Week 03 HW lab-automation:

from opentrons import types
import subprocess, sys
import numpy as np


metadata = {    # see https://docs.opentrons.com/v2/tutorial.html#tutorial-metadata
    'author': 'Vivien',
    'protocolName': 'Gray-Scott Reaction-Diffusion Pattern',
    'description': 'Generates a reaction-diffusion pattern on an agar plate using the Gray-Scott model, pipetting where the B concentration is high.',
    'source': 'HTGAA 2026 Opentrons Lab',
    'apiLevel': '2.20'
}

##############################################################################
###   Gray-Scott Model Global Parameters and Functions
##############################################################################

# Gray-Scott Model Parameters
GS_DA = 1.0
GS_DB = 0.5
GS_F = 0.014 # Feed rate. Experiment with values like 0.035 (spots), 0.014 (worms), 0.062 (moving spots)
GS_K = 0.045 # Kill rate. Experiment with values like 0.065 (spots), 0.045 (worms), 0.061 (moving spots)
GS_DT = 1.0

#1. Mitosis / spots : f=0.0367, k=0.0649
#2. Solitions / worms : f=0.030, k=0.062
#3. Coral / dense : f=0.0545, k=0.062
#4. Vibe : f = 0.029, K = 0.057
#5. labyrythn : f = 0.060, K = 0.063

def laplace(Z):
    """Weighted 3x3 Laplacian (Karl Sims style) using numpy.roll."""
    return (
        -1.0 * Z +
        0.2 * (np.roll(Z,  1, axis=0) + np.roll(Z, -1, axis=0) +
               np.roll(Z,  1, axis=1) + np.roll(Z, -1, axis=1)) +
        0.05 * (np.roll(np.roll(Z,  1, axis=0),  1, axis=1) +
                np.roll(np.roll(Z,  1, axis=0), -1, axis=1) +
                np.roll(np.roll(Z, -1, axis=0),  1, axis=1) +
                np.roll(np.roll(Z, -1, axis=0), -1, axis=1))
    )

def simulate_gray_scott(A, B, num_iterations):
    """
    Runs the Gray-Scott simulation for a given number of iterations.
    Modifies A and B arrays in place.
    """
    for _ in range(num_iterations):
        lapA = laplace(A)
        lapB = laplace(B)

        reaction = A * B * B
        A += (GS_DA * lapA - reaction + GS_F * (1 - A)) * GS_DT
        B += (GS_DB * lapB + reaction - (GS_K + GS_F) * B) * GS_DT
    return A, B # Return A and B for clarity, though they are modified in-place

##############################################################################
###   Robot deck setup constants - don't change these
##############################################################################

TIP_RACK_DECK_SLOT = 9
COLORS_DECK_SLOT = 6
AGAR_DECK_SLOT = 5
PIPETTE_STARTING_TIP_WELL = 'A1'

well_colors = {
    'A1' : 'Red',
    'B1' : 'Green',
    'C1' : 'Orange'
}


def run(protocol):
  ##############################################################################
  ###   Load labware, modules and pipettes
  ##############################################################################

  # Tips
  tips_20ul = protocol.load_labware('opentrons_96_tiprack_20ul', TIP_RACK_DECK_SLOT, 'Opentrons 20uL Tips')

  # Pipettes
  pipette_20ul = protocol.load_instrument("p20_single_gen2", "right", [tips_20ul])

  # Modules
  temperature_module = protocol.load_module('temperature module gen2', COLORS_DECK_SLOT)

  # Temperature Module Plate
  temperature_plate = temperature_module.load_labware('opentrons_96_aluminumblock_generic_pcr_strip_200ul',
                                                      'Cold Plate')
  # Choose where to take the colors from
  color_plate = temperature_plate

  # Agar Plate
  agar_plate = protocol.load_labware('htgaa_agar_plate', AGAR_DECK_SLOT, 'Agar Plate')  ## TA MUST CALIBRATE EACH PLATE!
  # Get the top-center of the plate, make sure the plate was calibrated before running this
  center_location = agar_plate['A1'].top()

  pipette_20ul.starting_tip = tips_20ul.well(PIPETTE_STARTING_TIP_WELL)

  ##############################################################################
  ###   Patterning
##############################################################################

