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 supersed the individual channel fields above.

label_channel_fiducial

in future this field will contain the ch for the label fiducial. This parameter will supersed 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 alignement.

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 3D local alignment; value needs to be a power of 2

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

tesselation

Allow tesselation 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