3- Alignment Evaluation

This section guides you through the process of evaluating the alignment in the ExSeq-Toolbox. It involves measuring alignment accuracy and calculating confidence intervals to ensure reliable data processing.

Step 1: Load Configuration Settings

Begin by loading the configuration settings for the toolbox.

from exm.args.args import Args

# Create a new Config object instance.
args = Args()

# Provide the path to the configuration file.
args_file_path = '/path/to/your/parameter/file.json'

# Load the configuration settings from the specified file.
args.load_config(args_file_path)

Step 2: Additional Configuration for Alignment Evaluation

Configure additional parameters specific to alignment evaluation.

# Set various parameters for alignment evaluation
args.nonzero_thresh = .2 * 2048 * 2048 * 80
args.N = 1000
args.subvol_dim = 100
args.xystep = 0.1625/40  # check value
args.zstep = 0.4/40  # check value
args.pct_thresh = 99

Step 3: Alignment Measurement

Measure the alignment for specified codes and FOVs.

from exm.align.align_eval import measure_round_alignment_NCC

# Define the list of Codes and Fovs for alignment evaluation
codes_to_analyze = args.codes
fovs_to_analyze = args.fovs  # e.g., [1, 3].

# Extract the coordinates and measure alignment
for fov in fovs_to_analyze:
    for code in codes_to_analyze:
        measure_round_alignment_NCC(args=args, code=code, fov=fov)

Step 4: Alignment Evaluation and Confidence Interval Calculation

Evaluate alignment accuracy and calculate confidence intervals.

from exm.align.align_eval import plot_alignment_evaluation, calculate_alignment_evaluation_ci

# Define CI and percentile parameters
ci_percentage = 95
percentile_filter_value = 95

for fov in fovs_to_analyze:
    # Plot and calculate CI for alignment evaluation
    plot_alignment_evaluation(args, fov, percentile=percentile_filter_value, save_fig=True)
    calculate_alignment_evaluation_ci(args, fov, ci=ci_percentage, percentile_filter=percentile_filter_value)

Next Steps

After assessing the alignment, the subsequent step in the ExSeq-Toolbox pipeline is Puncta Extraction. This step is crucial for identifying and analyzing specific biological structures in the data. For detailed instructions on Puncta Extraction, refer to the Puncta Extraction section of this guide.