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Tillberg Lab / Publications
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14 Publications

Showing 1-10 of 14 results
11/13/22 | Brain-wide measurement of protein turnover with high spatial and temporal resolution
Boaz Mohar , Jonathan B. Grimm , Ronak Patel , Timothy A. Brown , Paul Tillberg , Luke D. Lavis , Nelson Spruston , Karel Svoboda
bioRxiv. 2022 Nov 13:. doi: 10.1101/2022.11.12.516226

Cells regulate function by synthesizing and degrading proteins. This turnover ranges from minutes to weeks, as it varies across proteins, cellular compartments, cell types, and tissues. Current methods for tracking protein turnover lack the spatial and temporal resolution needed to investigate these processes, especially in the intact brain, which presents unique challenges. We describe a pulse-chase method (DELTA) for measuring protein turnover with high spatial and temporal resolution throughout the body, including the brain. DELTA relies on rapid covalent capture by HaloTag of fluorophores that were optimized for bioavailability in vivo. The nuclear protein MeCP2 showed brain region- and cell type-specific turnover. The synaptic protein PSD95 was destabilized in specific brain regions by behavioral enrichment. A novel variant of expansion microscopy further facilitated turnover measurements at individual synapses. DELTA enables studies of adaptive and maladaptive plasticity in brain-wide neural circuits.

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10/26/22 | Rapid reconstruction of neural circuits using tissue expansion and lattice light sheet microscopy
Joshua L. Lillvis , Hideo Otsuna , Xiaoyu Ding , Igor Pisarev , Takashi Kawase , Jennifer Colonell , Konrad Rokicki , Cristian Goina , Ruixuan Gao , Amy Hu , Kaiyu Wang , John Bogovic , Daniel E. Milkie , Edward S. Boyden , Stephan Saalfeld , Paul W. Tillberg , Barry J. Dickson
eLife. 2022 Oct 26:. doi: 10.7554/eLife.81248

Electron microscopy (EM) allows for the reconstruction of dense neuronal connectomes but suffers from low throughput, limiting its application to small numbers of reference specimens. We developed a protocol and analysis pipeline using tissue expansion and lattice light-sheet microscopy (ExLLSM) to rapidly reconstruct selected circuits across many samples with single synapse resolution and molecular contrast. We validate this approach in Drosophila, demonstrating that it yields synaptic counts similar to those obtained by EM, can be used to compare counts across sex and experience, and to correlate structural connectivity with functional connectivity. This approach fills a critical methodological gap in studying variability in the structure and function of neural circuits across individuals within and between species.

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10/22/22 | Multimodal mapping of cell types and projections in the central nucleus of the amygdala
Yuhan Wang , Sabine Krabbe , Mark Eddison , Fredrick E. Henry , Greg Fleishman , Andrew L. Lemire , Lihua Wang , Wyatt Korff , Paul W. Tillberg , Andreas Lüthi , Scott M. Sternson
bioRxiv. 2022 Oct 22:. doi: 10.1101/2022.10.19.512845

The central nucleus of the amygdala (CEA) is a brain region that integrates external and internal sensory information and executes innate and adaptive behaviors through distinct output pathways. Despite its complex functions, the diversity of molecularly defined neuronal types in the CEA and their contributions to major axonal projection targets have not been examined systematically. Here, we performed single-cell RNA-sequencing (scRNA-Seq) to classify molecularly defined cell types in the CEA and identified marker-genes to map the location of these neuronal types using expansion assisted iterative fluorescence in situ hybridization (EASI-FISH). We developed new methods to integrate EASI-FISH with 5-plex retrograde axonal labeling to determine the spatial, morphological, and connectivity properties of ∼30,000 molecularly defined CEA neurons. Our study revealed spatio-molecular organization of the CEA, with medial and lateral CEA associated with distinct cell families. We also found a long-range axon projection network from the CEA, where target regions receive inputs from multiple molecularly defined cell types. Axon collateralization was found primarily among projections to hindbrain targets, which are distinct from forebrain projections. This resource reports marker-gene combinations for molecularly defined cell types and axon-projection types, which will be useful for selective interrogation of these neuronal populations to study their contributions to the diverse functions of the CEA.

