vaughan group
Research![6](https://sites.uw.edu/vgroup/files/2023/09/6.jpg)
We develop new tools for high resolution fluorescence microscopy and we use these tools to understand the molecular-scale organization of biological systems in their native cellular and tissue contexts. Many of our projects make use of super-resolution fluorescence microscopy methods such as expansion microscopy and STORM in order to peer beneath the ~250 nm diffraction-limited resolution of traditional fluorescence microscopy and to interrogate nanoscale features of biological specimens with rich molecular detail. On the tool development side, we are creating chemistries for fluorescent labeling and super-resolution imaging with microscopy-based assays for single-cell epigenetic profiling. On the application side, we have partnered with biologists to apply imaging tools to aging in kidney and to immune cell development. We use an interdisciplinary approach for our research that spans chemistry, biology, and optics.
Fluorescent Labeling of Abundant Reactive Entities (FLARE) uses conventional and commercially available fluorescent dyes for rapid and intense covalent labeling of proteins and carbohydrates in super-resolution (expansion) microscopy and cleared tissue microscopy. This simple and robust method produces stains that are modern equivalents of classic small-molecule histology stains. FLARE efficiently reveals a wealth of key landmarks in cells and tissues under different fixation or sample processing conditions and is compatible with immunolabeling of proteins and in situ hybridization labeling of nucleic acids. This protocol allows for efficient and effective labeling of carbohydrates, proteins, and nucleic acids in thick tissue specimens and reveals physiology at the nanoscale. We mainly use FLARE to study glomeruli in the kidney, mapping out their 3D structures and measuring changes in their nanoscale features. Another example of this method can be seen in the header video, which shows the filtration barrier going up through a healthy glomerulus at a resolution of ~70 nm.
![nprotocols_Cover_MAR22_print](https://sites.uw.edu/vgroup/files/2023/08/nprotocols_Cover_MAR22_print.jpg)
![Composite_R-ColIV_G-PDPL_B-CD45_Gy-Ren_M-Calb_Cy-CD13_Y-PODXL](https://sites.uw.edu/vgroup/files/2023/08/Composite_R-ColIV_G-PDPL_B-CD45_Gy-Ren_M-Calb_Cy-CD13_Y-PODXL-e1694467321729.jpg)
Spatial Transcriptomics and Highly Multiplexed Imaging: Together with the group of Prof. Daniel Chiu (UW Chemistry), we are developing a new method of highly multiplexed fluorescence microscopy using novel polymer dots (Pdots) and single round of immunostaining/imaging. A series of Pdots used to immunolabel can be excited at one wavelength to emit 5-10 distinct wavelengths, compared to conventional small-molecule dyes which are typically associated with one emission spectrum per excitation wavelength. With the capacity to design multiple of such Pdots series that contains unique combinations of absorption and emission spectra, we can simultaneously label and image up to 20 targets in a single sample.
Glomerular Nanoscale Spatial Atlas (GNSA): We are building a glomerular nanoscale spatial atlas (GNSA), which will include a detailed annotated collection of high spatial resolution (~50 nm) three-dimensional (3D) reconstructions of glomeruli and obtained from healthy, aged, and diseased kidney tissues in mouse and human. Kidney sample is stained with FLARE stain, expanded, and imaged using confocal microscopy. Key glomerular structures and different glomerular cell types are segmented, reconstructed, and quantitatively analyzed. The establishment of the GNSA will aid in revealing the global linkages among glomerular structures and supply a more robust understanding of the glomerulus as a unity that could doubtless deliver novel facets of glomerular diseases that have not been explored.
![IMG_0852](https://sites.uw.edu/vgroup/files/2023/08/IMG_0852.jpeg)
![Screenshot 2023-09-08 104147](https://sites.uw.edu/vgroup/files/2023/09/Screenshot-2023-09-08-104147.png)
Epigenetic profiling with Single Cell Evaluation of Post-TRanslational Epigenetic Encoding (SCEPTRE) is a new tool for probing the chromatin state of individual genes at the single cell level. SCEPTRE uses Expansion Microscopy to preserve and better resolve immunostained histone marks at DNA fluorescence in situ hybridization (FISH) labeled genomic loci. SCEPTRE reveals extensive variability in epigenetic states between individual gene loci hidden from current population-averaged measurements, emphasizing the need for single cell methods when defining the chromatin state of genes. SCEPTRE was developed jointly with the group of Prof. Hao Yuan Kueh (UW Bioengineering), where we seek to use SCEPTRE to uncover the heterogeneity of chromatin states of key developmental genes in blood cell progenitors, and how this heterogeneity impacts cell-fate decision making. Additional applications of SCEPTRE from the group can be seen below:
Immune Cell: Hematopoietic stem cells (HSCs) can differentiate into all immune (e.g., B & T-cells) in the human body through lineage decisions. We are investigating the heterogeneity of histone marks at key-lineage specifying genes to identify active/repressive cell states and its effects on differentiation pathways using a combinatorial method of SCEPTRE and lineage tracking.
Tissue and Aging: We aim to utilize SCEPTRE in wide applications including optimization for tissue applications to aid cell categorization based on their epigenetic profile. We are also focused on studying cellular senescence, which is generally associated with aging and has epigenetic markers that differ from healthy aged cells. We can use SCEPTRE to probe for these marks to study their localization to post-translational modified histones in super-resolution to help further understand how senescence spreads within tissue and leads to various aging phenotypes.
![Untitled (1)](https://sites.uw.edu/vgroup/files/2023/09/Untitled-1.png)