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Microscopy CV Toolkit β classical computer-vision tools for microscopy image QC.
No ML models, pure OpenCV + NumPy + SciPy.
"""
import cv2
import numpy as np
import gradio as gr
import matplotlib
matplotlib.use("Agg")
import matplotlib.pyplot as plt
from matplotlib.colors import Normalize
from scipy import ndimage
from PIL import Image
import io
# ---------------------------------------------------------------------------
# Utilities
# ---------------------------------------------------------------------------
def _to_gray(img: np.ndarray) -> np.ndarray:
if len(img.shape) == 2:
return img
return cv2.cvtColor(img, cv2.COLOR_RGB2GRAY)
def _fig_to_image(fig) -> np.ndarray:
buf = io.BytesIO()
fig.savefig(buf, format="png", bbox_inches="tight", dpi=120)
plt.close(fig)
buf.seek(0)
return np.array(Image.open(buf))
# ---------------------------------------------------------------------------
# Tab 1 β Focus Quality
# ---------------------------------------------------------------------------
def _tenengrad(gray: np.ndarray) -> float:
gx = cv2.Sobel(gray, cv2.CV_64F, 1, 0, ksize=3)
gy = cv2.Sobel(gray, cv2.CV_64F, 0, 1, ksize=3)
return float(np.mean(gx ** 2 + gy ** 2))
def _laplacian_variance(gray: np.ndarray) -> float:
lap = cv2.Laplacian(gray, cv2.CV_64F)
return float(lap.var())
def _normalized_variance(gray: np.ndarray) -> float:
mean = gray.mean()
if mean < 1e-6:
return 0.0
return float(gray.astype(np.float64).var() / mean)
def _vollath_f4(gray: np.ndarray) -> float:
g = gray.astype(np.float64)
h, w = g.shape
t1 = np.sum(g[:, :w - 1] * g[:, 1:w])
t2 = np.sum(g[:, :w - 2] * g[:, 2:w])
return float(t1 - t2)
def _focus_heatmap(gray: np.ndarray, block: int = 64) -> np.ndarray:
h, w = gray.shape
rows = h // block
cols = w // block
hmap = np.zeros((rows, cols), dtype=np.float64)
for r in range(rows):
for c in range(cols):
patch = gray[r * block:(r + 1) * block, c * block:(c + 1) * block]
lap = cv2.Laplacian(patch, cv2.CV_64F)
hmap[r, c] = lap.var()
return hmap
def _score_label(val: float, low: float, high: float) -> str:
if val >= high:
return "PASS (sharp)"
elif val >= low:
return "MARGINAL"
return "FAIL (blurry)"
def analyze_focus(image: np.ndarray):
if image is None:
return None, "Upload an image first."
gray = _to_gray(image)
tenen = _tenengrad(gray)
lap_var = _laplacian_variance(gray)
norm_var = _normalized_variance(gray)
vollath = _vollath_f4(gray)
# Thresholds (heuristic, tuned for typical microscopy)
tenen_verdict = _score_label(tenen, 200, 1000)
lap_verdict = _score_label(lap_var, 50, 300)
norm_verdict = _score_label(norm_var, 5, 20)
vollath_verdict = _score_label(vollath, 1e5, 1e6)
overall_sharp = sum([
tenen >= 1000,
lap_var >= 300,
norm_var >= 20,
vollath >= 1e6,
])
if overall_sharp >= 3:
overall = "PASS β image is in focus"
elif overall_sharp >= 1:
overall = "MARGINAL β some metrics indicate softness"
else:
overall = "FAIL β image appears out of focus"
report = (
f"## Focus Quality Report\n\n"
f"| Metric | Value | Verdict |\n"
f"|--------|-------|---------|\n"
f"| Tenengrad | {tenen:.1f} | {tenen_verdict} |\n"
f"| Laplacian Variance | {lap_var:.1f} | {lap_verdict} |\n"
f"| Normalized Variance | {norm_var:.2f} | {norm_verdict} |\n"
f"| Vollath F4 | {vollath:.0f} | {vollath_verdict} |\n\n"
f"**Overall: {overall}**"
)
# Heatmap overlay
hmap = _focus_heatmap(gray, block=max(32, min(gray.shape) // 16))
fig, axes = plt.subplots(1, 2, figsize=(14, 5))
axes[0].imshow(cv2.cvtColor(image, cv2.COLOR_RGB2BGR) if len(image.shape) == 3 else gray, cmap="gray")
axes[0].set_title("Original", fontsize=12, fontweight="bold")
axes[0].axis("off")
im = axes[1].imshow(hmap, cmap="inferno", interpolation="bilinear")
axes[1].set_title("Focus Heatmap (Laplacian Var per block)", fontsize=12, fontweight="bold")
axes[1].axis("off")
fig.colorbar(im, ax=axes[1], fraction=0.046, pad=0.04, label="Sharpness")
fig.tight_layout()
overlay_img = _fig_to_image(fig)
return overlay_img, report
# ---------------------------------------------------------------------------
# Tab 2 β Illumination Analysis
# ---------------------------------------------------------------------------
def _nine_zone_map(gray: np.ndarray) -> np.ndarray:
h, w = gray.shape
rh, rw = h // 3, w // 3
zones = np.zeros((3, 3), dtype=np.float64)
for r in range(3):
for c in range(3):
patch = gray[r * rh:(r + 1) * rh, c * rw:(c + 1) * rw]
zones[r, c] = patch.mean()
return zones
def analyze_illumination(image: np.ndarray):
if image is None:
return None, "Upload an image first."
