Imaging: stacks, sections and cultures

Preprocessing and tools

• Motion correction (rigid): every frame is aligned to the mean image by FFT phase correlation with a sub-pixel peak fit (two passes) and shifted back (bilinear). Translation only.
• Detect cells: local correlation image (mean correlation of each pixel with its 8 neighbours), an automatic threshold (median + 4 robust SD, at least 0.2), connected components of 20–1000 px that are not elongated, holes filled; cells numbered left to right.
• Several ROIs: drawn, detected or loaded from roiMask / roiMasks; each has a name, colour and area.
• Optional B&W 256 levels, Gaussian smoothing (sigma 2 px) and per-frame normalisation to 0–1 (not for Brightness or ΔF/F), applied in that order.

• Data: demo_imaging.mat, 96 × 96 px, 150 frames at 10 Hz (15 s). A dark vertical vessel at x = 60 whose diameter oscillates 12 ± 3 px (9–15 px) every 5 s; a bright red blood cell moving down 2 px/frame (20 px/s); a cell at (24, 30), radius 6 px with calcium transients (ΔF/F ≈ 1) at 3, 7 and 11 s. The cell's roiMask is in the file.
• The demo selects ΔF/F with that mask (baseline = first 30 frames) and a line across the vessel from (45, 70) to (75, 70).
• ΔF/F: flat ~0 until 3 s, then three peaks of ~0.2–0.3 at 3, 7 and 11 s, each decaying in ~1–2 s. (The simulated calcium signal has ΔF/F ≈ 1, but it is added on top of the tissue background inside the ROI, so the measured ΔF/F of the ROI is smaller.)
• Vessel diameter (same line): a sine between ~9 and ~15 px with a 5 s period. Kymograph along the vessel (e.g. from (60, 5) to (60, 90)): slanted streaks with a slope of 2 px per frame.
• Advanced demo (Try advanced demo (motion, 3 cells)): 96 × 96 px, 150 frames at 10 Hz. Every frame is shifted by up to ±3 px (smooth random walk); three cells: cell 1 at (22, 24) with events at 4, 8.5, 13 s, cell 2 at (26, 78) at 5.5, 10.5 s, cell 3 at (82, 30) at 7, 12 s; the vessel at x = 60 (12 ± 3 px, period 5 s) and a bright red blood cell that crosses the diameter line (35, 64)–(85, 64) about every 3 s.
• Motion correction: the Motion correction tab shows dy and dx following the dashed true shifts (error < 0.3 px), max shift ≈ 3 px.
• Show → Correlation image: the three cells are bright disks (correlation ≈ 0.9), the vessel a bright band; Detect cells adds exactly Cell 1–3 (the vessel is rejected as too elongated).
• ΔF/F (Run): three traces, each peaking only at its own cell's event times.
• Vessel diameter: without Robust diameter the trace jumps to ~50 px whenever the blood cell crosses the line; with Robust diameter it follows the 9–15 px sine (error < 2.5 px) and the replaced frames are circled.

What it does

• Load TIFF (every page is a channel), PNG / JPG (the colours are channels) or .mat. The pixel size is read from ImageJ TIFFs, otherwise typed; sizes are given in µm² and densities per mm².
• Align (optional): the channels onto channel 1 (the colour shift of many microscopes), and images 2, 3 … onto image 1, either by an automatic shift (the same field imaged again) or by landmarks clicked in both images (serial sections: shift, rotation, scaling and shear).
• Count cells in the nuclear channel: background subtraction (uneven illumination), an automatic threshold (Otsu's method^[24], never below 3 noise SD), touching cells split at their narrow waist, and size and shape limits that leave out debris and fibres.
• Markers: a cell is positive when enough of it (default 50%) is bright in the marker channel.
• Regions: polygons clicked on the image give counts, % positive and cells per mm² per region.
• A Checks tab says in plain words where the pixel size came from, how well the alignment worked, which threshold was used and what was not counted.

• Data: demo_histology.mat (or Try demo data): two images of the same culture field, Culture, day 1 and Culture, day 3, 400 × 400 px at 1 µm per pixel (0.16 mm²). Channel 1 Nuclei (DAPI), channel 2 Marker (GFP).
• 60 nuclei in each image (48 single and 6 touching pairs): 30 in the left half and 30 in the right half. Count cells should give 60 in both images (375 cells per mm²); the pairs are split into two cells each.
• Marker-positive: 24 of 60 on day 1 and 39 of 60 on day 3 (every day-1 positive cell stays positive).
• Not counted: 20 small specks of debris (under 15 µm², removed by Min size) and 1 long fibre (removed by Max elongation). Uneven illumination is removed by the background step.
• Alignment: day 3 was imaged after the dish went back on the stage, shifted by 6.4 px down and 9.2 px left ([dy dx] = [6.4 −9.2]); Shift (automatic) recovers it to about 0.2 px. The marker channel is shifted by [1 2] px from the nuclei in both images (Align channels corrects it).
• Regions: add Region A = left half (x ≤ 200.5) and Region B = right half: 30 cells each (375 per mm², 0.08 mm² each). Marker-positive per region: 12 and 12 on day 1, 18 and 21 on day 3 (after aligning). Without aligning, the Checks tab warns that the regions cover different tissue in the two images.
• Checks tab (aligned, default settings): pixel size read from the file; image 2 moved by about 9.2 px right and 6.4 px up; the marker channel was about 2.6 px off (corrected; listed as a row to check, so look at the composite: markers should sit on their nuclei); 20 small objects and 1 elongated object not counted; 6 extra cells found by splitting the touching pairs.