  ###
  ### Helper functions for this lab
  ###

  # pass this e.g. 'Red' and get back a Location which can be passed to aspirate()
  def location_of_color(color_string):
    for well,color in well_colors.items():
      if color.lower() == color_string.lower():
        return color_plate[well]
    raise ValueError(f"No well found with color {color_string}")

  # For this lab, instead of calling pipette.dispense(1, loc) use this: dispense_and_detach(pipette, 1, loc)
  def dispense_and_detach(pipette, volume, location):
      """
      Move laterally 5mm above the plate (to avoid smearing a drop); then drop down to the plate,
      dispense, move back up 5mm to detach drop, and stay high to be ready for next lateral move.
      5mm because a 4uL drop is 2mm diameter; and a 2deg tilt in the agar pour is >3mm difference across a plate.
      """
      assert(isinstance(volume, (int, float)))
      above_location = location.move(types.Point(z=location.point.z + 5))  # 5mm above
      pipette.move_to(above_location)       # Go to 5mm above the dispensing location
      pipette.dispense(volume, location)    # Go straight downwards and dispense
      pipette.move_to(above_location)       # Go straight up to detach drop and stay high

  ###
  ### YOUR CODE HERE to create your design (Gray-Scott Reaction-Diffusion Model)
  ###

  # Grid size for simulation
  size = 100 # Reduced size to make simulation faster and denser pattern on plate
  A = np.ones((size, size))
  B = np.zeros((size, size))

  # Create a central square seed for B
  r = 5
  A[size//2-r:size//2+r, size//2-r:size//2+r] = 0.5
  B[size//2-r:size//2+r, size//2-r:size//2+r] = 0.25

  # Add small random noise to the entire grid to introduce variation
  # This helps break symmetry and encourages diverse pattern formation
  A += np.random.rand(size, size) * 0.05 # Add noise up to 0.05
  B += np.random.rand(size, size) * 0.05 # Add noise up to 0.05

  B_min = float(B.min()); B_max = float(B.max());
  B_mean = float(B.mean())
  print("B stats:", B_min, B_mean, B_max)

  # Ensure values remain within bounds [0, 1] after adding noise
  A = np.clip(A, 0.0, 1.0)
  B = np.clip(B, 0.0, 1.0)

  print("Running Gray-Scott simulation...")

  # Define the number of simulation iterations here
  SIMULATION_ITERATIONS = 8000 # Initial run for 400 iterations

  A, B = simulate_gray_scott(A, B, SIMULATION_ITERATIONS)

  print(f"Gray-Scott simulation complete after {SIMULATION_ITERATIONS} iterations. Starting patterning.")
  print(f"To run more iterations, change the 'SIMULATION_ITERATIONS' variable in the code and re-run this cell and the visualization cell below.")


  # Patterning Parameters for Opentrons
  PIPETTE_VOLUME = 1 # 1uL per dot
  ASPIRATE_VOLUME = 16 # Aspirate up to this much at a time
  MAX_DOTS_PER_COLOR = 300 # Maximum number of dots to dispense per color

  # Colors to use for B components
  COLOR_FOR_PRIMARY_PATTERN = 'Green' # For the 'thick black lines' pattern
  COLOR_FOR_SECONDARY_PATTERN = 'Orange' # For other B concentrations
  COLOR_FOR_TERTIARY_PATTERN = 'Red' # For the highest B concentrations

  # Thresholds for B values
  # For 'thick black lines' (Green color)
  PRIMARY_PATTERN_B_LOWER_THRESHOLD = 0.40 * B_max
  PRIMARY_PATTERN_B_UPPER_THRESHOLD = 0.70 * B_max # This value will also define the lower bound for Red

  # For the secondary pattern (Orange color)
  # This will catch 'B' values that are above this, but not in the primary or tertiary pattern bands
  SECONDARY_PATTERN_B_THRESHOLD = 0.20 * B_max # Adjust this percentage as needed

  print("Using Red Pattern (Highest B) threshold: B >=", PRIMARY_PATTERN_B_UPPER_THRESHOLD)
  print("Using Green Pattern (Mid B - 'thick lines') B thresholds: (", PRIMARY_PATTERN_B_LOWER_THRESHOLD, ",", PRIMARY_PATTERN_B_UPPER_THRESHOLD, ")")
  print("Using Orange Pattern (Lower B) threshold: (", SECONDARY_PATTERN_B_THRESHOLD, ",", PRIMARY_PATTERN_B_LOWER_THRESHOLD, "]")


