Tutorial: Multi Plant Image Pipeline

PlantCV is composed of modular functions that can be arranged (or rearranged) and adjusted quickly and easily. Pipelines do not need to be linear (and often are not). Please see pipeline example below for more details. Every function has a optional debug mode that prints out the resulting image. The debug has two modes, either 'plot' or print' if set to 'print' then the function prints the image out, if using a jupyter notebook, you would set debug to plot to have the images plot images to the screen. Debug mode allows users to visualize and optimize each step on individual test images and small test sets before pipelines are deployed over whole data-sets.

For multi-plant pipelines, images with multiple plants are processed and result in individual pictures for each plant, allowing a secondary pipeline (see VIS Tutorial for example) to be used. The challenge of multi plant processing is that a single plant can be composed of several contours, therefore contours need to be sorted and clustered together in some way. There are several functions that help with multi-plant image processing. First, the current clustering functions work by asking the user to provide an approximation of the number of desired 'rows' and 'columns' that they would like to split the image into. There does not need to be a plant in each spot, but the grid is used as an approximate region to cluster contours within. The rotation and shift function allow the image to be moved to optimize accurate clustering. Major assumptions that are made are that plants grow but that the imaging position does not change drastically. Also, the clustering functions will not work properly once plants start overlapping, since contours would also start overlapping.

Workflow

  1. Optimize pipeline on individual image in debug mode.
  2. Run pipeline on small test set (ideally that spans time and/or treatments).
  3. Re-optimize pipelines on 'problem images' after manual inspection of test set.
  4. Deploy optimized pipeline over test set using parallelization script.

Running A Pipeline

To run a Multi Plant pipeline over a single VIS image there are two required inputs:

  1. Image: Images can be processed regardless of what type of VIS camera was used (High-throughput platform, digital camera, cell phone camera). Image processing will work with adjustments if images are well lit and free of background that is similar in color to plant material.
  2. Output directory: If debug mode is on output images from each step are produced, otherwise ~4 final output images are produced.

Optional inputs:

  • Names File path to txt file with names of genotypes to split images into (order of names would be top to bottom, left to right
  • Debug Flag: Prints an image at each step

Sample command to run a pipeline on a single image:

  • Always test pipelines (preferably with -D 'print' option for debug mode) before running over a full image set
./pipelinename.py -i multi-plant-img.png -o ./output-images -n names.txt -D 'print'

Walk Through A Sample Pipeline

Pipelines start by importing necessary packages, and by defining user inputs.

#!/usr/bin/python

import sys, traceback
import cv2
import os
import re
import numpy as np
import argparse
import string
from plantcv import plancv as pcv

### Parse command-line arguments
def options():
    parser = argparse.ArgumentParser(description="Imaging processing with opencv")
    parser.add_argument("-i", "--image", help="Input image file.", required=True)
    parser.add_argument("-o", "--outdir", help="Output directory for image files.", required=True)
    parser.add_argument("-n", "--names", help="path to txt file with names of genotypes to split images into", required =False)
    parser.add_argument("-D", "--debug", help="Turn on debug, prints intermediate images.", action=None)
    args = parser.parse_args()
    return args

Start of the Main/Customizable portion of the pipeline.

The image input by the user is read in. The device variable is just a counter so that each debug image is labeled in numerical order.

### Main pipeline
def main():
    # Get options
    args = options()

    # Read image
    img, path, filename = pcv.readimage(args.image)

    debug=args.debug 

    # Pipeline step
    device = 0

Figure 1. Original image. This particular image was captured by a raspberry pi camera, just to show that PlantCV works on images not captured on a high-throughput phenotyping system with idealized vis image capture conditions. In this dataset images were captured over time of a flat (throughout the day and night).

Screenshot

Check if this is an image captured at night.


# Step 1: Check if this is a night image, for some of these datasets images were captured
# at night, even if nothing is visible. To make sure that images are not taken at
# night we check that the image isn't mostly dark (0=black, 255=white).
# if it is a night image it throws a fatal error and stops the pipeline.

if np.average(img) < 50:
    pcv.fatal_error("Night Image")
else:
    pass

White balance the image so that color can be compared across images and so that image processing can be the same between images (ideally). For more information see white balance function here.


