Correlative Organelle Microscopy: fluorescence guided volume electron microscopy of intracellular processes

Intracellular processes depend on a strict spatial and temporal organization of proteins and organelles. Directly linking molecular to nanoscale ultrastructural information is therefore crucial to understand cellular physiology. Volume or 3-dimensional (3D) correlative light and electron microscopy (volume-CLEM) holds unique potential to explore cellular physiology at high-resolution ultrastructural detail across cell volumes. Application of volume-CLEM is however hampered by limitations in throughput and 3D correlation efficiency. Addressing these limitations, we here describe a novel pipeline for volume-CLEM that provides high-precision (<100nm) registration between 3D fluorescence microscopy (FM) and 3D electron microscopy (EM) data sets with significantly increased throughput. Using multi-modal fiducial nanoparticles that remain fluorescent in epoxy resins and a 3D confocal fluorescence microscope integrated in a Focused Ion Beam Scanning Electron Microscope (FIB.SEM), our approach uses FM to target extremely small volumes of even single organelles for imaging in volume-EM, and obviates the need for post correlation of big 3D datasets. We extend our targeted volume-CLEM approach to include live-cell imaging, adding information on the motility of intracellular membranes selected for volume-CLEM. We demonstrate the power of our approach by targeted imaging of rare and transient contact sites between endoplasmic reticulum (ER) and lysosomes within hours rather than days. Our data suggest that extensive ER-lysosome and mitochondria-lysosome interactions restrict lysosome motility, highlighting the unique capabilities of our integrated CLEM pipeline for linking molecular dynamic data to high-resolution ultrastructural detail in 3D.

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In situ Microfluidic Cryofixation for Cryo Focused Ion Beam Milling and Cryo Electron Tomography

Scientific Reports ◽

10.1038/s41598-019-55413-2 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 4

Author(s):

Marie Fuest ◽

Miroslava Schaffer ◽

Giovanni Marco Nocera ◽

Rodrigo I. Galilea-Kleinsteuber ◽

Jan-Erik Messling ◽

...

Keyword(s):

Electron Microscopy ◽

High Resolution ◽

Light Microscope ◽

Focused Ion Beam ◽

Electron Tomography ◽

Ion Beam ◽

Light And Electron Microscopy ◽

Live Imaging ◽

Cryo Electron Tomography

AbstractWe present a microfluidic platform for studying structure-function relationships at the cellular level by connecting video rate live cell imaging with in situ microfluidic cryofixation and cryo-electron tomography of near natively preserved, unstained specimens. Correlative light and electron microscopy (CLEM) has been limited by the time required to transfer live cells from the light microscope to dedicated cryofixation instruments, such as a plunge freezer or high-pressure freezer. We recently demonstrated a microfluidic based approach that enables sample cryofixation directly in the light microscope with millisecond time resolution, a speed improvement of up to three orders of magnitude. Here we show that this cryofixation method can be combined with cryo-electron tomography (cryo-ET) by using Focused Ion Beam milling at cryogenic temperatures (cryo-FIB) to prepare frozen hydrated electron transparent sections. To make cryo-FIB sectioning of rapidly frozen microfluidic channels achievable, we developed a sacrificial layer technique to fabricate microfluidic devices with a PDMS bottom wall <5 µm thick. We demonstrate the complete workflow by rapidly cryo-freezing Caenorhabditis elegans roundworms L1 larvae during live imaging in the light microscope, followed by cryo-FIB milling and lift out to produce thin, electron transparent sections for cryo-ET imaging. Cryo-ET analysis of initial results show that the structural preservation of the cryofixed C. elegans was suitable for high resolution cryo-ET work. The combination of cryofixation during live imaging enabled by microfluidic cryofixation with the molecular resolution capabilities of cryo-ET offers an exciting avenue to further advance space-time correlative light and electron microscopy (st-CLEM) for investigation of biological processes at high resolution in four dimensions.

