Correlative three-dimensional super-resolution and block-face electron microscopy of whole vitreously frozen cells - PubMed (original) (raw)

. 2020 Jan 17;367(6475):eaaz5357.

doi: 10.1126/science.aaz5357.

Gleb Shtengel 1, C Shan Xu 1, Kirby R Campbell 2, Melanie Freeman 1, Lei Wang 3 4 5, Daniel E Milkie 1, H Amalia Pasolli 1, Nirmala Iyer 1, John A Bogovic 1, Daniel R Stabley 6, Abbas Shirinifard 7, Song Pang 1, David Peale 1, Kathy Schaefer 1, Wim Pomp 3 4 5, Chi-Lun Chang 1, Jennifer Lippincott-Schwartz 1, Tom Kirchhausen 1 3 4 5, David J Solecki 2, Eric Betzig 8 9 10 11 12 13, Harald F Hess 8

Affiliations

Correlative three-dimensional super-resolution and block-face electron microscopy of whole vitreously frozen cells

David P Hoffman et al. Science. 2020.

Abstract

Within cells, the spatial compartmentalization of thousands of distinct proteins serves a multitude of diverse biochemical needs. Correlative super-resolution (SR) fluorescence and electron microscopy (EM) can elucidate protein spatial relationships to global ultrastructure, but has suffered from tradeoffs of structure preservation, fluorescence retention, resolution, and field of view. We developed a platform for three-dimensional cryogenic SR and focused ion beam-milled block-face EM across entire vitreously frozen cells. The approach preserves ultrastructure while enabling independent SR and EM workflow optimization. We discovered unexpected protein-ultrastructure relationships in mammalian cells including intranuclear vesicles containing endoplasmic reticulum-associated proteins, web-like adhesions between cultured neurons, and chromatin domains subclassified on the basis of transcriptional activity. Our findings illustrate the value of a comprehensive multimodal view of ultrastructural variability across whole cells.

Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

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Conflict of interest statement

Competing interests:

E.B. has a financial interest in LLSM. E.B. and H.F.H. have a financial interest in SMLM.

Figures

Figure 1.

Figure 1.. Cryogenic photophysical characterization of fluorophores for single molecule localization microscopy (SMLM)

(A) Dynamic and (B) static contrast ratios for six different fluorophores at ~8K (blue) and ~77K (orange) ordered by increasing emission wavelength. (C) Corresponding images of mitochondrial outer membrane protein TOMM20 with detailed insets shown below. (D) Comparison of mEmerald and JF525 in cryo-SMLM imaging. Top row: U2OS cell transiently expressing ER membrane marker mEmerald-Sec61β and mitochondrial membrane marker Halo-TOMM20 conjugated to JF525. Bottom row: U2OS cell transiently expressing mEmerald-TOMM20 and Halo/JF525-Sec61β. Scale bars: 5 μm and 0.5 μm in (C), first and second rows; 1 μm in (D).

Figure 2.

Figure 2.. High accuracy correlation of cryo-SMLM and FIB-SEM data using organelle landmarks.

(A) A perspective view of mitochondrial (spheres) and ER (cubes) landmarks used for registration along with the plasma (grey) and nuclear (pink) membranes as determined by FIB-SEM of two COS-7 cells. Arrows point in the direction of and are sized according to the magnitude of the non-linear component of the final displacement field. Arrows are color coded according to the magnitude of the local difference (ΔDF) between the displacement fields determined by the mitochondrial or the ER landmarks separately. The pink surface indicates the boundaries of the sub-volume containing sufficient landmarks of both types for quantitative comparison of their respective displacement fields. (B) XY orthoslice illustrating landmark selection and determination of the displacement vectors (fig. S14). Scale bar: 1 μm. (C) Histograms of the correlation accuracy, ε (cf. supplementary note 7), for the sub-volume bounded by the pink surface (magenta) and the 61 μm3 sub-volume defined by the red box (red), where density of both types of landmarks is higher.

Figure 3.

Figure 3.. Whole-cell correlative cryogenic single molecule localization and block face electron microscopy.

(A) Perspective overview of a cryo-SMLM and FIB-SEM (orange and grey) data set of a COS-7 cell transiently expressing mEmerald-ER3 (ER lumen marker, green) and Halo/JF525-TOMM20 (mitochondrial outer membrane marker, magenta) (Movie 2). Cyan, yellow, and white boxes indicate regions with ortho slices shown in panels (B-M) and inset. Inset: SMLM (left column), FIB-SEM (middle column), and correlative (right column) orthoslices in XY (top row) and XZ (bottom row) through an intranuclear ER-positive vesicle. Scale bar: 200 nm. (B, E) SMLM, (C, F) FIB-SEM and (D, G) correlated overlay of orthoslices in XY (B-D) and XZ (E-G) in a lamellipodial region. Scale bar: 1 μm. (H, K) SMLM, (I, L) FIB-SEM and (J, M) correlated overlay of orthoslices in XY (H-J) and XZ (K-M) in a thicker region with ER sheets. Scale bar: 1 μm. Red arrows: TOMM20-positive vesicles; orange arrows: varicosities in the ER.