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02/18/22 | Visualizing cellular and tissue ultrastructure using Ten-fold Robust Expansion Microscopy (TREx)
Hugo G.J. Damstra , Boaz Mohar , Mark Eddison , Anna Akhmanova , Lukas C. Kapitein , Paul W. Tillberg
eLife. 2022 Feb 18:. doi: https://doi.org/10.1101/2021.02.03.428837

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 polymer 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, for example using iterative expansion, 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 to carry out. We demonstrate that TREx gels expand ten-fold, can be handled easily, and can be applied to both thick tissue sections and cells enabling high-resolution subcellular imaging in a single expansion step. We show that applying TREx on antibody-stained samples can be combined with off-the-shelf small molecule stains for both total protein and membranes to provide ultrastructural context to subcellular protein localization.

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02/15/22 | Glutamate indicators with improved activation kinetics and localization for imaging synaptic transmission
Abhi Aggarwal , Rui Liu , Yang Chen , Amelia J Ralowicz , Samuel J Bergerson , Filip Tomaska , Timothy L Hanson , Jeremy P Hasseman , Daniel Reep , Getahun Tsegaye , Pantong Yao , Xiang Ji , Marinus Kloos , Deepika Walpita , Ronak Patel , Paul W Tilberg , Boaz Mohar , GENIE , Loren L Looger , Jonathan S Marvin , Michael B Hoppa , Arthur Konnerth , David Kleinfeld , Eric R Schreiter , Kaspar Podgorski
bioRxiv PrePrint. 2022 Feb 15:. doi: 10.1101/2022.02.13.480251

The fluorescent glutamate indicator iGluSnFR enables imaging of neurotransmission with genetic and molecular specificity. However, existing iGluSnFR variants exhibit saturating activation kinetics and are excluded from post-synaptic densities, limiting their ability to distinguish synaptic from extrasynaptic glutamate. Using a multi-assay screen in bacteria, soluble protein, and cultured neurons, we generated novel variants with improved kinetics and signal-to-noise ratios. We also developed surface display constructs that improve iGluSnFR’s nanoscopic localization to post-synapses. The resulting indicator, iGluSnFR3, exhibits rapid non-saturating activation kinetics and reports synaptic glutamate release with improved linearity and increased specificity versus extrasynaptic signals in cultured neurons. In mouse visual cortex, imaging of iGluSnFR3 at individual boutons reported single electrophysiologically-observed action potentials with high specificity versus non-synaptic transients. In vibrissal sensory cortex Layer 4, we used iGluSnFR3 to characterize distinct patterns of touch-evoked feedforward input from thalamocortical boutons and both feedforward and recurrent input onto L4 cortical neuron dendritic spines.

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Svoboda LabSaalfeld LabSternson LabTillberg Lab
12/01/21 | EASI-FISH for thick tissue defines lateral hypothalamus spatio-molecular organization.
Wang Y, Eddison M, Fleishman G, Weigert M, Xu S, Wang T, Rokicki K, Goina C, Henry FE, Lemire AL, Schmidt U, Yang H, Svoboda K, Myers EW, Saalfeld S, Korff W, Sternson SM, Tillberg PW
Cell. 2021 Dec 01;184(26):6361. doi: 10.1016/j.cell.2021.11.024