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Cryo-correlative light and electron microscopy workflow for cryo-focused ion beam milled adherent cells

Methods in Cell Biology - Correlative Light and Electron Microscopy IV ◽

10.1016/bs.mcb.2020.12.009 ◽

2021 ◽

pp. 273-302

Author(s):

Steffen Klein ◽

Moritz Wachsmuth-Melm ◽

Sophie L. Winter ◽

Androniki Kolovou ◽

Petr Chlanda

Keyword(s):

Electron Microscopy ◽

Focused Ion Beam ◽

Ion Beam ◽

Light And Electron Microscopy ◽

Adherent Cells

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Ultrastructural Characterization of Flashing Mitochondria

Contact ◽

10.1177/2515256418801423 ◽

2018 ◽

Vol 1 ◽

pp. 251525641880142

Author(s):

Manon Rosselin ◽

Paula Nunes-Hasler ◽

Nicolas Demaurex

Keyword(s):

Electron Microscopy ◽

Focused Ion Beam ◽

Ion Beam ◽

Light And Electron Microscopy ◽

Three Dimensional ◽

Volume Ratio ◽

Tubular Structures ◽

Membrane Contact Sites ◽

Contact Sites ◽

Membrane Contact

Mitochondria undergo spontaneous transient elevations in matrix pH associated with drops in mitochondrial membrane potential. These mitopHlashes require a functional respiratory chain and the profusion protein optic atrophy 1, but their mechanistic basis is unclear. To gain insight on the origin of these dynamic events, we resolved the ultrastructure of flashing mitochondria by correlative light and electron microscopy. HeLa cells expressing the matrix-targeted pH probe mitoSypHer were screened for mitopHlashes and fixed immediately after the occurrence of a flashing event. The cells were then processed for imaging by serial block face scanning electron microscopy using a focused ion beam to generate ∼1,200 slices of 10 nm thickness from a 28 µm × 15 µm cellular volume. Correlation of live/fixed fluorescence and electron microscopy images allowed the unambiguous identification of flashing and nonflashing mitochondria. Three-dimensional reconstruction and surface mapping revealed that each tomogram contained two flashing mitochondria of unequal sizes, one being much larger than the average mitochondrial volume. Flashing mitochondria were 10-fold larger than silent mitochondria but with a surface to volume ratio and a cristae volume similar to nonflashing mitochondria. Flashing mitochondria were connected by tubular structures, formed more membrane contact sites, and a constriction was observed at a junction between a flashing mitochondrion and a nonflashing mitochondrion. These data indicate that flashing mitochondria are structurally preserved and bioenergetically competent but form numerous membrane contact sites and are connected by tubular structures, consistent with our earlier suggestion that mitopHlashes might be triggered by the opening of fusion pores between contiguous mitochondria.

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Assessment of different focused ion beam milling conditions for the preparation of TEM cross-sections for high-resolution electron microscopy

Electron Microscopy and Analysis 2001 ◽

10.1201/9781482289510-112 ◽

2001 ◽

pp. 455-458

Keyword(s):

Electron Microscopy ◽

High Resolution ◽

Cross Sections ◽

Focused Ion Beam ◽

Ion Beam ◽

High Resolution Electron Microscopy ◽

Resolution Electron ◽

Focused Ion Beam Milling

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A modular platform for automated cryo-FIB workflows

eLife ◽

10.7554/elife.70506 ◽

2021 ◽

Vol 10 ◽

Author(s):

Sven Klumpe ◽

Herman K H Fung ◽

Sara K Goetz ◽

Ievgeniia Zagoriy ◽

Bernhard Hampoelz ◽

...

Keyword(s):

Focused Ion Beam ◽

Structural Integrity ◽

Electron Tomography ◽

Ion Beam ◽

Light And Electron Microscopy ◽

Software Tool ◽

3 Dimensional ◽

Improved Stability ◽

Modular Platform ◽

Beam Currents

Lamella micromachining by focused ion beam milling at cryogenic temperature (cryo-FIB) has matured into a preparation method widely used for cellular cryo-electron tomography. Due to the limited ablation rates of low Ga+ ion beam currents required to maintain the structural integrity of vitreous specimens, common preparation protocols are time-consuming and labor intensive. The improved stability of new generation cryo-FIB instruments now enables automated operations. Here, we present an open-source software tool, SerialFIB, for creating automated and customizable cryo-FIB preparation protocols. The software encompasses a graphical user interface for easy execution of routine lamellae preparations, a scripting module compatible with available Python packages, and interfaces with 3-dimensional correlative light and electron microscopy (CLEM) tools. SerialFIB enables the streamlining of advanced cryo-FIB protocols such as multi-modal imaging, CLEM-guided lamella preparation and in situ lamella lift-out procedures. Our software therefore provides a foundation for further development of advanced cryogenic imaging and sample preparation protocols.

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