges of the cell islands, where they can exceed the intracellular stresses caused by lateral deformations. Our data from nonpatterned monolayers suggests that biomechanical perturbations far away from monolayer edges also cause significant localized alterations in bending tension. The localized effect of bending-induced stresses may be important in processes like cellular extravasation, which are accompanied by significant normal deflections of a cell monolayer (i.e., the endothelium) and require localized changes in monolayer permeability.

Project description:Traditional histology is the gold standard for tissue studies, but it is intrinsically reliant on two-dimensional (2D) images. Study of volumetric tissue samples such as whole hearts produces a stack of misaligned and distorted 2D images that need to be reconstructed to recover a congruent volume with the original sample's shape. In this paper, we develop a mathematical framework called Transformation Diffusion (TD) for stack alignment refinement as a solution to the heat diffusion equation. This general framework does not require contour segmentation, is independent of the registration method used, and is trivially parallelizable. After the first stack sweep, we also replace registration operations by operations in the space of transformations, several orders of magnitude faster and less memory-consuming. Implementing TD with operations in the space of transformations produces our Transformation Diffusion Reconstruction (TDR) algorithm, applicable to general transformations that are closed under inversion and composition. In particular, we provide formulas for translation and affine transformations. We also propose an Approximated TDR (ATDR) algorithm that extends the same principles to tensor-product B-spline transformations. Using TDR and ATDR, we reconstruct a full mouse heart at pixel size 0.92µm×0.92µm, cut 10µm thick, spaced 20µm (84G). Our algorithms employ only local information from transformations between neighboring slices, but the TD framework allows theoretical analysis of the refinement as applying a global Gaussian low-pass filter to the unknown stack misalignments. We also show that reconstruction without an external reference produces large shape artifacts in a cardiac specimen while still optimizing slice-to-slice alignment. To overcome this problem, we use a pre-cutting blockface imaging process previously developed by our group that takes advantage of Brewster's angle and a polarizer to capture the outline of only the topmost layer of wax in the block containing embedded tissue for histological sectioning.

Dataset Information

High-density three-dimensional localization microscopy across large volumes.

Publications

High-density three-dimensional localization microscopy across large volumes.

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