gray = _to_gray(image)
h, w = gray.shape
mean_b = float(gray.mean())
std_b = float(gray.std())
min_b = int(gray.min())
max_b = int(gray.max())
dynamic_range = max_b - min_b
# Clipping detection
total_px = h * w
clipped_low = int(np.sum(gray <= 5))
clipped_high = int(np.sum(gray >= 250))
pct_low = 100.0 * clipped_low / total_px
pct_high = 100.0 * clipped_high / total_px
clip_warning = ""
if pct_low > 5:
clip_warning += f" - {pct_low:.1f}% pixels crushed to black (underexposed regions)\n"
if pct_high > 5:
clip_warning += f" - {pct_high:.1f}% pixels blown to white (overexposed regions)\n"
if not clip_warning:
clip_warning = " - No significant clipping detected\n"
# Vignetting: compare center vs corners
cy, cx = h // 2, w // 2
r = min(h, w) // 8
center_mean = float(gray[cy - r:cy + r, cx - r:cx + r].mean())
corner_vals = []
for yr, xr in [(0, 0), (0, w - r * 2), (h - r * 2, 0), (h - r * 2, w - r * 2)]:
corner_vals.append(float(gray[yr:yr + r * 2, xr:xr + r * 2].mean()))
corner_mean = np.mean(corner_vals)
if center_mean > 1e-3:
vig_ratio = corner_mean / center_mean
else:
vig_ratio = 1.0
if vig_ratio < 0.75:
vig_verdict = f"SIGNIFICANT vignetting (corner/center = {vig_ratio:.2f})"
elif vig_ratio < 0.90:
vig_verdict = f"Mild vignetting (corner/center = {vig_ratio:.2f})"
else:
vig_verdict = f"No significant vignetting (corner/center = {vig_ratio:.2f})"
zones = _nine_zone_map(gray)
# Build figure: histogram + zone map
fig, axes = plt.subplots(1, 3, figsize=(18, 5))
# Histogram
axes[0].hist(gray.ravel(), bins=256, range=(0, 256), color="#448AFF", alpha=0.85, edgecolor="none")
axes[0].axvline(mean_b, color="#FF1744", linestyle="--", linewidth=1.5, label=f"Mean={mean_b:.0f}")
axes[0].set_title("Brightness Histogram", fontsize=12, fontweight="bold")
axes[0].set_xlabel("Pixel value")
axes[0].set_ylabel("Count")
axes[0].legend()
# Zone brightness map
im = axes[1].imshow(zones, cmap="YlOrRd", vmin=0, vmax=255, interpolation="nearest")
for r in range(3):
for c in range(3):
axes[1].text(c, r, f"{zones[r, c]:.0f}", ha="center", va="center",
fontsize=14, fontweight="bold",
color="black" if zones[r, c] > 128 else "white")
axes[1].set_title("9-Zone Brightness Map", fontsize=12, fontweight="bold")
axes[1].set_xticks([0, 1, 2])
axes[1].set_xticklabels(["L", "C", "R"])
axes[1].set_yticks([0, 1, 2])
axes[1].set_yticklabels(["T", "M", "B"])
fig.colorbar(im, ax=axes[1], fraction=0.046, pad=0.04)
# Original image
axes[2].imshow(image if len(image.shape) == 3 else gray, cmap="gray")
axes[2].set_title("Original", fontsize=12, fontweight="bold")
axes[2].axis("off")
fig.tight_layout()
vis = _fig_to_image(fig)
report = (
f"## Illumination Analysis\n\n"
f"| Metric | Value |\n"
f"|--------|-------|\n"
f"| Mean Brightness | {mean_b:.1f} / 255 |\n"
f"| Std Dev | {std_b:.1f} |\n"
f"| Min / Max | {min_b} / {max_b} |\n"
f"| Dynamic Range | {dynamic_range} |\n"
f"| Clipped Low (<=5) | {clipped_low} px ({pct_low:.2f}%) |\n"
f"| Clipped High (>=250) | {clipped_high} px ({pct_high:.2f}%) |\n\n"
f"**Clipping:**\n{clip_warning}\n"
f"**Vignetting:** {vig_verdict}\n\n"
