Class: Cicada::CicadaMain

Inherits:
Object
  • Object
show all
Includes:
IATScripting
Defined in:
lib/cicada/cicada_main.rb

Overview

This class is the main entry point for running 3D high-resolution colocalization and CICADA.

Constant Summary collapse

REQUIRED_PARAMETERS =

parameters required by the methods in this class

[:dirname, :basename, :im_border_size, :half_z_size, :determine_correction, :pixelsize_nm, :z_sectionsize_nm, :num_wavelengths, :photons_per_greylevel]
OPTIONAL_PARAMETERS =

parmeters used but not required in this class or only required for optional functionality

[:precomputed_position_data, :max_threads, :darkcurrent_image, :residual_cutoff, :max_greylevel_cutoff, :distance_cutoff, :fit_error_cutoff, :determine_correction, :determine_tre, :output_positions_to_directory, :in_situ_aberr_corr_basename, :in_situ_aberr_corr_channel, :log_to_file, :log_detailed_messages]
FAILURE_REASONS = 
{r2: "R^2 value", edge: "Too close to image edge", sat: "Saturated pixels", sep: "Separation between channels too large", err: "Fit error too large"}

Instance Attribute Summary collapse

Class Method Summary collapse

Instance Method Summary collapse

Constructor Details

#initialize(p) ⇒ CicadaMain

Sets up the analysis from a parameter dictionary.

Parameters:

  • p (ParameterDictionary, Hash) —

    a parameter dictionary or other object with hash-like behavior containing all the parameters for the analysis.



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# File 'lib/cicada/cicada_main.rb', line 76

def initialize(p)
  @parameters = p
  #@parameters = RImageAnalysisTools.create_parameter_dictionary(p) unless @parameters.is_a? ParameterDictionary
  @failures = {r2: 0, edge: 0, sat: 0, sep: 0, err: 0}

  if @parameters[:darkcurrent_image] then
    @dark_image = FileInteraction.load_image(@parameters[:darkcurrent_image])
  end

  set_up_logging
end

Instance Attribute Details

#failures ⇒ Object

Returns the value of attribute failures.



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# File 'lib/cicada/cicada_main.rb', line 66

def failures
  @failures
end

#logger ⇒ Object

Returns the value of attribute logger.



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# File 'lib/cicada/cicada_main.rb', line 66

def logger
  @logger
end

#parameters ⇒ Object

Returns the value of attribute parameters.



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# File 'lib/cicada/cicada_main.rb', line 66

def parameters
  @parameters
end

Class Method Details

.parse_parameter_file(fn) ⇒ ParameterDictionary

Reads a parameters file and creates a parameter dictionary.

Parameters:

  • fn (String) —

    the filename of the parameter file

Returns:

  • (ParameterDictionary) —

    the parsed parameters



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# File 'lib/cicada/cicada_main.rb', line 499

def self.parse_parameter_file(fn)
  java_import Java::edu.stanford.cfuller.imageanalysistools.meta.AnalysisMetadataParserFactory
  parser = AnalysisMetadataParserFactory.createParserForFile(fn)
  parser.parseFileToParameterDictionary(fn)
end

.run_from_parameter_file(fn) ⇒ void

This method returns an undefined value.

Runs analysis using a specified parameter file.

Parameters:

  • fn (String) —

    the filename of the parameter file



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# File 'lib/cicada/cicada_main.rb', line 512

def self.run_from_parameter_file(fn)
  p = parse_parameter_file(fn)
  c = new(p)
  c.go
end

Instance Method Details

#check_edges(to_check) ⇒ Boolean

Checks whether the fitted position is too close to the image edges.

Parameters:

  • to_check (ImageObject) —

    the ImageObject to check for fitting success

Returns:

  • (Boolean) —

    whether the fitting was successful by this criterion.



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# File 'lib/cicada/cicada_main.rb', line 229

def check_edges(to_check)
  eps = 0.1
  border_size = @parameters[:im_border_size].to_f
  z_size = @parameters[:half_z_size].to_f

  range_x = border_size...(to_check.getParent.getDimensionSizes[:x] - border_size)
  range_y = border_size...(to_check.getParent.getDimensionSizes[:y] - border_size)
  range_z = z_size...(to_check.getParent.getDimensionSizes[:z] - z_size)

  to_check.getFitParametersByChannel.each do |fp|
    x = fp.getPosition(ImageCoordinate::X)
    y = fp.getPosition(ImageCoordinate::Y)
    z = fp.getPosition(ImageCoordinate::Z)
    
    ok = (range_x.include?(x) and range_y.include?(y) and (range_z.include?(z) or to_check.getParent.getDimensionSizes[:z] == 1))
    unless ok then 
      @failures[:edge] += 1
      @logger.debug { "check failed for object #{to_check.getLabel} position: #{x}, #{y}, #{z}" }
      return false
    end
  end

  true
end

#check_error(to_check) ⇒ Boolean

Checks whether the caluclated fitting error (summed in quadrature over all wavelengths) is larger than a specified cutoff.

