Fluorescence-based 3D targeting of FIB-SEM acquisition of small volumes in large samples

Abstract

Cells are three dimensional objects. Therefore, 3D electron microscopy is often crucial for correct interpretation of ultrastructural data. Today samples are frequently imaged in 3D at ultrastructural resolution using volume Scanning Electron Microscopy (SEM) methods such as Focused Ion Beam (FIB) SEM and Serial Block face SEM. While these imaging modalities allow for automated data acquisition, precise targeting of (small) volumes of interest within a large sample remains challenging. Here, we provide an easy and reliable approach to target FIB-SEM acquisition of fluorescently labelled cells or subcellular structures with micrometer precision. The strategy relies on fluorescence preservation during sample preparation and targeting based on confocal acquisition of the fluorescence signal in the resin block. Targeted trimming of the block exposes the cell of interest and laser branding of the surface after trimming creates landmarks to precisely position the FIB-SEM acquisition. Using this method, we acquired volumes of specific single cells within large tissues such as a 3D culture of mouse primary mammary gland organoids, tracheal terminal cells in Drosophila melanogaster larvae and ovarian follicular cells in adult Drosophila, discovering ultrastructural details that could not be appreciated before.SummaryRonchi et al. present a workflow to facilitate the precise targeting of three-dimensional (3D) Electron Microscopy acquisitions, guided by fluorescence. This method allows ultrastructural visualization of single cells within a millimeter-range large specimen, based on molecular identity characterized by fluorescence.

Correlative Organelle Microscopy: Fluorescence Guided Volume Electron Microscopy of Intracellular Processes

“…This is incompatible with most life science studies, which often require the analysis of small volumes of several independent samples. If the high-resolution acquisition could be limited to a pre-defined region of interest (ROI), the throughput of volume EM would be considerably enhanced ( Burel et al, 2018 ; Ronchi et al, 2021 ). With the introduction of correlative light electron microscopy (CLEM), an ROI can be highlighted/selected with FM (a common driver for CLEM).…”

“…Moreover, at the cellular level, state-of-the-art volume-CLEM studies visualize protein-ultrastructure relationships in three dimensions across whole cells by identification of morphologically complex structures within the crowded intracellular environment ( Hoffman et al, 2020 ). However, an accurate and reliable correlation between separate 3D-FM and 3D-EM platforms datasets is far from trivial because of the resolution mismatch and the sample transfer in between the two modalities ( Ronchi et al, 2021 ). Current efforts in volume-CLEM development focus on improving its accuracy, throughput, and accessibility.…”

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

“…This is incompatible with most life sciences studies, which often require analysis of small volumes of several independent samples. If the high-resolution acquisition could be limited to a pre-defined region of interest (ROI), throughput of volume-EM would be considerably enhanced (Burel et al, 2018;Ronchi et al, 2021). With the introduction of correlative light electron microscopy (CLEM), a ROI can be highlighted/selected with FM (a common driver for CLEM).…”

“…Also in cellular level, state-of-art volume-CLEM studies visualize protein-ultrastructure relationships in three dimensions across whole cells by identification of morphologically complex structures within the crowded intracellular environment (Hoffman et al, 2020). However, an accurate and reliable correlation between separate 3D-FM and 3D-EM platforms data sets is far from trivial because of the resolution mismatch and the sample transfer in between the two modalities (Ronchi et al, 2021). Current efforts in volume-CLEM development focus around improving its accuracy, throughput and accessibility.…”

Light and electron microscopy continuum-resolution imaging of 3D cell cultures

“…For room temperature EM, the cryo-fixed samples are then subjected to freeze-substitution and stained with heavy metals to provide contrast in EM imaging (Kellenberger, 1987). A variation of this procedure reduces the amount of metals in the freeze-substitution cocktail to only 0.1% uranyl acetate, allowing preservation of endogenous fluorescent signal for targeting of specific regions of interest (ROI) in large sample volumes by fluorescence imaging after embedding (Kukulski et al, 2011; Nixon et al, 2009), while still providing enough contrast for FIB-SEM imaging (Ronchi et al, 2021). Such preparation methods are easily applicable for specimens that are amenable to manual handling for cryo-fixation, including small model organisms or dissected tissues.…”