Protein-retention expansion microscopy for visualizing subcellular organelles in fixed brain tissue

Campbell, Logan A.; Pannoni, Katy E.; Savory, Niesha A.; Lal, Dinesh; Farris, Shannon

doi:10.1016/j.jneumeth.2021.109285

Cited by 15 publications

(31 citation statements)

References 43 publications

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“…Expansion Microscopy (ExM) (Chen et al, 2015; Gallagher and Zhao, 2021), which physically expands tissue samples ∼4 to 20-fold by embedding them in swellable hydrogels, has resolved subcellular protein distributions of synaptic targets in mouse brain tissue (Chen et al, 2015; Tillberg et al, 2016; Ku et al, 2016; Chozinski et al, 2016; Chang et al, 2017; Murakami et al, 2018; Truckenbrodt et al, 2019; Park et al, 2019; Shen et al, 2020; Campbell et al, 2021; Gao et al, 2021; Park et al, 2021; Damstra et al, 2022), as well as map RNA transcripts within axons and dendrites in the brain (Alon et al, 2021). However, while the expansion-related decrowding avoids fluorescence quenching, ExM has traditionally capitalized almost exclusively on antibody labelings, and no ExM method has demonstrated correlative molecular and contextual imaging with synaptic resolution so far.…”

Section: Introductionmentioning

confidence: 99%

All-optical visualization of specific molecules in the ultrastructural context of brain tissue

M’Saad

Kasula

Kondratiuk

et al. 2022

Preprint

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SummaryUnderstanding the molecular anatomy and neural connectivity of the brain requires imaging technologies that can map the 3D nanoscale distribution of specific proteins in the context of brain ultrastructure. Light and electron microscopy (EM) enable visualization of either specific labels or anatomical ultrastructure, but combining molecular specificity with anatomical context is challenging. Here, we present pan-Expansion Microscopy of tissue (pan-ExM-t), an all-optical mouse brain imaging method that combines ∼24-fold linear expansion of biological samples with fluorescent pan-staining of protein densities (providing EM-like ultrastructural context), and immunolabeling of protein targets (for molecular imaging). We demonstrate the versatility of this approach by imaging the established synaptic markers Homer1, Bassoon, PSD-95, Synaptophysin, the astrocytic protein GFAP, myelin basic protein (MBP), and anti-GFP antibodies in dissociated neuron cultures and mouse brain tissue sections. pan-ExM-t reveals these markers in the context of ultrastructural features such as pre and postsynaptic densities, 3D nanoarchitecture of neuropil, and the fine structures of cellular organelles. pan-ExM-t is adoptable in any neurobiological laboratory with access to a confocal microscope and has therefore broad applicability in the research community.Highlightspan-ExM-t visualizes proteins in the context of synaptic ultrastructureLipid labeling in pan-ExM-t reveals organellar and cellular membranesAll-optical, easily accessible alternative to correlative light/electron microscopyHigh potential for high throughput connectomics studies

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Section: Introductionmentioning

confidence: 99%

All-optical visualization of specific molecules in the ultrastructural context of brain tissue

M’Saad

Kasula

Kondratiuk

et al. 2022

Preprint

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Section: Methodsmentioning

confidence: 99%

“…2H) labeled mitochondria in CA2 SLM alongside a novel pan-mitochondrial marker MT-5 (Kalyuzhny et al, 2021). We used protein retention expansion microscopy (ProExM) to visualize densely packed mitochondria (Campbell et al, 2021). In order to compare MCU and COX4 labeling across similar mitochondrial populations, we used ProExM images with similar MT-5 labeling and CA2 SLM dendrite labeling.…”