  # Agar plate dimensions (estimated for a standard 90mm round agar plate,
  # patterning in a 70x70mm square area roughly) to fit the pattern
  PATTERN_AREA_WIDTH_MM = 55 # Reduced from 85mm to fit within 40mm radius safe area
  PATTERN_AREA_HEIGHT_MM = 55 # Reduced from 85mm to fit within 40mm radius safe area

  # Calculate scaling factor to map grid coordinates to millimeters on the plate
  scale_x = PATTERN_AREA_WIDTH_MM / size
  scale_y = PATTERN_AREA_HEIGHT_MM / size

  # Add sampling step for clearer dots
  SAMPLING_STEP = 4 # Pipette every Nth pixel to create distinct dots
  DOT_SPACING_MM = SAMPLING_STEP * scale_x # Actual physical spacing between dot centers
  print(f"Desired dot spacing for distinct patterns: {DOT_SPACING_MM:.2f} mm (approx. 2mm drop diameter).")

  pipetted_count_primary = 0 # Green
  pipetted_count_secondary = 0 # Orange
  pipetted_count_tertiary = 0 # Red

  # --- Pipetting for Tertiary Pattern (Red component - highest B concentration) ---
  pipette_20ul.pick_up_tip()
  pipette_20ul.aspirate(ASPIRATE_VOLUME, location_of_color(COLOR_FOR_TERTIARY_PATTERN))
  current_pipette_volume = ASPIRATE_VOLUME

  print(f"Starting patterning for Tertiary Pattern with {COLOR_FOR_TERTIARY_PATTERN}.")

  for y in range(0, size, SAMPLING_STEP):
      for x in range(0, size, SAMPLING_STEP):
          if B[y, x] >= PRIMARY_PATTERN_B_UPPER_THRESHOLD:
              if current_pipette_volume < PIPETTE_VOLUME:
                  pipette_20ul.aspirate(ASPIRATE_VOLUME, location_of_color(COLOR_FOR_TERTIARY_PATTERN))
                  current_pipette_volume += ASPIRATE_VOLUME

              x_offset_mm = (x - size / 2) * scale_x
              y_offset_mm = (y - size / 2) * scale_y

              adjusted_location = center_location.move(types.Point(x=x_offset_mm, y=y_offset_mm))
              dispense_and_detach(pipette_20ul, PIPETTE_VOLUME, adjusted_location)
              current_pipette_volume -= PIPETTE_VOLUME
              pipetted_count_tertiary += 1
          if pipetted_count_tertiary >= MAX_DOTS_PER_COLOR:
              break
      if pipetted_count_tertiary >= MAX_DOTS_PER_COLOR:
          break
  pipette_20ul.drop_tip()
  print(f"Total {pipetted_count_tertiary} drops pipetted for Tertiary Pattern using {COLOR_FOR_TERTIARY_PATTERN}.")


  # --- Pipetting for Primary Pattern (Green component - 'thick lines') ---
  pipette_20ul.pick_up_tip()
  pipette_20ul.aspirate(ASPIRATE_VOLUME, location_of_color(COLOR_FOR_PRIMARY_PATTERN))
  current_pipette_volume = ASPIRATE_VOLUME

  print(f"Starting patterning for Primary Pattern with {COLOR_FOR_PRIMARY_PATTERN}.")

  for y in range(0, size, SAMPLING_STEP):
      for x in range(0, size, SAMPLING_STEP):
          if (B[y, x] > PRIMARY_PATTERN_B_LOWER_THRESHOLD) and (B[y, x] < PRIMARY_PATTERN_B_UPPER_THRESHOLD):
              if current_pipette_volume < PIPETTE_VOLUME:
                  pipette_20ul.aspirate(ASPIRATE_VOLUME, location_of_color(COLOR_FOR_PRIMARY_PATTERN))
                  current_pipette_volume += ASPIRATE_VOLUME

              x_offset_mm = (x - size / 2) * scale_x
              y_offset_mm = (y - size / 2) * scale_y

              adjusted_location = center_location.move(types.Point(x=x_offset_mm, y=y_offset_mm))
              dispense_and_detach(pipette_20ul, PIPETTE_VOLUME, adjusted_location)
              current_pipette_volume -= PIPETTE_VOLUME
              pipetted_count_primary += 1
          if pipetted_count_primary >= MAX_DOTS_PER_COLOR:
              break
      if pipetted_count_primary >= MAX_DOTS_PER_COLOR:
          break
  pipette_20ul.drop_tip()
  print(f"Total {pipetted_count_primary} drops pipetted for Primary Pattern using {COLOR_FOR_PRIMARY_PATTERN}.")

