@article {69457, title = {Deep-Tissue Spatial Omics: Imaging Whole-Embryo Transcriptomics and Subcellular Structures at High Spatial Resolution}, journal = {bioRxiv}, year = {2024}, month = {2024 May 17}, abstract = {

The inherent limitations of fluorescence microscopy, notably the restricted number of color channels, have long constrained comprehensive spatial analysis in biological specimens. Here, we introduce cycleHCR technology that leverages multicycle DNA barcoding and Hybridization Chain Reaction (HCR) to surpass the conventional color barrier. cycleHCR facilitates high-specificity, single-shot imaging per target for RNA and protein species within thick specimens, mitigating the molecular crowding issues encountered with other imaging-based spatial omics techniques. We demonstrate whole-mount transcriptomics imaging of 254 genes within an E6.5\~7.0 mouse embryo, achieving precise three-dimensional gene expression and cell fate mapping across a specimen depth of \~ 310 {\textmu}m. Utilizing expansion microscopy alongside protein cycleHCR, we unveil the complex network of 10 subcellular structures in primary mouse embryonic fibroblasts. Furthermore, in mouse hippocampal slice, we image 8 protein targets and profile the transcriptome of 120 genes, uncovering complex gene expression gradients and cell-type specific nuclear structural variances. cycleHCR provides a unifying framework for multiplex RNA and protein imaging, offering a quantitative solution for elucidating spatial regulations in deep tissue contexts for research and potentially diagnostic applications.

}, doi = {10.1101/2024.05.17.594641}, url = {https://www.biorxiv.org/content/early/2024/05/17/2024.05.17.594641}, author = {Gandin, Valentina and Kim, Jun and Yang, Liang-Zhong and Lian, Yumin and Kawase, Takashi and Hu, Amy and Rokicki, Konrad and Fleishman, Greg and Tillberg, Paul and Aguilera Castrejon, Alejandro and Stringer, Carsen and Preibisch, Stephan and Liu, Zhe J.} } @article {67300, title = {Protein-Retention Expansion Microscopy (ExM): Scalable and Convenient Super-Resolution Microscopy.}, journal = {Methods in Molecular Biology}, volume = {2304}, year = {2021}, month = {2021 May 28}, pages = {147-156}, abstract = {

Expansion microscopy (ExM) is a method to expand biological specimens ~fourfold in each dimension by embedding in a hyper-swellable gel material. The expansion is uniform across observable length scales, enabling imaging of structures previously too small to resolve. ExM is compatible with any microscope and does not require expensive materials or specialized software, offering effectively sub-diffraction-limited imaging capabilities to labs that are not equipped to use traditional super-resolution imaging methods. Expanded specimens are ~99\% water, resulting in strongly reduced optical scattering and enabling imaging of sub-diffraction-limited structures throughout specimens up to several hundred microns in (pre-expansion) thickness.

}, issn = {1940-6029}, doi = {10.1007/978-1-0716-1402-0_7}, author = {Tillberg, Paul} } @article {65079, title = {BigStitcher: reconstructing high-resolution image datasets of cleared and expanded samples.}, journal = {Nature Methods}, volume = {16}, year = {2019}, month = {2019 Sep}, pages = {870-74}, abstract = {

Light-sheet imaging of cleared and expanded samples creates terabyte-sized datasets that consist of many unaligned three-dimensional image tiles, which must be reconstructed before analysis. We developed the BigStitcher software to address this challenge. BigStitcher enables interactive visualization, fast and precise alignment, spatially resolved quality estimation, real-time fusion and deconvolution of dual-illumination, multitile, multiview datasets. The software also compensates for optical effects, thereby improving accuracy and enabling subsequent biological analysis.

}, issn = {1548-7091}, doi = {10.1038/s41592-019-0501-0}, url = {http://www.nature.com/articles/s41592-019-0501-0http://www.nature.com/articles/s41592-019-0501-0.pdfhttp://www.nature.com/articles/s41592-019-0501-0.pdfhttp://www.nature.com/articles/s41592-019-0501-0}, author = {H{\"o}rl, David and Rojas Rusak, Fabio and Preusser, Friedrich and Tillberg, Paul and Randel, Nadine and Chhetri, Raghav K. and Cardona, Albert and Keller, Philipp J. and Harz, Hartmann and Leonhardt, Heinrich and Treier, Mathias and Preibisch, Stephan} } @article {65227, title = {Nitric oxide acts as a cotransmitter in a subset of dopaminergic neurons to diversify memory dynamics.}, journal = {eLife}, volume = {8}, year = {2019}, month = {2019 Nov 14}, abstract = {

Animals employ diverse learning rules and synaptic plasticity dynamics to record temporal and statistical information about the world. However, the molecular mechanisms underlying this diversity are poorly understood. The anatomically defined compartments of the insect mushroom body function as parallel units of associative learning, with different learning rates, memory decay dynamics and flexibility (Aso \& Rubin 2016). Here we show that nitric oxide (NO) acts as a neurotransmitter in a subset of dopaminergic neurons in . NO{\textquoteright}s effects develop more slowly than those of dopamine and depend on soluble guanylate cyclase in postsynaptic Kenyon cells. NO acts antagonistically to dopamine; it shortens memory retention and facilitates the rapid updating of memories. The interplay of NO and dopamine enables memories stored in local domains along Kenyon cell axons to be specialized for predicting the value of odors based only on recent events. Our results provide key mechanistic insights into how diverse memory dynamics are established in parallel memory systems.

}, issn = {2050-084X}, doi = {10.7554/eLife.49257}, author = {Aso, Yoshinori and Ray, Robert P and Long, Xi and Bushey, Daniel and Cichewicz, Karol and Ngo, Teri-Tb and Sharp, Brandi and Christoforou, Christina and Hu, Amy and Lemire, Andrew L and Tillberg, Paul and Hirsh, Jay and Litwin-Kumar, Ashok and Rubin, Gerald M} } @article {47602, title = {Protein-retention expansion microscopy of cells and tissues labeled using standard fluorescent proteins and antibodies.}, journal = {Nature Biotechnology}, volume = {34}, year = {2016}, month = {2016 Jul 4}, pages = {987-92}, abstract = {

Expansion microscopy (ExM) enables imaging of preserved specimens with nanoscale precision on diffraction-limited instead of specialized super-resolution microscopes. ExM works by physically separating fluorescent probes after anchoring them to a swellable gel. The first ExM method did not result in the retention of native proteins in the gel and relied on custom-made reagents that are not widely available. Here we describe protein retention ExM (proExM), a variant of ExM in which proteins are anchored to the swellable gel, allowing the use of conventional fluorescently labeled antibodies and streptavidin, and fluorescent proteins. We validated and demonstrated the utility of proExM for multicolor super-resolution (\~{}70 nm) imaging of cells and mammalian tissues on conventional microscopes.

}, issn = {1546-1696}, doi = {10.1038/nbt.3625}, author = {Tillberg, Paul and Chen, Fei and Piatkevich, Kiryl D and Zhao, Yongxin and Yu, Chih-Chieh Jay and English, Brian P and Gao, Linyi and Martorell, Anthony and Suk, Ho-Jun and Yoshida, Fumiaki and DeGennaro, Ellen M and Roossien, Douglas H and Gong, Guanyu and Seneviratne, Uthpala and Tannenbaum, Steven R and Desimone, Robert and Cai, Dawen and Boyden, Edward S} }