# Step 2: Normalize the white color so you can later
# compare color between images.
# Inputs:
# device = device number. Used to count steps in the workflow
# img = image object, RGB colorspace
# debug = None, print, or plot. Print = save to file, Plot = print to screen.
# roi = region for white reference, if none uses the whole image,
# otherwise (x position, y position, box width, box height)

#white balance image based on white toughspot
device,img1=pcv.white_balance(device,img,debug,roi=(400,800,200,200))

Figure 2. White balance the image so that later image processing is easier.

Screenshot Screenshot Screenshot

Rotate image slighly so that plants line up with grid (later step) For more information see rotate function here.


# Step 3: Rotate the image 

device, rotate_img = pcv.rotate(img1, -1, device, debug)

Figure 3. Rotated image

Screenshot

Shift image slighly so that plants line up with grid (later step) For more information see shift function here.


#Step 4: Shift image. This step is important for clustering later on.
# For this image it also allows you to push the green raspberry pi camera
# out of the image. This step might not be necessary for all images.
# The resulting image is the same size as the original.
# Input:
# img = image object
# device = device number. Used to count steps in the workflow
# number = integer, number of pixels to move image
# side = direction to move from "top", "bottom", "right","left"
# debug = None, print, or plot. Print = save to file, Plot = print to screen.

device, shift1 = pcv.shift_img(img1, device, 300, 'top', debug)
img1 = shift1

Figure 4. Shifted image

Screenshot

Select single color channel that has contrast between the target object and background For more information see shift function here.


# STEP 5: Convert image from RGB colorspace to LAB colorspace
# Keep only the green-magenta channel (grayscale)
# Inputs:
#    img     = image object, RGB colorspace
#    channel = color subchannel (l = lightness, a = green-magenta , b = blue-yellow)
#    device  = device number. Used to count steps in the workflow
#    debug   = None, print, or plot. Print = save to file, Plot = print to screen.
device, a = pcv.rgb2gray_lab(img1, 'a', device, debug)

Figure 5. (Top) Green-magenta channel from LAB colorspace from original image.

Screenshot

Threshold green-magenta image. For more information see binary threshold function here.


# STEP 6: Set a binary threshold on the Saturation channel image
# Inputs:
#    img         = img object, grayscale
#    threshold   = threshold value (0-255)
#    maxValue    = value to apply above threshold (usually 255 = white)
#    object_type = light or dark
#                  - If object is light then standard thresholding is done
#                  - If object is dark then inverse thresholding is done
#    device      = device number. Used to count steps in the pipeline
#    debug       = None, print, or plot. Print = save to file, Plot = print to screen.
device, img_binary = pcv.binary_threshold(a, 120, 255, 'dark', device, debug)
#                                            ^
#                                            |
#                                           adjust this value

Figure 6. Thresholded image.

Screenshot

Fill noise. For more information on this function see here


# STEP 7: Fill in small objects (speckles)
# Inputs:
#    img    = image object, grayscale. img will be returned after filling
#    mask   = image object, grayscale. This image will be used to identify contours
#    size   = minimum object area size in pixels (integer)
#    device = device number. Used to count steps in the pipeline
#    debug  = None, print, or plot. Print = save to file, Plot = print to screen.
mask = np.copy(img_binary)
device, fill_image = pcv.fill(img_binary, mask, 100, device, debug)
#                                               ^
#                                               |
#                                               adjust this value

Figure 7. Fill noise.

Screenshot

Dilate binary image. For more information on this function see here


# STEP 8: Dilate so that you don't lose leaves (just in case)
# Inputs:
#    img     = input image
#    kernel  = integer
#    i       = interations, i.e. number of consecutive filtering passes
#    device  = device number. Used to count steps in the pipeline
#    debug   = None, print, or plot. Print = save to file, Plot = print to screen.

device, dilated = pcv.dilate(fill_image, 1, 1, device, debug)

Figure 8. Dilated image.

Screenshot

Find objects within the image, for more information see here)


# STEP 9: Find objects (contours: black-white boundaries)
# Inputs:
#    img       = image that the objects will be overlayed
#    mask      = what is used for object detection
#    device    = device number.  Used to count steps in the pipeline
#    debug     = None, print, or plot. Print = save to file, Plot = print to screen.
device, id_objects, obj_hierarchy = pcv.find_objects(img1, dilated, device, debug)

Figure 9. Identified objects.