f"**Zone Brightness (3x3 grid):**\n"
f"```\n"
f" {zones[0,0]:6.1f} {zones[0,1]:6.1f} {zones[0,2]:6.1f}\n"
f" {zones[1,0]:6.1f} {zones[1,1]:6.1f} {zones[1,2]:6.1f}\n"
f" {zones[2,0]:6.1f} {zones[2,1]:6.1f} {zones[2,2]:6.1f}\n"
f"```"
)
return vis, report
# ---------------------------------------------------------------------------
# Tab 3 β Microscopy Type Detection
# ---------------------------------------------------------------------------
def _histogram_features(gray: np.ndarray) -> dict:
hist = cv2.calcHist([gray], [0], None, [256], [0, 256]).ravel()
hist_norm = hist / hist.sum()
mean_int = float(gray.mean())
std_int = float(gray.std())
median_int = float(np.median(gray))
# Skewness
if std_int > 1e-6:
skew = float(np.mean(((gray.astype(np.float64) - mean_int) / std_int) ** 3))
else:
skew = 0.0
# Peak count (modes)
from scipy.signal import find_peaks
smoothed = ndimage.gaussian_filter1d(hist_norm, sigma=5)
peaks, props = find_peaks(smoothed, height=0.002, distance=20)
n_peaks = len(peaks)
# Edge density
edges = cv2.Canny(gray, 50, 150)
edge_density = float(np.sum(edges > 0)) / (gray.shape[0] * gray.shape[1])
# Fraction of dark pixels
dark_frac = float(np.sum(gray < 40)) / gray.size
bright_frac = float(np.sum(gray > 215)) / gray.size
return {
"mean": mean_int,
"std": std_int,
"median": median_int,
"skew": skew,
"n_peaks": n_peaks,
"edge_density": edge_density,
"dark_frac": dark_frac,
"bright_frac": bright_frac,
"hist_norm": hist_norm,
}
_MODALITIES = ["Brightfield", "Darkfield", "Phase Contrast", "Fluorescence", "Polarized Light"]
def _classify_modality(feats: dict) -> list[tuple[str, float]]:
scores = {m: 0.0 for m in _MODALITIES}
mean = feats["mean"]
std = feats["std"]
skew = feats["skew"]
dark_frac = feats["dark_frac"]
bright_frac = feats["bright_frac"]
edge_density = feats["edge_density"]
n_peaks = feats["n_peaks"]
# Brightfield: medium-high mean, moderate std, near-zero skew, low dark fraction
if 80 < mean < 200:
scores["Brightfield"] += 2.0
if std < 60:
scores["Brightfield"] += 1.0
if abs(skew) < 1.0:
scores["Brightfield"] += 1.0
if dark_frac < 0.15:
scores["Brightfield"] += 1.5
# Darkfield: low mean, high dark fraction, positive skew
if mean < 60:
scores["Darkfield"] += 2.5
if dark_frac > 0.5:
scores["Darkfield"] += 2.0
if skew > 1.0:
scores["Darkfield"] += 1.5
if bright_frac < 0.05:
scores["Darkfield"] += 0.5
# Phase contrast: bimodal histogram, halos (high edge density), medium mean
if n_peaks >= 2:
scores["Phase Contrast"] += 2.0
if edge_density > 0.08:
scores["Phase Contrast"] += 2.0
if 50 < mean < 160:
scores["Phase Contrast"] += 1.0
if std > 40:
scores["Phase Contrast"] += 0.5
# Fluorescence: very dark background, sparse bright spots, very high skew
if mean < 40:
scores["Fluorescence"] += 2.0
if dark_frac > 0.7:
scores["Fluorescence"] += 2.0
if skew > 2.0:
scores["Fluorescence"] += 2.5
if bright_frac > 0.001 and bright_frac < 0.15:
scores["Fluorescence"] += 1.0
# Polarized: high contrast, possible birefringence colors (high std in color)
if std > 50:
scores["Polarized Light"] += 1.0
if n_peaks >= 2:
scores["Polarized Light"] += 0.5
