The napari-bigfish plugin
=========================

Start napari and run ``Detect FISH spots (napari-bigfish)`` from the menu ``Plugins``. Open the image containing the FISH-spots. If you also want to count spots per cell and per category nucleus/cytoplasm, open an image with the cell labels and one with the nuclei mask or labels.

.. image:: https://dev.mri.cnrs.fr/attachments/download/2945/detect_FISH_spots.png
		:alt: the gui of the napari-bigfish plugin

The plugin comes with an example image that you can open via ``File>Open Sample>BigFISH smFISH Analysis (napari-bigfish)``. This is a synthetic image that contains the FISH-Spots in the background and in 4 "cells" each one with one "nucleus".

.. image:: https://dev.mri.cnrs.fr/attachments/download/2944/bigfish_data.png
		:alt: the example input image

When using your own images, make sure that the scale in the image (pixel size in x, y and z) is correctly set. The plugin requires the scale to be in nanometers (nm). If you use the `naparj-j <https://www.napari-hub.org/plugins/napari-J>`_ plugin to transfer images from FIJI/ImageJ to napari, you can set the scale in the properties dialog available via ``Image>Properties...``. 

.. image:: https://dev.mri.cnrs.fr/attachments/download/2947/properties.png
		:alt: the properties of the image in FIJI/ImageJ

In napari you can check and modify the scale attribute of a layer from the integrated IPython console:

.. code-block::

	In [1]: viewer.layers.selection.active.scale
	Out[1]: array([1000.    ,  108.3424,  108.3424])
 

Background Subtraction
======================

.. image:: https://dev.mri.cnrs.fr/attachments/download/2948/background_subtraction.png
		:alt: napari-bigfish subtract background

The operation removes the background by subtracting a blurred version of the image from the original image. A new image layer with the result image will be created. 

**sigma xy**
  The standard deviation of the Gaussian kernel in the xy-plane.
**sigma z**
  The standard deviation of the Gaussian kernel in the z-dimension.

See also:
  `bigfish.stack.remove_background_gaussian <https://big-fish.readthedocs.io/en/stable/stack/preprocessing.html#bigfish.stack.remove_background_gaussian>`_

Spot Detection
==============

.. image:: https://dev.mri.cnrs.fr/attachments/download/2949/spot_detection.png
		:alt: napari-bigfish spot detection

The operation uses a LoG-filter and a local maximum detection to detect spots. A new points-layer containing the detected spots will be added to the viewer.

**threshold**
  The local threshold used for the spot detection. If ``find threshold`` is selected, this value will be ignored and the auto-detected threshold value will be used instead. The field will be updated with the detected threshold value.
**spot radius xy**
  The radius of a spot in the xy-plane in nanometer.
**spot radius z**
  The radius of a spot in the z-dimension in nanometer.
**remove duplicates**
  Remove potential duplicate coordinates for the same spots. The option slows the execution.
**find threshold**
  If selected the threshold is automatically detected, otherwise the value from the threshold-field is used.

See also:
  `bigfish.detection.detect_spots <https://big-fish.readthedocs.io/en/stable/detection/spots.html#bigfish.detection.detect_spots>`_

Decompose Dense Regions
=======================

.. image:: https://dev.mri.cnrs.fr/attachments/download/2950/decompose_dense.png
		:alt: napari-bigfish decompose dense regions

The operation breaks up clustered regions by building a reference spot and fitting it as many times as possible into the region. A new points-layer with the resulting spots will be added to the viewer.

**spots**
  The points layer of the spots from a previous detection to which the decomposition shall be applied.
**spot radius xy**  
  The radius of a spot in the xy-plane in nanometer.
**spot radius z**
  The radius of a spot in the z-dimension in nanometer.
**alpha**
  Intensity percentile used to compute the reference spot, between 0 and 1. The higher, the brighter are the spots simulated in the dense regions. Consequently, a high intensity score reduces the number of spots added. Default is 0.5, meaning the reference spot considered is the median spot.
**beta**
  Multiplicative factor for the intensity threshold of a dense region. Default is 1. 
**gamma**
  Multiplicative factor used to set the gaussian kernel size, for a gaussian background removal. A large gamma increases the scale of the gaussian filter and smooth the estimated background. To decompose very large bright areas, a larger gamma should be set.

See also:
  `bigfish.detection.decompose_dense <https://big-fish.readthedocs.io/en/stable/detection/dense.html#bigfish.detection.decompose_dense>`_

Spot Counting
=============

.. image:: https://dev.mri.cnrs.fr/attachments/download/2951/Spot_Counting.png
		:alt: napari-bigfish count spots

Counts the spots in the image. If a label image for cells is given the spots are counted per cell. If in addition a label image or a binary mask of the nuclei is given the spots within the cytoplasm and the nuclei are counted separately per cell. A table containing the results will be opened in the viewer. The results from the table can be exported to a spreadsheet program using copy and paste.

**spots**
  The previously detected spots in the form of a points-layer.
**cytoplasm labels**
  A labels layer with labels of the cells. The background should have the label 0.
**nuclei labels of mask**
  A labels layer in which the background has the label 0 and voxels belonging to a nucleus have a value bigger than 0.

.. image:: https://dev.mri.cnrs.fr/attachments/download/2952/napari-bigfish-results.png
		:alt: napari-bigfish results table

To select the whole table click into the upper-left corner of the table. To copy the selected data use either ``ctrl+c`` or the contex-menu of the table.