Spatial Transcriptomics

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Core Idea

Spatial transcriptomics measures gene expression while preserving the spatial location of each measurement within a tissue section. Sequencing-based methods (10x Visium, Slide-seq) capture mRNA on spatially barcoded arrays, providing transcriptome-wide coverage at defined locations. Imaging-based methods (MERFISH, seqFISH) use multiplexed fluorescence in situ hybridization to detect hundreds to thousands of genes at single-molecule resolution within intact tissue. By retaining spatial context that scRNA-seq loses during dissociation, spatial transcriptomics reveals how gene expression varies across tissue architecture, identifies spatial domains and niches, and maps cell-cell communication patterns.

How It's Best Learned

Analyze a 10x Visium dataset from a mouse brain section using Squidpy or Scanpy: visualize gene expression overlaid on the tissue image, identify spatially variable genes, and map clusters to anatomical regions. Compare to a dissociated scRNA-seq dataset from the same tissue and note what spatial information was lost in the scRNA-seq.

Common Misconceptions

10x Visium spots are not single cells — each 55-micrometer spot captures RNA from approximately 1-10 cells, requiring computational deconvolution to estimate cell type composition per spot.
Spatial transcriptomics does not yet provide the same transcriptome-wide coverage at single-cell resolution in a single technology — there is a tradeoff between the number of genes measured and spatial resolution.

Explainer

Single-cell RNA sequencing revealed that tissues are composed of diverse cell types with distinct transcriptional programs. But by dissociating tissue into single cells, scRNA-seq destroys the very thing that makes a tissue a tissue: the spatial organization of cells. Which cells are next to which? How does gene expression change from the center to the edge of a tumor? Where exactly in the brain is a particular gene expressed? Spatial transcriptomics answers these questions by measuring gene expression in situ — within intact tissue sections.

Sequencing-based methods work by placing a tissue section onto a surface printed with spatially barcoded oligonucleotides. When RNA is released from the tissue (by permeabilization), it hybridizes to the barcoded probes, which capture it at known locations. After reverse transcription and sequencing, each read carries both the gene identity and the spatial barcode, enabling reconstruction of a spatial gene expression map. 10x Genomics Visium, the most widely used platform, prints ~5,000 spots (each 55 micrometers in diameter, spaced 100 micrometers apart) on a capture area. This provides transcriptome-wide coverage but at multi-cell resolution — each spot captures RNA from several cells. Newer methods like Slide-seq (10-micrometer beads) and Stereo-seq (sub-cellular resolution) are pushing spatial resolution finer.

Imaging-based methods take the opposite approach: they visualize individual RNA molecules in situ using multiplexed FISH. MERFISH (Multiplexed Error-Robust Fluorescence In Situ Hybridization) encodes each RNA species with a unique combination of fluorescent probes across multiple rounds of imaging. A gene detected in rounds 1, 3, and 5 (but not 2, 4, and 6) has a unique binary barcode that identifies it. Error-robust encoding schemes tolerate missed or false hybridizations. seqFISH uses a sequential barcoding strategy for similar results. These methods achieve single-molecule, subcellular resolution but are limited in gene number (hundreds to low thousands) and require specialized microscopy.

The analytical challenge is integrating spatial and molecular information. Spatially variable gene detection identifies genes whose expression shows significant spatial patterns (gradients, hot spots, domain boundaries). Spatial domain identification segments the tissue into regions with coherent expression programs, often corresponding to anatomical structures. Deconvolution (for multi-cell-resolution data like Visium) estimates the cell type composition of each spot by combining the spatial data with a scRNA-seq reference. Cell-cell communication analysis maps ligand-receptor interactions between spatially adjacent cells. These analyses are producing spatial cell atlases of organs in health and disease, revealing how tissue microenvironments shape cell behavior in ways that dissociated studies could never capture.