Figure 4.

Figure 4.. Diversity of peroxisome morphologies and peroxisome-organelle interactions.

(A-D) FIB-SEM segmentations (top) of four peroxisomal targeting signal (SKL) containing peroxisomes (magenta) and corresponding orthoslices (bottom) with cryo-SMLM overlays of SKL (green) from two HeLa cells expressing mEmerald-SKL. (E-G) Three examples of peroxisome/organelle interactions, showing both segmentations (top) and orthoslices (bottom) with overlaid contours of matching colors. Scale bars: 200 nm. (H) Surface-to-volume relationship for 466 peroxisomes (fig. S17), with the specific peroxisomes in (A)-(G) indicated, showing the increasing deviation from spherical shape with increasing volume. See also Movie 3.

Figure 5.

Figure 5.. Cryo-SIM/FIB-SEM accurately identifies endosomal compartments and reveals their diverse morphologies at the nanoscale.

(A) Volume rendered FIB-SEM overview (interior, orange; plasma membrane, cyan) of a SUM159 cell, with cutaway correlative cryo-SIM showing endolysosomal compartments containing AF647-conjugated transferrin (green). (B) Segmented Tfn-AF647 containing compartments (colored surfaces) with superimposed 3D cryo-SIM data (green voxels) in the 13 μm3 subvolume denoted by the red box in (A). (C) XY (top) and ZY (bottom) orthoslices of the same region in (B) showing the FIB-SEM (left) overlaid with segmentations of transferrin labeled compartments (middle) and cryo-SIM of Tfn-AF647 (right). (D, E) Same as (B) and (C) for the 19.5 μm3 subvolume denoted by the yellow box in (A). Scale bars, (A, inset) 10 μm, (C, E) 1 μm.

Figure 6.

Figure 6.. Membrane proteins correlate to membrane ultrastructure at cell-cell adhesions.

(A) Cryo-SIM volume of cultured mouse cerebellar granule neurons transiently expressing JF549i/SNAP-JAM-C (green) and 2x-mVenus-Drebrin (magenta). (B) MIP through an ~3 μm thick slab (white box in (A)) centered on the contact zone between two cell bodies. (C) FIB-SEM volume of the same region in (A), with plasma membrane (cyan), intracellular content (orange), and segmented electron dense regions of the contact zone (white). (D) FIB-SEM MIP through the same region in (B), after masking the nuclei. (E) Single FIB-SEM slice through the contact zone at the central vertical line in (D). (F) same as (E), with more (blue) and less (red) electron dense membranes traced; (G) same as (E) overlaid with the JAM-C signal. Scale bar: 500 nm. (H) Histograms of the curvedness (supplemental note 9) for the high (blue) and low (red) electron density membrane regions. (I) Partial segmentation of the cells’ membranes in the contact zone, color coded according to curvedness, with brighter colors indicating larger values. Note the high correlation between JAM-C (B), electron density (D), and membrane curvedness (I) (Movie 5).

Figure 7.

Figure 7.. Cryo-SIM/FIB-SEM reveals nuclear rearrangements associated with cerebellar granule neuron progenitor (GNP) differentiation.

(A) Live-cell lattice light sheet time-lapse images showing an EGFP-Atoh1 positive GNP (top row) condensing its nuclear size to that of a CGN while the size of an EGFP-Atoh1 negative CGN nucleus (bottom row) remains constant. Scale bar: 3 μm. (B) Quantification of GNP and CGN nuclear volume for both static (histograms at left, 85 CGNs and 71 GNPs) and time lapse imaging (box plots at right, 5 GNPs and 5 CGNs), showing that, on average, GNPs are 40% larger than CGNs and condense their nuclei to the size of CGNs in approximately 2 hours. (C) Top: FIB-SEM (left) and SIM (right) volume renderings of a group of GNPs. Bottom: One such GNP nucleus (orange boxes at top), with cutaway, showing color-coded chromatin territories (heterochromatin, euchromatin or nucleoli) as identified on the basis of the EM data alone. (D-F) HP1α (green) or H3.3 (magenta) Cryo-SIM, FIB-SEM and correlative XZ ortho slices of the plane bordered in cyan in (C). Arrowheads indicate different types of labeled chromatin domains, see legend. Scalebar, 1 μm. (G-J) Same as (C-F) but for a representative CGN nucleus (Movie 6). (K) Quantification of EM segmented and (L) cryo-SIM defined chromatin domains and their correlation for 7 GNP and 9 CGN nuclei.

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