Methods
We development experimental methods to investigate molecular states of each cell in its original spatial context.
Deep Dye Drop
The Dye Drop Assay is a versatile, low-cost, highly reproducible multiplexed microscopy method for obtaining detailed single-cell viability and cell cycle information from perturbation experiments. Dye Drop is composed of two main parts: i) an experimental method where cells in multi-well plates are perturbed in high throughput, stained, and fixed using sequential density displacement, then imaged, and ii) a set of associated computational tools that assign cell cycle state and calculate growth rate metrics. Dye Drop can be combined with CyCIF to yield further molecular and spatial information.
CyCIF
CyCIF (cyclic immunofluorescence) is a robust and inexpensive method for highly multiplexed immunofluorescence imaging using standard instruments and reagents. The concept of repeatedly staining and imaging slides has been around for many years and most commonly involves antibody stripping using denaturants. Gerdes et al (2013) described an approach in which fluorophores are chemically inactivated after each of several rounds of immunofluorescence. The t-CyCIF (Tissue-based cyclic immunofluorescence) method by Lin et al (2018) builds on this and related approaches.
t-CyCIF uses formalin-fixed, paraffin-embedded (FFPE) tumor and tissue specimens mounted on glass slides. These are the most widely used specimens for histopathological diagnosis of cancer and other diseases. t-CyCIF generates multiplexed images of FFPE samples using an iterative process (a cycle) in which conventional low-plex fluorescence images are repeatedly collected from the same sample and then assembled into a high dimensional representation. Several variants are possible using direct and indirect immunofluorescence.
Pick-Seq
Pick-Seq is a form of micro-region sequencing in which small regions of tissue, containing 5-20 cells, are mechanically isolated on a microscope and then sequenced. Pick-Seq is compatible with several different fixed and frozen human specimens. It provides deep transcriptional profiling in the spatial contex and can be integrated with other spatial omics methods to achieve a hollistic view of the biology.