SparcFISH: Democratising spatial transcriptomics

Context Spatial transcriptomics is a rapidly developing transformational technology that detects genome-wide RNA expression in cells, tissues and organs at intracellular spatial resolution. Challenges Current state-of-the-art off-the-shelf technologies are not democratized, due to very costly proprietary reagents and untransparent data, instrument pipelines and mostly their limitation to mouse and human tissue sections. The majority also compromise imaging sensitivity and speed in favour of high genomic coverage (plex) using many rounds of hybridisation with slow automated microfluidic fluid exchange. Aims and objectives Our aim is to overcome these limitations using SparcFISH, our novel open, affordable and rapid high-plex spatial-transcriptomics method. SparcFISH is based on the gold-standard low-plex spatial-transcriptomics method, single molecule FISH (smFISH), which our lab has applied effectively in complex tissues and organs in 3D and at super-resolution. All commercial platforms use 4-5 colours and many rounds of hybridisation. In contrast, SparcFISH will image many transcripts in one round of hybridisation by using unique colour combinations to detect different mRNA species. We will follow the same principles of our published single molecule FISH (smFISH) methods to detect at ultra-high sensitivity all the individual mRNA molecules in thick tissues in 3D. Most commercial platforms need to reduce sensitivity to avoid crowding of spots at high-plex because they use low-resolution low-magnification objectives to detect amplified signal. In contrast, like smFISH, SparcFISH will use high-resolution and high-magnification objectives to detect smaller unamplified mRNA spots with absolute (>80%) sensitivity. Moreover, this makes the nascent transcript foci highly visible, so the rate of transcription and decay can be precisely estimated, as previously published by us. Our objectives are: Demonstrate the power of SparcFISH by doing 120-plex detection, using 3-colour combinations from a palette of 10 colours. We will make use of SpectraPlex, Leica’s recently launched convenient turnkey 15-channel spectral unmixing pipeline, already installed on our lab’s Leica Stellaris 8 confocal microscope. We will use the other 5 channels to detect DNA, cell membranes, the cytoskeleton, general cytoplasmic markers and/or cell identity markers, to segment individual cells and the definition of the tissue context in 3D. Apply SparcFISH to different specimen, starting with simple samples like Drosophila macrophage-like cells, and then moving on to complex 3D tissues like neuromuscular junctions (NMJs) and larval brains. We will work with our collaborators to apply SparcFISH to mouse marrow bone macrophages and brains slices. Beside the protocols and methodology behind SparcFISH, we will develop all the software tools and other resources required to demystify spatial transcriptomics, to lower the activation energy needed for adoption, and to build a community. SparcFISH is deliberately designed to be highly scalable. If there is time, we will demonstrate 1000-plex SparcFISH or higher, e.g. by increasing the number of channels. Potential applications and benefits With SparcFISH, we will demonstrate that spatial transcriptomics can be highly multiplexed yet affordable, without compromising key parameters such as sensitivity and speed, and without restricting it to certain genomes. We believe that our thorough and open approach will increase the adoption of cutting-edge state-of-the-art tools and technologies, which are also accessible and easy to use. We envision SparcFISH, together with the tools and resources we build around it, to have a transformative impact on the broader research community.