Glossary

  • Anchor round - A single round taken, usually on a chosen "anchor channel" that has a high Signal-to-Noise Ratio (SNR). All genes of interest are given the same fluorescing dye probe so that every spot lights up. The anchor round is essential for detecting all spots at once in the same microscope image.

  • Background gene - Background genes refer to constant pixel intensity across all sequencing rounds in one channel. This is an indicator of an anomalous fluorescing artefact that is not a spot. No spot codes are made to be the same channel in all rounds. This way spots are not mistaken for background fluorescence and vice versa.

  • Bled code - Every gene has a unique bled code. It is a series of intensities expected in each sequencing round/channel. Its bled code is dependent on the gene's gene code and the bleed matrix.

  • Bleed matrix - How intense each dye is in each sequencing channel. This is estimated during the call spots stage.

  • Channel - A combination of excitation light of a certain wavelength and specific emission filter. We use multiple channels to distinguish every dye colour (almost always the number of channels is equal to the number of unique dyes). But, a dye can have "bleed through", i.e. brightness in multiple channels from the same dye.

  • DAPI - A dye that fluoresces the nuclei of all cells. It is used to register between sequencing rounds. The DAPI is also a background image in the Viewer.

  • Gene code - A sequence of dyes that are assigned to a gene for each sequencing round. Each gene has a unique gene code. For example, if the dyes are labelled 0, 1, 2 and there are 2 sequencing rounds, some example gene codes are 0, 1 (i.e. dye 0 in first round, dye 1 in second round), 1, 2, 0, 2.

  • Notebook - A write-once1 compressed file that stores all important outputs from coppafisher. The notebook is used to plot many diagnostics. The notebook contains notebook pages. There is at least one notebook page for each method section. A notebook can be loaded by from coppafisher import Notebook; nb = Notebook("path/to/notebook"). Variables from the notebook can be directly read. For example, you can read the use_tiles variable from the basic_info page by print(nb.basic_info.use_tiles). Each variable has a description, which can be printed. For example, nb.basic_info > "use_tiles".

  • OMP - Stands for Orthogonal Matching Pursuit. It is the final section of the coppafisher pipeline. It is coppafisher's most sophisticated algorithm for gene calling and is used as a way of untangling genes that overlap by assuming that the pixel intensity is a linear combination of each gene bled code. There is currently no reason to suspect that gene bled codes combine non-linearly.

  • Point cloud - A series of spatial pixel positions. Typically used to represent detected spot positions during find spots.

  • PSF - Stands for Point Spread Function and is used during image filtering. The Wiener deconvolution requires a PSF to remove blurring caused by frequencies with a low signal-to-noise ratio. See the filter overview and the Wikipedia article for more details.

  • Sequencing round - An image of the tissue, made up of multiple tiles and sequencing channels. Before each imaging round, the tissue is treated with various solutions to remove the previous DNA probes and then hybridise new ones. Each spot will bind to a specific bridge probe which then binds to a fluorescing dye probe, causing it to fluoresce in specific channel(s). The colour of each spot in each round is dictated by its gene identity (identities) and their corresponding gene code(s).

  • Spot - An amplified ball of DNA with a unique barcode specific to each gene. The gene can be determined by looking at the same spot in all sequencing rounds to reveal the gene code. Coppafisher takes the raw images of the spots as input and outputs the gene identity of each spot in situ.

  • Tile - A cuboid subset of the microscope image of pixel size \(n_z \times n_y \times n_x\) in z, y, and x, where \(n_y = n_x \sim 2000 \text{ pixels}\). Typically, \(n_z\sim10\text{s}\). Usually, all adjacent tiles overlap by \(10\%-15\%\) to give coppafisher sufficient information to align tiles (see stitch for details).

  1. There are some cases of notebooks being "rewritten", see advanced usage. ↩