Parameters:

  • to_check (ImageObject) —

    the ImageObject to check for fitting success

Returns:

  • (Boolean) —

    whether the fitting was successful by this criterion.



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# File 'lib/cicada/cicada_main.rb', line 322

def check_error(to_check)
  if @parameters[:fit_error_cutoff] then
    total_error = 0
    to_check.getFitErrorByChannel.each do |d|
      total_error += d**2
    end
    total_error = total_error**0.5

    if total_error > @parameters[:fit_error_cutoff].to_f or total_error.nan? then
      @failures[:err] += 1
      @logger.debug { "check failed for object #{to_check.getLabel} with total fitting error: #{total_error}" }
      return false
    end
  end

  true
end

#check_fit(to_check) ⇒ Boolean

Checks whether the fitting was successful for a given object according to several criteria: whether the fitting finished without error, whether the R^2 value of the fit is above the cutoff, whether the object is too close to the image edges, whether the camera is saturated in the object, whether the separation between channels is above some cutoff, and whether the calculated fitting error is too large. Cutoffs for all these criteria are specified in the parameters file.

Parameters:

  • to_check (ImageObject) —

    the ImageObject to check for fitting success

Returns:

  • (Boolean) —

    whether the fitting was successful by all criteria.



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# File 'lib/cicada/cicada_main.rb', line 199

def check_fit(to_check)
  checks = [:check_r2, :check_edges, :check_saturation, :check_separation, :check_error]
  to_check.finishedFitting and checks.all? { |c| self.send(c, to_check) }
end

#check_r2(to_check) ⇒ Boolean

Checks whether the fit R^2 value is below the specified cutoff.

Parameters:

  • to_check (ImageObject) —

    the ImageObject to check for fitting success

Returns:

  • (Boolean) —

    whether the fitting was successful by this criterion.



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# File 'lib/cicada/cicada_main.rb', line 210

def check_r2(to_check)
  return true unless @parameters[:residual_cutoff]

  to_check.getFitR2ByChannel.each do |r2|
    if r2 < @parameters[:residual_cutoff].to_f then       
      @failures[:r2] += 1
      @logger.debug { "check failed for object #{to_check.getLabel} R^2 = #{r2}" }
      return false
    end
  end

  true
end

#check_saturation(to_check) ⇒ Boolean

Checks whether the camera has saturated in the object.

Parameters:

  • to_check (ImageObject) —

    the ImageObject to check for fitting success

Returns:

  • (Boolean) —

    whether the fitting was successful by this criterion.



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# File 'lib/cicada/cicada_main.rb', line 259

def check_saturation(to_check)
  if @parameters[:max_greylevel_cutoff] then
    to_check.boxImages
    cutoff = @parameters[:max_greylevel_cutoff].to_f
    to_check.getParent.each do |ic|

      if to_check.getParent[ic] > cutoff then 
        to_check.unboxImages
        @failures[:sat] += 1
        @logger.debug { "check failed for object #{to_check.getLabel} greylevel: #{to_check.getParent[ic]}" }
        return false 
      end
    end
  end

  true
end

#check_separation(to_check) ⇒ Boolean

Checks whether the separation between channels is too large.

Note that this check can significantly skew the distance measurements if the cutoff is too small. This remains here because occasionally closely spaced objects are fit as a single object and produce ridiculous values.

Parameters:

  • to_check (ImageObject) —

    the ImageObject to check for fitting success

Returns:

  • (Boolean) —

    whether the fitting was successful by this criterion.



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# File 'lib/cicada/cicada_main.rb', line 287

def check_separation(to_check)
  if @parameters[:distance_cutoff] then
    size_c = to_check.getFitParametersByChannel.size
    xy_pixelsize_2 = @parameters[:pixelsize_nm].to_f**2
    z_sectionsize_2 = @parameters[:z_sectionsize_nm].to_f**2

    0.upto(size_c-1) do |ci|
      0.upto(size_c-1) do |cj|
        fp1 = to_check.getFitParametersByChannel.get(ci)
        fp2 = to_check.getFitParametersByChannel.get(cj)
        ijdist = xy_pixelsize_2 * (fp1.getPosition(ImageCoordinate::X) - fp2.getPosition(ImageCoordinate::X))**2 +
          xy_pixelsize_2 * (fp1.getPosition(ImageCoordinate::Y) - fp2.getPosition(ImageCoordinate::Y))**2 +
          z_sectionsize_2 * (fp1.getPosition(ImageCoordinate::Z) - fp2.getPosition(ImageCoordinate::Z))**2
        ijdist = ijdist**0.5

        if (ijdist > @parameters[:distance_cutoff].to_f) then
          @failures[:sep] += 1
          @logger.debug { "check failed for object #{to_check.getLabel} with distance: #{ijdist}" } 
          return false              
        end
      end
    end
  end

  true
end

#do_and_save_fits ⇒ List<ImageObject>

Fits all objects in an image or loads objects from disk if they have already been fit and refitting has not been requested.