if 40 < mean < 140:
scores["Polarized Light"] += 0.5
total = sum(scores.values())
if total < 1e-6:
return [(m, 1.0 / len(_MODALITIES)) for m in _MODALITIES]
confidences = [(m, scores[m] / total) for m in _MODALITIES]
confidences.sort(key=lambda x: -x[1])
return confidences
def detect_microscopy_type(image: np.ndarray):
if image is None:
return None, "Upload an image first."
gray = _to_gray(image)
feats = _histogram_features(gray)
confidences = _classify_modality(feats)
best_name, best_conf = confidences[0]
# Build histogram plot
fig, axes = plt.subplots(1, 2, figsize=(14, 5))
axes[0].bar(range(256), feats["hist_norm"], color="#448AFF", width=1.0, edgecolor="none")
axes[0].set_title("Intensity Histogram", fontsize=12, fontweight="bold")
axes[0].set_xlabel("Pixel value")
axes[0].set_ylabel("Normalized frequency")
# Confidence bar chart
names = [c[0] for c in confidences]
vals = [c[1] * 100 for c in confidences]
colors = ["#00E676" if i == 0 else "#448AFF" for i in range(len(names))]
bars = axes[1].barh(names[::-1], vals[::-1], color=colors[::-1], edgecolor="none")
axes[1].set_title("Modality Confidence", fontsize=12, fontweight="bold")
axes[1].set_xlabel("Confidence (%)")
axes[1].set_xlim(0, 100)
for bar, v in zip(bars, vals[::-1]):
axes[1].text(bar.get_width() + 1, bar.get_y() + bar.get_height() / 2,
f"{v:.1f}%", va="center", fontsize=10)
fig.tight_layout()
vis = _fig_to_image(fig)
report = (
f"## Microscopy Type Detection\n\n"
f"**Detected: {best_name}** (confidence: {best_conf * 100:.1f}%)\n\n"
f"| Modality | Confidence |\n"
f"|----------|------------|\n"
)
for name, conf in confidences:
marker = " <<" if name == best_name else ""
report += f"| {name} | {conf * 100:.1f}%{marker} |\n"
report += (
f"\n**Image Features:**\n"
f"- Mean intensity: {feats['mean']:.1f}\n"
f"- Std deviation: {feats['std']:.1f}\n"
f"- Skewness: {feats['skew']:.2f}\n"
f"- Histogram peaks: {feats['n_peaks']}\n"
f"- Edge density: {feats['edge_density']:.4f}\n"
f"- Dark pixel fraction: {feats['dark_frac']:.3f}\n"
f"- Bright pixel fraction: {feats['bright_frac']:.3f}\n"
)
return vis, report
# ---------------------------------------------------------------------------
# Tab 4 β Image Enhancement
# ---------------------------------------------------------------------------
def _apply_clahe(img: np.ndarray, clip_limit: float = 3.0, grid_size: int = 8) -> np.ndarray:
if len(img.shape) == 3:
lab = cv2.cvtColor(img, cv2.COLOR_RGB2LAB)
clahe = cv2.createCLAHE(clipLimit=clip_limit, tileGridSize=(grid_size, grid_size))
lab[:, :, 0] = clahe.apply(lab[:, :, 0])
return cv2.cvtColor(lab, cv2.COLOR_LAB2RGB)
else:
clahe = cv2.createCLAHE(clipLimit=clip_limit, tileGridSize=(grid_size, grid_size))
return clahe.apply(img)
def _apply_unsharp(img: np.ndarray, sigma: float = 2.0, strength: float = 1.5) -> np.ndarray:
blurred = cv2.GaussianBlur(img, (0, 0), sigma)
sharpened = cv2.addWeighted(img, 1.0 + strength, blurred, -strength, 0)
return np.clip(sharpened, 0, 255).astype(np.uint8)
def _apply_denoise(img: np.ndarray, h: float = 10.0) -> np.ndarray:
if len(img.shape) == 3:
