hdl73-signal-isolation

name	hdl73-signal-isolation
description	Batch signal isolation for Phenocycler spleen channels using matched blank pairs and kintsugi.signal
author	smith6jt
date	"2026-02-25T00:00:00.000Z"

HDL73 Signal Isolation - Research Notes

Experiment Overview

Item	Details
Date	2026-02-25
Goal	Process all 28 signal channels from HDL73 spleen sample with autofluorescence subtraction using matched blank pairs
Environment	KINTSUGI conda env, kintsugi.signal module, Python 3.x, tifffile, numpy
Status	Success

Context

HDL73 spleen sample from HiperGator had 28 signal markers but only 5 had been manually signal-isolated (CD11c, CD20, CD3e, CD4, CD8). The remaining 23 markers needed autofluorescence subtraction before the OME-TIFF could be rebuilt with all channels. Existing param files covered 9 channels (including the 5 already done), so 19 channels needed auto-analysis.

Verified Workflow

1. Channel-to-blank mapping from channelnames.txt

The Phenocycler cycle layout in channelnames.txt encodes 3 positions per cycle (a/b/c). Blanks at cycles 1 and 13 provide matched autofluorescence references:

POSITION_A = ["CD20", "CD31", "CD34", "CD35", "Lyve1", "PanCK", "SMActin"]
POSITION_B = ["CD8", "CD15", "CD21", "CD44", "CD45RO", "CD5", "CollagenIV", "ECAD", "FoxP3", "Ki67", "Podoplanin"]
POSITION_C = ["CD3e", "CD4", "CD11c", "CD107a", "CD163", "CD1c", "CD45", "CD68", "HLADR", "Vimentin"]

2. Blank averaging

blank_avg = ((blank1.astype(np.float32) + blank13.astype(np.float32)) / 2.0).astype(np.uint16)

3. Parameter resolution (file or auto)

from kintsugi.signal import analyze_for_subtraction, subtract_autofluorescence, compute_subtraction_quality

# If param file exists, parse blank_clip_factor and background_scale_factor
# If not, auto-analyze:
params = analyze_for_subtraction(signal, blank_avg, tissue_type="spleen", marker_name=name)

4. Subtraction and quality check

result = subtract_autofluorescence(signal, blank_avg,
    blank_clip_factor=params["blank_clip_factor"],
    blank_scale_factor=params["blank_scale_factor"])
quality = compute_subtraction_quality(signal, result, blank_avg)

5. OME-TIFF rebuild

python scripts/convert_to_ome_tiff.py FromHipergator/HDL73_SPL_Processed/ImageJ Images/HDL073_PC29.ome.tiff

Failed Attempts (Critical)

Attempt	Why it Failed	Lesson Learned
conda run -n KINTSUGI python script.py	stdout completely buffered — appeared to hang with no output for 10+ minutes	Use direct python path: $HOME/miniconda3/envs/KINTSUGI/bin/python -u
conda run --no-banner flag	Not supported in this conda version	Stick with direct python invocation
Importing kintsugi.signal_isolation	Module doesn't exist	Correct path is kintsugi.signal (check kintsugi._SUBMODULE_MAPPING)
f-strings with nested braces in inline python	SyntaxError on older Python	Use .format() for complex formatting in -c one-liners

Final Parameters

Channels with existing param files

CD11c:  clip=7000,  scale=1.4
CD15:   clip=9000,  scale=0.7
CD1c:   clip=5000,  scale=1.8  # failed marker
CD20:   clip=7000,  scale=0.2
CD21:   clip=3000,  scale=1.6
CD3e:   clip=12000, scale=1.9  # param file also contains ImageJ macros
CD4:    clip=7000,  scale=2.0
CD5:    clip=10000, scale=1.4  # failed marker
CD8:    clip=8000,  scale=1.5

Blank medians by position

position_a: 14526  # highest autofluorescence
position_b: 6708
position_c: 4341   # lowest autofluorescence

Output

• 29 files in ImageJ/ (28 signal + DAPI copy)
• OME-TIFF: 2.69 GB, pyramidal 5-level, 512x512 tiles, DEFLATE

Key Insights

• CD3e param file contained ImageJ macro commands mixed with subtraction params — must parse carefully, only extract blank_clip_factor and background_scale_factor
• DAPI requires no subtraction — just copy directly
• analyze_for_subtraction() with tissue_type="spleen" works well for most channels but PanCK, Podoplanin, SMActin got low signal preservation (<0.3) — may need manual parameter tuning
• CD1c and CD5 were listed in Failed_markers.txt — processed anyway but results should be visually validated
• Position a blanks have ~3x higher median than position c — blank intensity varies significantly across positions

References

• scripts/process_hdl73_channels.py — the processing script
• scripts/convert_to_ome_tiff.py — OME-TIFF builder
• FromHipergator/HDL73_SPL_meta/channelnames.txt — cycle layout source
• FromHipergator/HDL73_SPL_Processed/Processing_parameters/ — existing param files