Saves fits to disk in the parameter-specified data directory.

Returns:

  • (List<ImageObject>) —

    the fitted or loaded image objects



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# File 'lib/cicada/cicada_main.rb', line 412

def do_and_save_fits
  image_objects = load_or_fit_image_objects
  FileInteraction.write_position_data(image_objects, @parameters)
  image_objects
end

#fit_objects_in_single_image(im_set) ⇒ Array<ImageObject>

Fits all the image objects in a single supplied image.

Does not check whether the fitting was successful.

Parameters:

  • im_set (OpenStruct, #image, #mask) —

    An object that references the image and the mask from which the objects will be fit. Should respond to #image and #mask.

Returns:

  • (Array<ImageObject>) —

    an array containing all the image objects in the image (one per unique greylevel in the mask).



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# File 'lib/cicada/cicada_main.rb', line 154

def fit_objects_in_single_image(im_set)
  objs = []
  load_and_dark_correct_image(im_set)

  unless im_set.image and im_set.mask then
    logger.error { "Unable to process image #{im_set.image_fn}." }
    return objs
  end

  h = Histogram.new(im_set.mask)
  max_threads = 1
  if @parameters[:max_threads] then
    max_threads = @parameters[:max_threads].to_i
  end
  thread_queue = Executors.newFixedThreadPool(max_threads)

  1.upto(h.getMaxValue) do |i|
    obj = GaussianImageObject.new(i, image_shallow_copy(im_set.mask), image_shallow_copy(im_set.image), ParameterDictionary.new(@parameters))
    obj.setImageID(im_set.image_fn)
    objs << obj
  end

  objs.each do |obj|
    submit_single_object(obj, thread_queue)
  end

  thread_queue.shutdown
  until thread_queue.isTerminated do
    sleep 0.4
  end

  objs
end

#get_scalar_diffs_from_vector(vector_diffs) ⇒ Array

Converts an array of vectors to an array of scalars by taking their 2-norm.

Parameters:

  • vector_diffs (Enumerable< Enumerable<Numeric> >) —

    an array of arrays (vectors, etc.) each of which will be normed.

Returns:

  • (Array) —

    an array of the norms of the vectors provided.



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# File 'lib/cicada/cicada_main.rb', line 486

def get_scalar_diffs_from_vector(vector_diffs)
  vector_diffs.map do |vd|
    Math.sqrt(Math.sum(vd) { |e| e**2 })
  end
end

#go ⇒ void

This method returns an undefined value.

Runs the analysis.



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# File 'lib/cicada/cicada_main.rb', line 424

def go
  image_objects = do_and_save_fits  
  pc = PositionCorrector.new(@parameters)
  pc.logger= @logger

  if @parameters[:correct_images] then
    c = pc.generate_correction(image_objects)

    tre = 0.0
    if @parameters[:determine_tre] and @parameters[:determine_correction] then       
      self.logger.info("calculating tre")      
      tre = pc.determine_tre(image_objects)
      c.tre= tre
    else
      tre = c.tre
    end
    c.write_to_file(FileInteraction.correction_filename(@parameters))
  end
  
  diffs = pc.apply_correction(c, image_objects)
  corrected_image_objects = []
  image_objects.each do |iobj|
    if iobj.getCorrectionSuccessful or not @parameters[:correct_images] then   
      corrected_image_objects << iobj
    end
  end
  FileInteraction.write_position_data(corrected_image_objects, @parameters)
  image_objects = corrected_image_objects
  
  df= P3DFitter.new(@parameters)
  fitparams = df.fit(image_objects, diffs)
  @logger.info { "p3d fit parameters: #{fitparams.join(', ')}" }