Section: Cox4 Labels a Broader But Overlapping Population Of Mitochondria Than Mcumentioning

confidence: 99%

“…Gels were then placed in digestion solution containing proteinase K (8U/mL, New England BioLabs, P8107S) and digested for 4 hours at room temperature. For the AAV experiments, gels were placed in an alkaline buffer (100 mM Tris base, 5% (w/v) Triton X-100, 1% SDS) and digested with heat using an autoclave on the liquid cycle for 1 h at 121 C, which is a milder alternative to enzymatic digestion with Proteinase K, prior to antibody staining (Campbell et al 2021, Asano et al 2018. Proteinase K digested gels were immunostained prior to digestion instead.…”

Section: Protein-retention Expansion Microscopy (Proexm)mentioning

confidence: 99%

“…40μm horizontal mouse brain sections were expanded with 4X protein expansion microscopy (ProExM) as previously described in Campbell et al 2021. All solutions were prepared as described by Asano et al 2018. Sections to be expanded were incubated overnight in Acryloyl-X stock/PBS (1:100, ThermoFisher, A20770) at room temperature in the dark.…”

Section: Analysis Of Mitotag Mitochondria In Ca2mentioning

confidence: 99%

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Layer-specific mitochondrial diversity across hippocampal CA2 dendrites

Pannoni

Gil

Campbell

et al. 2021

Preprint

Self Cite

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CA2 is an understudied subregion of the hippocampus that is critical for social memory. Previous studies identified multiple components of the mitochondrial calcium uniporter (MCU) complex as selectively enriched in CA2, however the functional significance of this enrichment remains unclear. The MCU complex regulates calcium entry into mitochondria, which in turn regulates mitochondrial transport and localization to active synapses. We found that MCU is strikingly enriched in CA2 distal apical dendrites, precisely where CA2 neurons receive entorhinal cortical input carrying social information. Further, MCU-enriched mitochondria in CA2 distal dendrites are larger compared to mitochondria in CA2 proximal apical dendrites and neighboring CA1 apical dendrites. Genetic knockdown of MCU in CA2 resulted in smaller mitochondria in CA2 distal dendrites, indicating that MCU expression plays a role in regulating mitochondrial mass in CA2. MCU overexpression in neighboring CA1 led to larger mitochondria preferentially in proximal dendrites compared to distal dendrites and GFP controls. Our findings demonstrate that mitochondria are molecularly and structurally diverse across hippocampal cell types and circuits, and that MCU expression cell-autonomously regulates mitochondrial mass, but layer-specific dendritic localization depends on cell type. Our data support the idea that CA2 mitochondria are functionally distinct from CA1 mitochondria, which may confer unique synaptic and circuit properties underlying CA2 function in social memory.

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Expansion Microscopy‐based imaging for visualization of mitochondria in Drosophila ovarian germline stem cells

Lin

Hsu

et al. 2022

FEBS Open Bio

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Recent studies have shown that mitochondrial morphology can modulate organelle function and greatly affect stem cell behavior, thus affecting tissue homeostasis. As such, we previously showed that the accumulation of fragmented mitochondria in aged Drosophila ovarian germline stem cells (GSCs) contributes to age‐dependent GSC loss. However, standard immunofluorescence methods to examine mitochondrial morphology yield images with insufficient resolution for rigorous analysis, while 3‐dimensional electron microscopy examination of mitochondrial morphology is labor intensive and allows only limited sampling of mitochondria. To overcome these issues, we utilized the expansion microscopy technique to expand GSC samples by 4‐fold in combination with mitochondrial immunofluorescence labeling. Here, we present a simple, inexpensive method for nanoscale optical imaging of mitochondria in the germline. This protocol may be beneficial for studies that require visualization of mitochondria or other fine subcellular structures in the Drosophila ovary.

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Protein-retention expansion microscopy for visualizing subcellular organelles in fixed brain tissue

Cited by 15 publications

References 43 publications

All-optical visualization of specific molecules in the ultrastructural context of brain tissue

All-optical visualization of specific molecules in the ultrastructural context of brain tissue

Layer-specific mitochondrial diversity across hippocampal CA2 dendrites

Expansion Microscopy‐based imaging for visualization of mitochondria in Drosophila ovarian germline stem cells

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