  # --- Pipetting for Secondary Pattern (Orange component) ---
  pipette_20ul.pick_up_tip()
  pipette_20ul.aspirate(ASPIRATE_VOLUME, location_of_color(COLOR_FOR_SECONDARY_PATTERN))
  current_pipette_volume = ASPIRATE_VOLUME

  print(f"Starting patterning for Secondary Pattern with {COLOR_FOR_SECONDARY_PATTERN}.")

  for y in range(0, size, SAMPLING_STEP):
      for x in range(0, size, SAMPLING_STEP):
          # Dispense Orange if B is above its threshold, AND NOT in the Green or Red band
          if (B[y, x] > SECONDARY_PATTERN_B_THRESHOLD) and (B[y, x] <= PRIMARY_PATTERN_B_LOWER_THRESHOLD):
              if current_pipette_volume < PIPETTE_VOLUME:
                  pipette_20ul.aspirate(ASPIRATE_VOLUME, location_of_color(COLOR_FOR_SECONDARY_PATTERN))
                  current_pipette_volume += ASPIRATE_VOLUME

              x_offset_mm = (x - size / 2) * scale_x
              y_offset_mm = (y - size / 2) * scale_y

              adjusted_location = center_location.move(types.Point(x=x_offset_mm, y=y_offset_mm))
              dispense_and_detach(pipette_20ul, PIPETTE_VOLUME, adjusted_location)
              current_pipette_volume -= PIPETTE_VOLUME
              pipetted_count_secondary += 1
          if pipetted_count_secondary >= MAX_DOTS_PER_COLOR:
              break
      if pipetted_count_secondary >= MAX_DOTS_PER_COLOR:
          break
  pipette_20ul.drop_tip()
  print(f"Total {pipetted_count_secondary} drops pipetted for Secondary Pattern using {COLOR_FOR_SECONDARY_PATTERN}.")

# Execute Simulation / Visualization -- don't change this code block
protocol = OpentronsMock(well_colors)
run(protocol)
protocol.visualize()

 B stats: 1.1769677182305039e-07 0.02738330028607403 0.2993330786497384
Running Gray-Scott simulation...
Gray-Scott simulation complete after 8000 iterations. Starting patterning.
To run more iterations, change the 'SIMULATION_ITERATIONS' variable in the code and re-run this cell and the visualization cell below.
Using Red Pattern (Highest B) threshold: B >= 0.20953315505481687
Using Green Pattern (Mid B - 'thick lines') B thresholds: ( 0.11973323145989537 , 0.20953315505481687 )
Using Orange Pattern (Lower B) threshold: ( 0.059866615729947684 , 0.11973323145989537 ]
Desired dot spacing for distinct patterns: 2.20 mm (approx. 2mm drop diameter).
Starting patterning for Tertiary Pattern with Red.
Total 0 drops pipetted for Tertiary Pattern using Red.
Starting patterning for Primary Pattern with Green.
Total 0 drops pipetted for Primary Pattern using Green.
Starting patterning for Secondary Pattern with Orange.
Total 0 drops pipetted for Secondary Pattern using Orange.

=== VOLUME TOTALS BY COLOR ===
	Orange:		 aspirated 16	 dispensed 0		##### WASTING BIO-INK : more aspirated than dispensed!
	Green:		 aspirated 16	 dispensed 0		##### WASTING BIO-INK : more aspirated than dispensed!
	Red:		 aspirated 16	 dispensed 0		##### WASTING BIO-INK : more aspirated than dispensed!
	[all colors]:	[aspirated 48]	[dispensed 0]

=== TIP COUNT ===
	 Used 3 tip(s)  (ideally exactly one per unique color)