GelMap: intrinsic calibration and deformation mapping for expansion microscopy.

Expansion microscopy (ExM) is a powerful technique to overcome the diffraction limit of light microscopy by physically expanding biological specimen in three dimensions. Nonetheless, using ExM for quantitative or diagnostic applications requires robust quality control methods to precisely determine expansion factors and to map deformations due to anisotropic expansion. Here we present GelMap, a flexible workflow to introduce a fluorescent grid into pre-expanded hydrogels that scales with expansion and reports deformations. We demonstrate that GelMap can be used to precisely determine the local expansion factor and to correct for deformations without the use of cellular reference structures or pre-expansion ground-truth images. Moreover, we show that GelMap aids sample navigation for correlative uses of expansion microscopy. Finally, we show that GelMap is compatible with expansion of tissue and can be readily implemented as a quality control step into existing ExM workflows.

Ten-fold Robust Expansion Microscopy.

Expansion microscopy (ExM) is a powerful technique to overcome the diffraction limit of light microscopy that can be applied in both tissues and cells. In ExM, samples are embedded in a swellable polymer gel to physically expand the sample and isotropically increase resolution in x, y, and z. By systematic exploration of the ExM recipe space, we developed a novel ExM method termed Ten-fold Robust Expansion Microscopy (TREx) that, as the original ExM method, requires no specialized equipment or procedures. TREx enables ten-fold expansion of both thick mouse brain tissue sections and cultured human cells, can be handled easily, and enables high-resolution subcellular imaging with a single expansion step. Furthermore, TREx can provide ultrastructural context to subcellular protein localization by combining antibody-stained samples with off-the-shelf small molecule stains for both total protein and membranes.

Taxanes trigger cancer cell killing in vivo by inducing non-canonical T cell cytotoxicity.

Although treatment with taxanes does not always lead to clinical benefit, all patients are at risk of their detrimental side effects such as peripheral neuropathy. Understanding the in vivo mode of action of taxanes can help design improved treatment regimens. Here, we demonstrate that in vivo, taxanes directly trigger T cells to selectively kill cancer cells in a non-canonical, T cell receptor-independent manner. Mechanistically, taxanes induce T cells to release cytotoxic extracellular vesicles, which lead to apoptosis specifically in tumor cells while leaving healthy epithelial cells intact. We exploit these findings to develop an effective therapeutic approach, based on transfer of T cells pre-treated with taxanes ex vivo, thereby avoiding toxicity of systemic treatment. Our study reveals a different in vivo mode of action of one of the most commonly used chemotherapies, and opens avenues to harness T cell-dependent anti-tumor effects of taxanes while avoiding systemic toxicity.

Visualizing cellular and tissue ultrastructure using Ten-fold Robust Expansion Microscopy (TREx).

Expansion microscopy (ExM) is a powerful technique to overcome the diffraction limit of light microscopy that can be applied in both tissues and cells. In ExM, samples are embedded in a swellable polymer gel to physically expand the sample and isotropically increase resolution in x, y, and z. The maximum resolution increase is limited by the expansion factor of the gel, which is four-fold for the original ExM protocol. Variations on the original ExM method have been reported that allow for greater expansion factors but at the cost of ease of adoption or versatility. Here, we systematically explore the ExM recipe space and present a novel method termed Ten-fold Robust Expansion Microscopy (TREx) that, like the original ExM method, requires no specialized equipment or procedures. We demonstrate that TREx gels expand 10-fold, can be handled easily, and can be applied to both thick mouse brain tissue sections and cultured human cells enabling high-resolution subcellular imaging with a single expansion step. Furthermore, we show that TREx can provide ultrastructural context to subcellular protein localization by combining antibody-stained samples with off-the-shelf small-molecule stains for both total protein and membranes.

Dynein self-organizes while translocating the centrosome in T-cells.

T-cells massively restructure their internal architecture upon reaching an antigen-presenting cell (APC) to form the immunological synapse (IS), a cell-cell interface necessary for efficient elimination of the APC. This reorganization occurs through tight coordination of cytoskeletal processes: actin forms a peripheral ring, and dynein motors translocate the centrosome toward the IS. A recent study proposed that centrosome translocation involves a microtubule (MT) bundle that connects the centrosome perpendicularly to dynein at the synapse center: the "stalk." The synapse center, however, is actin-depleted, while actin was assumed to anchor dynein. We propose that dynein is attached to mobile membrane anchors, and investigate this model with computer simulations. We find that dynein organizes into a cluster in the synapse when translocating the centrosome, aligning MTs into a stalk. By implementing both a MT-capture-shrinkage and a MT-sliding mechanism, we explicitly demonstrate that this organization occurs in both systems. However, results obtained with MT-sliding dynein are more robust and display a stalk morphology consistent with our experimental data obtained with expansion microscopy. Thus, our simulations suggest that actin organization in T-cells during activation defines a specific geometry in which MT-sliding dynein can self-organize into a cluster and cause stalk formation.