Localization of albumin with correlative super resolution light- and electron microscopy in the kidney

TL;DR

This study uses advanced microscopy to precisely locate albumin in the kidney's filtration barrier, helping understand how proteins leak in disease.

Contribution

A workflow for nanometer-precise albumin localization using correlative super-resolution light and electron microscopy in kidney tissue.

Findings

Albumin localization reveals a linear reduction gradient across the filtration barrier.

Post-embedding fluorescence is preserved after high-pressure freezing and freeze-substitution.

The slit diaphragm is likely responsible for kidney leakiness.

Abstract

Workflow for the nanometer precise localization of albumin within the murine glomerular filtration barrier.

Workflow for the nanometer precise localization of albumin within the murine glomerular filtration barrier.

•Super resolution correlative light- and electron microscopy allows for the precise tracing of albumin in kidney tissue.•The slit diaphragm (SD) is most likely responsible for the kidney leakiness.•Post-embedding fluorescence is preserved after high-pressure freezing and freeze-substitution of kidney.

Super resolution correlative light- and electron microscopy allows for the precise tracing of albumin in kidney tissue.

The slit diaphragm (SD) is most likely responsible for the kidney leakiness.

Post-embedding fluorescence is preserved after high-pressure freezing and freeze-substitution of kidney.

The functioning of vertebrate life relies on renal filtration of surplus fluid and elimination of low-molecular-weight waste products, while keeping serum proteins in the blood. In disease, however, there is leak of serum proteins and tracing them to identify the leaking position within tissue with a nanometer resolution poses a significant challenge. Correlative microscopy integrates the specificity of fluorescent protein labeling into high-resolution electron micrographs. Using chemical tagging of albumin with synthetic fluorophores we achieve protein-specific labeling that preserve their post-embedding fluorescence after high-pressure freezing and freeze-substitution of murine kidney tissue. Using advanced registration techniques for super-resolution correlative light and electron microscopy, we can localize the labeled albumin with a high precision in the x-y plane of electron micrographs and cartograph its distribution. Thereby we can quantify the albumin concentration and measure a linear reduction gradient across the kidney filtration barrier. Our study shows the feasibility of combining different microscopy contrasts for tracing fluorescently labeled protein markers with super resolution in various tissue samples and opens new perspectives for correlative imaging in volume electron microscopy.

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