Parameters

parameters.json file, previously named infoList.json (DEPRECATED)

Global structure

Each section in common represents a step of pyHiM processing. Parameters are defined by default in common in their corresponding sections. If you want to change a parameter just for one channel, go to labels and you can overwrite the parameter value in the channel of your choice (an example here).

Parameter overview by section

Parameters are sort by alphabetical order.

1. acquisition

Parameter name

Used to

barcode_channel

List the files to process; label of barcode channel

DAPI_channel

List the files to process; label of DAPI channel

fiducialBarcode_channel

List the files to process; label of fiducial channel for barcode cycles

fiducialDAPI_channel

List the files to process; label of fiducial channel for the DAPI/RNA cycles

fiducialMask_channel

List the files to process; label of fiducial channel for mask cycles

fileNameRegExp

Extraction information from filename

label

Know which label is currently being processed (added in convertsParameterFile method)

label_channel

in future this field will contain the ch for the label. This parameter will superseded the individual channel fields above.

label_channel_fiducial

in future this field will contain the ch for the label fiducial. This parameter will superseded the individual channel fields above.

mask_channel

List the files to process, label of mask channel

pixelSizeXY

Get lateral pixel size in nm; compute voxel size for 3D gaussian fitting

pixelSizeZ

Get axial pixel size in nm; compute voxel size for 3D gaussian fitting

RNA_channel

List the files to process; Label of RNA channel

zBinning

Speed up processing time; binning in z-axis. A z-binning of 2 will skip every other plane. A z-binning of 1 will keep all planes.

2. zProject

Parameter name

Used to

blockSize

Split image into blocks for laplacian mode

display

Save output 2D projection as PNG; show image

folder

Give a name of output folder to save output data of zPorject features

mode

Select projection mode between full, manual, automatic, laplacian

windowSecurity

Remove the lowest and highest Z-plans for automatic mode

zmax

Set a maximum on Z axis for manual mode

zmin

Set a minimum on Z axis for manual mode

zProjectOption

Select projection option between sum of all concerned planes (sum) or takes the maximum intensity projection (MIP)

zwindows

Set a margin to find best focal place for both automatic and laplacian modes

3. alignImages

Parameter name

Used to

alignByBlock

True will perform block alignment. False will do global alignment.

background_sigma

Remove inhomogeneous background; set the number of standard deviations to use for both the lower and upper clipping limit (astropy.stats.SigmaClip)

blockSize

Define size in (X,Y) of block for global XY block alignment

globalAlignment

Select global registration output mode: 2D (default) or 3D

sliceSize

Target width (in pixels) of X/Y slices used for Z-shift polling when globalAlignment=3D

zMinSignalFraction

Minimum fraction of above-background pixels required for a slice to be used in Z-shift polling

zMinSignalFractionAuto

If True, automatically relaxes zMinSignalFraction when no valid slices are found

folder

Give a name of output folder to save output data of register_global features

higher_threshold

Set higher threshold to adjust image intensity levels before xcorrelation for alignment in 2D

localAlignment

Select mode between global alignment (None), 2D local alignment (mask2D) and 3D local alignment ( block3D)

lower_threshold

Set lower threshold to adjust image intensity levels before xcorrelation for alignment in 2D

outputFile

Set a base name for output files

referenceFiducial

Set name of the reference fiducial cycle

tolerance

Determine the % of error tolerated for blockAlignment mode

3D_higher_threshold

Set higher threshold to adjust image intensity levels before xcorrelation for Alignment3D

3D_lower_threshold

Set lower threshold to adjust image intensity levels before xcorrelation for Alignment3D

