327 - An introduction to Single Molecule Fluorescence In Situ Hybridization (smFISH​)

327 - An introduction to Single Molecule Fluorescence In Situ Hybridization (smFISH​)

smFISH uses a set of short, fluorescently labeled oligonucleotide probes that bind to specific target RNA molecules. Each probe is designed to hybridize to a specific sequence within the target RNA. The probes are designed to be complementary to the target RNA, allowing for specific and precise binding. Upon hybridization, the fluorescent probes generate a signal that can be visualized using fluorescence microscopy.

Why smFISH for studying gene expression?

High sensitivity: smFISH enables the detection of individual RNA molecules, providing high sensitivity and resolution.
Single-cell resolution: It allows the examination of gene expression patterns at the single-cell level, providing insights into cellular heterogeneity.
Quantitative analysis: smFISH enables the quantification of gene expression levels in individual cells or tissues.
Spatial information: It provides spatial localization of RNA molecules within cells, allowing for the investigation of subcellular distribution patterns.

smFISH experimental workflow – at a high level
Sample preparation: Cells or tissues are fixed, permeabilized, and then hybridized with fluorescently labeled probes.
Imaging: The samples are imaged using fluorescence microscopy, capturing the signals emitted by the labeled probes.
Image analysis: The acquired images are processed and analyzed to extract quantitative data on gene expression patterns.

Analyzing smFISH data
Specific analysis approach depends on the research question and the desired insights. However, a general workflow typically involves a few key steps:

Spot and Cluster detection: Detect individual spots or clusters corresponding to the bound fluorescent probes.
Cellular and nuclear segmentation: crucial for assigning spots to specific cellular compartments and extracting cell-specific information.
Statistical analysis: such as average distance of mRNA spots from the cell boundary, proportion of mRNA spots within the nucleus versus the cytoplasm. Note that multiplex analysis involves additional steps such as colocalization
Visualization and interpretation: heatmaps or scatter plots to interpret the analyzed data effectively.

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