Screenshot

Define region of interest in the image, for more information see here.


# STEP 10: Define region of interest (ROI)
# Inputs:
#    img       = img to overlay roi
#    roi       = default (None) or user input ROI image, object area should be white and background should be black,
#                has not been optimized for more than one ROI
#    roi_input = type of file roi_base is, either 'binary', 'rgb', or 'default' (no ROI inputted)
#    shape     = desired shape of final roi, either 'rectangle' or 'circle', if  user inputs rectangular roi but chooses
#                'circle' for shape then a circle is fitted around rectangular roi (and vice versa)
#    device    = device number.  Used to count steps in the pipeline
#    debug     = None, print, or plot. Print = save to file, Plot = print to screen.
#    adjust    = either 'True' or 'False', if 'True' allows user to adjust ROI
#    x_adj     = adjust center along x axis
#    y_adj     = adjust center along y axis
#    w_adj     = adjust width
#    h_adj     = adjust height
device, roi, roi_hierarchy = pcv.define_roi(img1, 'rectangle', device, None, 'default', debug, True, 
                                             10, 500, -10, -100)
#                                            ^                ^
#                                            |________________|
#                                            adjust these four values

Figure 10. Define ROI.

Screenshot

Once the region of interest is defined you can decide to keep all of the contained and overlapping with that region of interest or cut the objects to the shape of the region of interest. For more information see here.


# STEP 11: Keep objects that overlap with the ROI
# Inputs:
#    img            = img to display kept objects
#    roi_type       = 'cutto' or 'partial' (for partially inside)
#    roi_contour    = contour of roi, output from "View and Ajust ROI" function
#    roi_hierarchy  = contour of roi, output from "View and Ajust ROI" function
#    object_contour = contours of objects, output from "Identifying Objects" fuction
#    obj_hierarchy  = hierarchy of objects, output from "Identifying Objects" fuction
#    device         = device number.  Used to count steps in the pipeline
#    debug          = None, print, or plot. Print = save to file, Plot = print to screen.
device, roi_objects, roi_obj_hierarchy, kept_mask, obj_area = pcv.roi_objects(img1, 'partial', roi, roi_hierarchy,
                                                                           id_objects, obj_hierarchy, device,
                                                                           debug)

Figure 11. Define ROI.

Screenshot Screenshot Screenshot

Cluster plants based on defined grid, for more info see here).


#Step 12: This function take a image with multiple contours and 
# clusters them based on user input of rows and columns

#Inputs:
#    img - An RGB image array
#    roi_objects - object contours in an image that are needed to be clustered.
#    nrow - number of rows to cluster (this should be the approximate  number of desired rows in the entire image (even if there isn't a literal row of plants)
#    ncol - number of columns to cluster (this should be the approximate number of desired columns in the entire image (even if there isn't a literal row of plants)
#    file -  output of filename from read_image function
#    filenames - input txt file with list of filenames in order from top to bottom left to right
#    debug - print debugging images

device, clusters_i, contours = pcv.cluster_contours(device, img1, roi_objects, 4, 6, debug)

Figure 12. Cluster contours

Screenshot

Split the images


#Step 13:This function takes clustered contours and splits them into multiple images, 
#also does a check to make sure that the number of inputted filenames matches the number
#of clustered contours. If no filenames are given then the objects are just numbered

#Inputs:
#    img - ideally a masked RGB image.
#    grouped_contour_indexes - output of cluster_contours, indexes of clusters of contours
#    contours - contours to cluster, output of cluster_contours
plantcv
#    filenames - input txt file with list of filenames in order from top to bottom left to right (likely list of genotypes)
#    debug - print debugging images

out = args.outdir
names = args.names
device, output_path = pcv.cluster_contour_splitimg(device, img1, clusters_i, contours, out, file=filename, filenames=names, debug)

Figure 13. Split image based on clustering.

Screenshot

Screenshot

Screenshot

Screenshot

Screenshot

Screenshot

Screenshot

Screenshot

Screenshot

Screenshot

Screenshot

Screenshot

Screenshot

Screenshot

Screenshot

Screenshot

Screenshot

Screenshot

To deploy a pipeline over a full image set please see tutorial on Pipeline Parallelization here.