4. segmentedObjects

Parameter name

Used to

area_max

Set max area to keep 2D segmented object (in pixels)

area_min

Set min area to keep 2D segmented object (in pixels)

background_method

Select method to process background for barcode, DAPI and mask labels, between flat, inhomogeneous and (just for DAPI and mask) stardist method

background_sigma

Remove inhomogeneous background; set the number of standard deviations to use for both the lower and upper clipping limit (astropy.stats.SigmaClip)

brightest

Set max number of objects segmented per FOV (only for barcodes!); “Number of brightest objects to keep after sorting the full object list. If brightest is set to None, all objects will be selected” (photutils.DAOStarFinder)

centroidDifference_max

Set max difference between z centroid position determined by moment and by gaussian fitting

folder

Give a name of output folder to save output data of segmentedObjects features

fwhm

Set source size (in pixels); “The full-width half-maximum (FWHM) of the major axis of the Gaussian kernel in units of pixels.” (photutils.DAOStarFinder)

intensity_max

Set max intensity to keep object

intensity_min

Set min intensity to keep object

operation

Select 2D or 3D method of segmentation for DAPI and barcode label; Process secondary masks in process_snd_channel script for RNA label

outputFile

Set a base name for output files

residual_max

Set maximum difference between axial spot intensity and gaussian fit

sigma_max

Set maximum gaussian fit sigma allowed (axial spot intensity)

stardist_basename

Set path name of AI models for 2D segmentation with StarDist

stardist_network

Set network name for 2D DAPI and mask segmentation with StarDist

stardist_network3D

Set network name for 3D barcode, DAPI and mask segmentation with StarDist

tessellation

Allow tessellation to segment DAPI and mask if value is True

threshold_over_std

Set threshold used to detect sources

3D_area_max

Set max area to keep 3D segmented object (in pixels)

3D_area_min

Set min area to keep 3D segmented object (in pixels)

3D_boxSize

Set size of box used for block decomposition

3D_contrast

“The fraction of the total (blended) source flux that a local peak must have (at any one of the multi-thresholds) to be considered as a separate object.” (photutils.segmentation.deblend_sources)

3D_lower_threshold

Set lower threshold for adjusting image levels

3D_nlevels

“The number of multi-thresholding levels to use.” (photutils.segmentation.deblend_sources)

3D_psf_yx

“Radius of the spot, in nanometer. One value per spatial dimension (zyx or yx dimensions). If it’s a scalar, the same radius is applied to every dimensions.” (apifish.detection.spot_modeling.fit_subpixel)

3D_psf_z

“Radius of the spot, in nanometer. One value per spatial dimension (zyx or yx dimensions). If it’s a scalar, the same radius is applied to every dimensions.” (apifish.detection.spot_modeling.fit_subpixel)

3D_sigma

instance a 2D Gaussian filter kernel (astropy.convolution.Gaussian2DKernel)

3D_threshold_over_std

Set threshold used to detect 3D sources

3D_higher_threshold

Set higher threshold for adjusting image levels

3dAP_brightest

Set number of sources sought in each YZ plane; “Number of brightest objects to keep after sorting the full object list. If brightest is set to None, all objects will be selected” (photutils.DAOStarFinder)

3dAP_distTolerance

Set distance (in pixels) to attribute a source localized in YZ to one localized in XY

3dAP_flux_min

Set threshold to keep a source detected in YZ

3dAP_window

construct a YZ image by summing from xPlane-window:xPlane+window

3DGaussianfitWindow

Set size of window in xy to extract 3D subVolume, in px. 3 means subvolume will be 7x7.

3Dmethod

Select which segmentation method to use for the 3D source segmentation between stardist or just by thresholding

5. buildsPWDmatrix

Parameter name

Used to

colormaps

Set colormaps used for plotting matrices

flux_min

Set minimum flux per spot for 2D. If flux is smaller, localization will be discarded

flux_min_3D

Set minimum flux per spot for 3D localizations. If flux is smaller, localization will be discarded

folder

Give a name of output folder to save output data of buildsPWDmatrix features

KDtree_distance_threshold_mum

Set distance threshold used to build KDtree

mask_expansion

Set number of pixels masks will be expanded to assign localizations

toleranceDrift

Set tolerance used for block drift correction (in pixels)

tracing_method

Set list of methods it will use