return cv2.fastNlMeansDenoisingColored(img, None, h, h, 7, 21)
else:
return cv2.fastNlMeansDenoising(img, None, h, 7, 21)
def _apply_white_balance(img: np.ndarray) -> np.ndarray:
if len(img.shape) != 3:
return img
result = img.copy().astype(np.float64)
for c in range(3):
ch = result[:, :, c]
low = np.percentile(ch, 1)
high = np.percentile(ch, 99)
if high - low < 1:
continue
ch = (ch - low) / (high - low) * 255.0
result[:, :, c] = ch
return np.clip(result, 0, 255).astype(np.uint8)
def enhance_image(image: np.ndarray, method: str,
clahe_clip: float = 3.0, clahe_grid: int = 8,
unsharp_sigma: float = 2.0, unsharp_strength: float = 1.5,
denoise_h: float = 10.0):
if image is None:
return None, "Upload an image first."
if method == "CLAHE (Contrast Enhancement)":
enhanced = _apply_clahe(image, clip_limit=clahe_clip, grid_size=int(clahe_grid))
desc = f"CLAHE β clipLimit={clahe_clip}, gridSize={int(clahe_grid)}"
elif method == "Unsharp Mask (Sharpening)":
enhanced = _apply_unsharp(image, sigma=unsharp_sigma, strength=unsharp_strength)
desc = f"Unsharp Mask β sigma={unsharp_sigma}, strength={unsharp_strength}"
elif method == "NLM Denoising":
enhanced = _apply_denoise(image, h=denoise_h)
desc = f"Non-Local Means Denoising β h={denoise_h}"
elif method == "Auto White Balance":
enhanced = _apply_white_balance(image)
desc = "Auto White Balance (percentile stretch per channel)"
else:
enhanced = image
desc = "No method selected"
# Side-by-side
fig, axes = plt.subplots(1, 2, figsize=(14, 6))
axes[0].imshow(image)
axes[0].set_title("Before", fontsize=14, fontweight="bold")
axes[0].axis("off")
axes[1].imshow(enhanced)
axes[1].set_title("After", fontsize=14, fontweight="bold")
axes[1].axis("off")
fig.suptitle(desc, fontsize=12, y=0.02)
fig.tight_layout()
comparison = _fig_to_image(fig)
report = (
f"## Enhancement Applied\n\n"
f"**Method:** {desc}\n\n"
f"| Metric | Before | After |\n"
f"|--------|--------|-------|\n"
)
for label, a, b in [
("Mean", image, enhanced),
("Std", image, enhanced),
]:
ga = _to_gray(a).astype(np.float64)
gb = _to_gray(b).astype(np.float64)
if label == "Mean":
report += f"| Mean Brightness | {ga.mean():.1f} | {gb.mean():.1f} |\n"
else:
report += f"| Std Dev | {ga.std():.1f} | {gb.std():.1f} |\n"
# Sharpness comparison
ga = _to_gray(image)
gb = _to_gray(enhanced)
lap_before = cv2.Laplacian(ga, cv2.CV_64F).var()
lap_after = cv2.Laplacian(gb, cv2.CV_64F).var()
report += f"| Laplacian Var (sharpness) | {lap_before:.1f} | {lap_after:.1f} |\n"
return comparison, enhanced, report
# ---------------------------------------------------------------------------
# Gradio UI
# ---------------------------------------------------------------------------
css = """
.gr-block { border-radius: 12px !important; }
footer { display: none !important; }
"""
with gr.Blocks(title="Microscopy CV Toolkit", css=css, theme=gr.themes.Base()) as demo:
gr.Markdown(
"# Microscopy CV Toolkit\n"
"Classical computer-vision tools for microscopy image quality analysis. "
"No ML models β pure OpenCV, NumPy, SciPy. Upload any microscopy image to get started."