  if @parameters[:in_situ_aberr_corr_basename_set] and @parameters[:in_situ_aberr_corr_channel] then
    isc_iobjs = FileInteraction.read_in_situ_corr_data(@parameters)
    isc = InSituCorrection.new(@parameters[:reference_channel].to_i,
                               @parameters[:in_situ_aberr_corr_channel].to_i,
                               @parameters[:channel_to_correct].to_i,
                               isc_iobjs,
                               @parameters[:disable_in_situ_corr_constant_offset])
    vector_diffs = isc.apply(image_objects)
    vector_diffs.map! { |v| pc.apply_scale(v) }
    scalar_diffs = get_scalar_diffs_from_vector(vector_diffs)
    corr_fit_params = df.fit(image_objects, scalar_diffs)
    FileInteraction.write_differences(scalar_diffs, @parameters)

    if corr_fit_params then
      @logger.info { "p3d fit parameters after in situ correction: #{corr_fit_params.join(', ') }" }       
    else
      @logger.info { "unable to fit after in situ correction" } 
    end
  end
end

#load_and_dark_correct_image(im_set) ⇒ void

This method returns an undefined value.

Loads the image and mask from an image and mask pair and darkcurrent corrects the image if specified in the parameters.

Parameters:

  • im_set (OpenStruct, #image_fn, #mask_fn, #image=, #mask=) —

    An object that specified the filename of image and mask and can store the loaded image and mask. Should respond to #image_fn, #mask_fn, #image=, and #mask= for getting the filenames and setting the loaded images, respectively.



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# File 'lib/cicada/cicada_main.rb', line 113

def load_and_dark_correct_image(im_set)
  im_set.image = FileInteraction.load_image(im_set.image_fn)
  im_set.mask = FileInteraction.load_image(im_set.mask_fn)
  
  if (@dark_image) then
    im_set.image = im_set.image.writableInstance
    isf = ImageSubtractionFilter.new
    isf.setSubtractPlanarImage(true)
    isf.setReferenceImage(@dark_image)
    isf.apply(im_set.image)
  end     
end

#load_or_fit_image_objects ⇒ Array<ImageObject>

Loads previously existing image objects for the current images or fits them anew if they don’t exist or this is requested in the parameters.

Returns:

  • (Array<ImageObject>) —

    The image objects that have been loaded or fit. Only successfully fit objects that have passed all checks are included.



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# File 'lib/cicada/cicada_main.rb', line 367

def load_or_fit_image_objects
  image_objects = load_position_data
  unless image_objects then
    image_objects = []
    to_process = FileInteraction.list_files(@parameters)
    
    to_process.each do |im_set|      
      objs = fit_objects_in_single_image(im_set)
      objs.each do |o| 
        if check_fit(o) then
          image_objects << o
        end
        o.nullifyImages
      end
    end

    log_fitting_failures
  end

  puts "number of image objects: #{image_objects.size}"
  image_objects
end

#load_position_data ⇒ Array<ImageObject>

Load the position data from disk if this is requested in the specified parameters. If this has not been requested or if the position data file does not exist, returns nil.

Returns:

  • (Array<ImageObject>) —

    the image objects, complete with their fitted positions, or nil if this should be recalculated or if the file cannot be found.



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# File 'lib/cicada/cicada_main.rb', line 95

def load_position_data
  if @parameters[:precomputed_position_data] and FileInteraction.position_file_exists?(@parameters) then
    return FileInteraction.read_position_data(@parameters)
  end

  nil
end

#log_fitting_failures ⇒ void

This method returns an undefined value.

Formats the fitting failures using their description strings and logs them



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# File 'lib/cicada/cicada_main.rb', line 396

def log_fitting_failures
  @logger.info { "fitting failures by type:" }
  @failures.each_key do |k|
    @logger.info { FAILURE_REASONS[k] + ": " + @failures[k].to_s }
  end
end

#set_up_logging ⇒ void

This method returns an undefined value.

Sets up a logger to either standard output or a file with appropriate detail level as specified in the parameters



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# File 'lib/cicada/cicada_main.rb', line 346

def set_up_logging
  if @parameters[:log_to_file] then
    @logger = Logger.new(@parameters[:log_to_file])
  else
    @logger = Logger.new(STDOUT)
  end

  if @parameters[:log_detailed_messages] then
    @logger.sev_threshold = Logger::DEBUG
  else
    @logger.sev_threshold = Logger::INFO
  end
end

#submit_single_object(obj, queue) ⇒ void

This method returns an undefined value.

Submits a single object to a thread queue for fitting.

Parameters:

  • obj (ImageObject) —

    the image object to fit

  • queue (ExecutorService) —

    the thread queue



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# File 'lib/cicada/cicada_main.rb', line 134

def submit_single_object(obj, queue)
  queue.submit do 
      @logger.debug { "Processing object #{obj.getLabel}" }
      begin
        obj.fitPosition(@parameters)
      rescue => e
        logger.error { "error while processing object #{obj.label}: #{e.message}" }
      end
  end
end