Determining the spatial organization and morphological characteristics of molecularly defined cell types is a major bottleneck for characterizing the architecture underpinning brain function. We developed Expansion-Assisted Iterative Fluorescence In Situ Hybridization (EASI-FISH) to survey gene expression in brain tissue, as well as a turnkey computational pipeline to rapidly process large EASI-FISH image datasets. EASI-FISH was optimized for thick brain sections (300 μm) to facilitate reconstruction of spatio-molecular domains that generalize across brains. Using the EASI-FISH pipeline, we investigated the spatial distribution of dozens of molecularly defined cell types in the lateral hypothalamic area (LHA), a brain region with poorly defined anatomical organization. Mapping cell types in the LHA revealed nine spatially and molecularly defined subregions. EASI-FISH also facilitates iterative reanalysis of scRNA-seq datasets to determine marker-genes that further dissociated spatial and morphological heterogeneity. The EASI-FISH pipeline democratizes mapping molecularly defined cell types, enabling discoveries about brain organization.

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05/28/21 | Protein-Retention Expansion Microscopy (ExM): Scalable and Convenient Super-Resolution Microscopy.
Tillberg P
Methods in Molecular Biology. 2021 May 28;2304:147-156. doi: 10.1007/978-1-0716-1402-0_7

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.

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03/08/21 | Expansion-Assisted Iterative-FISH defines lateral hypothalamus spatio-molecular organization
Yuhan Wang , Mark Eddison , Greg Fleishman , Martin Weigert , Shengjin Xu , Frederick E. Henry , Tim Wang , Andrew L. Lemire , Uwe Schmidt , Hui Yang , Konrad Rokicki , Cristian Goina , Karel Svoboda , Eugene W. Myers , Stephan Saalfeld , Wyatt Korff , Scott M. Sternson , Paul W. Tillberg
bioRxiv. 2021 Mar 8:. doi: 10.1101/2021.03.08.434304

Determining the spatial organization and morphological characteristics of molecularly defined cell types is a major bottleneck for characterizing the architecture underpinning brain function. We developed Expansion-Assisted Iterative Fluorescence In Situ Hybridization (EASI-FISH) to survey gene expression in brain tissue, as well as a turnkey computational pipeline to rapidly process large EASI-FISH image datasets. EASI-FISH was optimized for thick brain sections (300 µm) to facilitate reconstruction of spatio-molecular domains that generalize across brains. Using the EASI-FISH pipeline, we investigated the spatial distribution of dozens of molecularly defined cell types in the lateral hypothalamic area (LHA), a brain region with poorly defined anatomical organization. Mapping cell types in the LHA revealed nine novel spatially and molecularly defined subregions. EASI-FISH also facilitates iterative re-analysis of scRNA-Seq datasets to determine marker-genes that further dissociated spatial and morphological heterogeneity. The EASI-FISH pipeline democratizes mapping molecularly defined cell types, enabling discoveries about brain organization.

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10/06/19 | Expansion microscopy: scalable and convenient super-resolution microscopy.
Tillberg PW, Chen F
Annual Review of Cell and Developmental Biology. 2019 Oct 6;35:683-701. doi: 10.1146/annurev-cellbio-100818-125320

Expansion microscopy (ExM) is a physical form of magnification that increases the effective resolving power of any microscope. Here, we describe the fundamental principles of ExM, as well as how recently developed ExM variants build upon and apply those principles. We examine applications of ExM in cell and developmental biology for the study of nanoscale structures as well as ExM's potential for scalable mapping of nanoscale structures across large sample volumes. Finally, we explore how the unique anchoring and hydrogel embedding properties enable postexpansion molecular interrogation in a purified chemical environment. ExM promises to play an important role complementary to emerging live-cell imaging techniques, because of its relative ease of adoption and modification and its compatibility with tissue specimens up to at least 200 μm thick. Expected final online publication date for the , Volume 35 is October 7, 2019. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

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09/01/19 | BigStitcher: reconstructing high-resolution image datasets of cleared and expanded samples.
Hörl D, Rojas Rusak F, Preusser F, Tillberg P, Randel N, Chhetri RK, Cardona A, Keller PJ, Harz H, Leonhardt H, Treier M, Preibisch S
Nature Methods. 2019 Sep;16(9):870-74. doi: 10.1038/s41592-019-0501-0

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.

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