)
with gr.Tabs():
# ---- Tab 1: Focus Quality ----
with gr.Tab("Focus Quality"):
gr.Markdown(
"Measures image sharpness using four complementary metrics. "
"The heatmap shows per-block focus quality across the field of view."
)
with gr.Row():
with gr.Column(scale=1):
focus_input = gr.Image(label="Upload Image", type="numpy")
focus_btn = gr.Button("Analyze Focus", variant="primary")
with gr.Column(scale=2):
focus_output = gr.Image(label="Focus Heatmap", type="numpy")
focus_report = gr.Markdown()
focus_btn.click(
fn=analyze_focus,
inputs=[focus_input],
outputs=[focus_output, focus_report],
)
# ---- Tab 2: Illumination Analysis ----
with gr.Tab("Illumination Analysis"):
gr.Markdown(
"Checks brightness distribution, clipping, dynamic range, vignetting, "
"and displays a 9-zone brightness map for Kohler illumination assessment."
)
with gr.Row():
with gr.Column(scale=1):
illum_input = gr.Image(label="Upload Image", type="numpy")
illum_btn = gr.Button("Analyze Illumination", variant="primary")
with gr.Column(scale=2):
illum_output = gr.Image(label="Analysis", type="numpy")
illum_report = gr.Markdown()
illum_btn.click(
fn=analyze_illumination,
inputs=[illum_input],
outputs=[illum_output, illum_report],
)
# ---- Tab 3: Microscopy Type Detection ----
with gr.Tab("Microscopy Type Detection"):
gr.Markdown(
"Auto-detects imaging modality based on histogram shape, intensity statistics, "
"contrast, and edge density. Works best on standard preparations."
)
with gr.Row():
with gr.Column(scale=1):
type_input = gr.Image(label="Upload Image", type="numpy")
type_btn = gr.Button("Detect Type", variant="primary")
with gr.Column(scale=2):
type_output = gr.Image(label="Analysis", type="numpy")
type_report = gr.Markdown()
type_btn.click(
fn=detect_microscopy_type,
inputs=[type_input],
outputs=[type_output, type_report],
)
# ---- Tab 4: Image Enhancement ----
with gr.Tab("Image Enhancement"):
gr.Markdown(
"Apply classical enhancement techniques. Adjust parameters and compare side-by-side."
)
with gr.Row():
with gr.Column(scale=1):
enhance_input = gr.Image(label="Upload Image", type="numpy")
enhance_method = gr.Radio(
choices=[
"CLAHE (Contrast Enhancement)",
"Unsharp Mask (Sharpening)",
"NLM Denoising",
"Auto White Balance",
],
value="CLAHE (Contrast Enhancement)",
label="Enhancement Method",
)
with gr.Accordion("Parameters"):
clahe_clip = gr.Slider(0.5, 10.0, value=3.0, step=0.5, label="CLAHE Clip Limit")
clahe_grid = gr.Slider(2, 16, value=8, step=1, label="CLAHE Grid Size")
unsharp_sigma = gr.Slider(0.5, 5.0, value=2.0, step=0.5, label="Unsharp Sigma")
unsharp_strength = gr.Slider(0.5, 5.0, value=1.5, step=0.5, label="Unsharp Strength")
denoise_h = gr.Slider(1.0, 30.0, value=10.0, step=1.0, label="Denoise Strength (h)")
enhance_btn = gr.Button("Enhance", variant="primary")
with gr.Column(scale=2):
enhance_comparison = gr.Image(label="Before / After", type="numpy")
enhance_result = gr.Image(label="Enhanced Image (downloadable)", type="numpy")
enhance_report = gr.Markdown()
enhance_btn.click(
fn=enhance_image,
inputs=[enhance_input, enhance_method,
clahe_clip, clahe_grid,
unsharp_sigma, unsharp_strength,
denoise_h],
outputs=[enhance_comparison, enhance_result, enhance_report],
)
gr.Markdown(
"<center style='color:#888;font-size:0.85em;'>"
"Microscopy CV Toolkit | Pure OpenCV, no ML models | "
"<a href='https://huggingface.co/spaces/Laborator/microscopy-cv-toolkit'>HuggingFace Space</a>"
"</center>"
)
if __name__ == "__main__":
demo.launch()
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