Practice Questions 3 questions

Prerequisite Chain

Counting to 10 → Counting to 20 → Understanding Zero → The Number Zero → Counting to Five → One-to-One Correspondence → Combining Small Groups Within 5 → Addition Within 10 → Addition Within 20 → Two-Digit Addition Without Regrouping → Two-Digit Addition with Regrouping → Addition Within 100 → Repeated Addition as Multiplication → Multiplication Facts Within 100 → Division as Equal Sharing → Division as Grouping (Measurement Division) → Division: Grouping (Repeated Subtraction) Model → Division: Fair Sharing Model → Division as Equal Sharing → Division as Grouping → Basic Division Facts → Division Facts Within 100 → Two-Digit by One-Digit Division → Division with Remainders → Remainders and Quotients in Division → Division Word Problems → Introduction to Long Division → Factors and Multiples → Prime and Composite Numbers → Equivalent Fractions → Relating Fractions and Decimals → Decimal Place Value → Reading and Writing Decimals → Comparing and Ordering Decimals → Adding and Subtracting Decimals → Multiplying Decimals → Dividing Decimals → Dividing Fractions → Mixed Number Arithmetic → Order of Operations → Integer Order of Operations → Variable Expressions → Combining Like Terms → One-Step Equations → Two-Step Equations → Solving Multi-Step Equations → Equations with Variables on Both Sides → Angle Pairs: Complementary, Supplementary, and Vertical → Parallel Lines and Transversals → Corresponding Angles → Alternate Interior Angles → Triangle Angle Sum Theorem → Exterior Angle Theorem → Triangle Inequality Theorem → Similar Triangles: AA Similarity → Similar Triangles: SSS and SAS Similarity → Proportions in Similar Triangles → Right Triangle Trigonometry Introduction → Trigonometric Ratios Review → Radian Measure → Converting Between Degrees and Radians → The Unit Circle → Graphing Sine and Cosine → Graphing Tangent and Reciprocal Trigonometric Functions → Derivatives of Trigonometric Functions → Antiderivatives → Iterated Integrals and Fubini's Theorem → Double Integrals in Cartesian Coordinates → Double Integrals over Rectangular Regions → Double Integrals in Polar Coordinates → Double Integrals: Definition and Setup → Iterated Integrals and Fubini's Theorem → Double Integrals over Rectangular Regions → Double Integrals over General Regions → Applications of Double Integrals: Area, Mass, and Moments → Triple Integrals in Cartesian Coordinates → Triple Integrals in Cylindrical and Spherical Coordinates → Change of Variables and the Jacobian Determinant → Applications of Triple Integrals: Volume and Mass → Vector Fields and Their Representations → Line Integrals of Vector Fields → Green's Theorem → Surface Integrals and Flux of Vector Fields → Surface Integrals and Flux of Vector Fields → Divergence Theorem: Flux and Outflow → Divergence Theorem → Electric Flux → Gauss's Law → Conductors in Electrostatic Equilibrium → Capacitance and Capacitors → Dielectrics → Dielectric Constant and Relative Permittivity → Electric Field Inside Dielectric Materials → Dielectric Materials and Polarization → Dielectric Susceptibility and Permittivity → Energy Density in Electric Fields → Electric Current and Current Density → Electrical Resistance and Resistivity → Ohm's Law and Circuit Elements → Electromotive Force (EMF) and Batteries → Kirchhoff's Circuit Laws: Voltage and Current → DC Circuit Network Analysis Methods → Transient Response in RC Circuits → RC Circuits → LC and RLC Circuits → AC Circuits: Fundamentals → Impedance and Reactance → AC Power and Resonance → Electromagnetic Waves → The Electromagnetic Spectrum → Blackbody Radiation and Planck's Law → Photoelectric Effect → The Photon: Light as Quanta → Compton Scattering → Wave-Particle Duality → de Broglie Wavelength → Heisenberg Uncertainty Principle → Wavefunction and the Born Rule → The Schrödinger Equation → State Vectors and Wavefunctions → Quantum Superposition → Quantum Entanglement → Bell Theorem and Bell Inequalities → Postulates of Quantum Mechanics → Scattering Theory → Introduction to Scattering Theory → Partial Wave Analysis in Scattering → Spin Angular Momentum → Electron Spin and Intrinsic Magnetic Moment → Stern-Gerlach Experiment: Spin Quantization and Measurement → Electron Diffraction and Matter Wave Properties → Davisson-Germer Experiment: Crystal Diffraction of Electrons → Electron Diffraction and Matter Wave Interference → Wavefunctions and Probability Density Interpretation → Quantum Superposition and Linear Combinations of States → Quantum Operators and Observables → Canonical Commutation Relations and Uncertainty → Heisenberg Uncertainty Principle and Measurement Limits → Time-Independent Schrödinger Equation and Eigenvalues → Hydrogen Atom in Quantum Mechanics → Spectral Lines and Energy Transitions → Selection Rules for Atomic Transitions → LS and jj Coupling Schemes in Multi-Electron Atoms → Pauli Exclusion Principle and Antisymmetric Wavefunctions → Electron Configuration and the Aufbau Principle → The Periodic Table and Atomic Electronic Structure → The Periodic Table → Electron Configuration → Periodic Trends → Ionization Energy → Ionic Bonding → Lewis Structures → Resonance Structures and Delocalized Electrons → Resonance and Formal Charge → Molecular Polarity and Dipole Moments → Intermolecular Forces → States of Matter and Phase Changes: Melting, Boiling, and Sublimation → Gas Laws and the Ideal Gas Equation → Gas Stoichiometry and Volume-Volume Calculations → Thermochemistry and Enthalpy → Heat Capacity and Calorimetry → Entropy and Molecular Disorder → Spontaneity and ΔG → Entropy and Gibbs Free Energy → Chemical Equilibrium → Chemical Kinetics → Rate Law Determination → Enzyme Kinetics → Cell Cycle Regulation and Checkpoints → Mitosis → Cytokinesis → Meiosis → Chromosomal Theory of Inheritance → Mendelian Genetics → Dominance, Recessiveness, and Allelic Interactions → Sex-Linked Inheritance → Non-Mendelian Inheritance Patterns → Population Genetics and Hardy-Weinberg Equilibrium → Natural Selection → Genetic Drift → Evolutionary Genetics Foundations → Allele Frequency Change and Evolutionary Dynamics → Gene Flow and Population Structure → Gene Flow and Selection: Opposing Forces → Gene Flow → Hardy-Weinberg Equilibrium → Speciation → Phylogenetics and Evolutionary Trees → Molecular Evolution and Molecular Clocks → Pairwise Sequence Alignment → Gene Prediction and Annotation → RNA-seq Analysis Pipeline → Differential Gene Expression Analysis → Single-Cell RNA Sequencing → Spatial Transcriptomics

Longest path: 195 steps · 1035 total prerequisite topics

Prerequisites (2)

Single-Cell RNA Sequencinghard RNA-seq Analysis Pipelinehard

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