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Getting Started

The fastest way to gain familiarity with FlyEye Analysis is to start with a working example. Please feel free to use the data from our study of Pnt and Yan expression during eye development.

We recommend reading the sections below before working with your own microscopy data.

Preparing Images

FlyEye Analysis supports analysis of expression data contained in .silhouette files. Each of these files corresponds to a single eye disc that has been marked with fluorescent reporters, dissected, imaged, segmented, and annotated. See the FlyEye Silhouette documentation for tips on generating .silhouette files from your microscopy data.

Note

The initial release of NU FlyEye Silhouette only supports three-channel (e.g. RGB) microscopy data. One of these reporter colors must be reserved for a nuclear marker in order to facilitate segmentation. This leaves at most two reporter channels available for measuring gene expression in any one experiment. However, in anticipation of future versions FlyEye Silhouette supporting additional channels, FlyEye Analysis was designed to support any number of reporter colors. Individual channels are therefore referred to by zero-indexed integer values (e.g. 0, 1, 2) rather than color names. For convenience, any user-provided string values of red, green, and blue (as well as r, g, and b) are automatically mapped to integer values 0, 1, and 2 respectively.

The .silhouette filetype includes reporter level measurements and an image for each layer in a 3D image stack. A single file containing all measurement annotations is also included. The contents are structured as:

example.silhouette
├── feud.json        # image stack metadata
├── feed.json        # measurement annotations
├── 0.json           # reporter level measurements for first layer
├── 0.png            # image of first layer
|
├── ... N.json       # reporter level measurements for Nth layer
└── ... N.png        # image of Nth layer

Each <layer_number>.json file contains all reporter levels measured during segmentation of the corresponding layer. Measured reporter levels reflect the mean pixel intensity within each nuclear contour, evaluated across all reporter wavelengths. These values are raw measurements; all subsequent normalization and processing by FlyEye Analysis are performed in local memory.

The feud.json file contains all user-assigned contour labels. FlyEye Analysis automatically pairs measurements with their corresponding labels upon import of a .sihouette file. Unlabeled contours are ignored.

The <layer_number>.png images are compressed versions of the original microscopy. They provide a visual representation of the original images, but they are not suitable for expression quantification.

Each .sihouette file must adhere to some basic requirements before using FlyEye Analysis:
  • R8 cells must be fully annotated within a locally contiguous region.
  • R8 cells must be labeled ‘r8’ or ‘R8’.
  • Only one contour should be labeled per cell that appears in the 3-D image stack.

Organizing Data

FlyEye Analysis offers three levels of organization for accessing measurement data. At the highest level, measurements are combined between eye discs collected under similar experimental conditions. We recommend organizing your .silhouette files in an equivalent manner by creating a separate directory for each experiment:

data
├── experiment_A
|   ├── eye0.silhouette
|   |
|   └── ... eyeN.silhouette
|
└── ... experiment_Z

Loading Data

Measurement data must be loaded as data.discs.Disc instances prior to analysis. Several important operations are automatically triggered upon instantiation of a Disc:

  1. Each cell is assigned a developmental age based on its proximity to the furrow
  2. Expression levels are normalized against the level of the reporter used to mark cell nuclei
  3. The expression ratio between the two remaining reporters is evaluated
  4. Moving average expression trends are evaluated for each labeled cell type

These operations are governed by a handful of user-specified parameters such as furrow velocity and the reporter color used to mark cell nuclei. These parameters must be specified in accordance with your particular dataset.

Note

FlyEye Analysis assumes that one of the three available reporter colors was reserved for a nuclear marker. The expression ratio assigned to each cell is evaluated using the two remaining reporter colors.

To load an individual .silhouette file:

>>> from flyeye.data import Disc
>>> path_to_disc = './data/experiment_A/eye0.silhouette'
>>> disc = Disc.from_silhouette(path_to_disc)

Alternatively, the experiments.Experiment constructor will automatically load and combine all discs within a specified directory:

>>> from flyeye.data import Experiment
>>> path_to_experiment = './data/experiment_A'
>>> experiment = Experiment(path_to_experiment)

Your data are now ready for analysis!