def mapping_automatic(
file_tetra,
tf1_matrix,
show=0,
f=None,
bg=None,
tol=1): #'E:\CMJ trace analysis\\autopick\\tetraspeck.tif'
#print(tf1_matrix)
root, name = os.path.split(file_tetra)
save_fn = os.path.join(root, name[:-4] + '-P.map')
if os.path.isfile(save_fn):
#print('loading automatic mapping')
P = np.zeros((4, 4))
Q = np.zeros((4, 4))
tm = np.zeros((3, 3))
with open(save_fn, 'r') as infile:
for ii in range(0, 16):
P[ii // 4, ii % 4] = float(infile.readline())
for ii in range(0, 16):
Q[ii // 4, ii % 4] = float(infile.readline())
tm[0, 2] = P[0, 0]
tm[0, 1] = P[0, 1]
tm[0, 0] = P[1, 0]
tm[1, 2] = Q[0, 0]
tm[1, 1] = Q[0, 1]
tm[1, 0] = Q[1, 0]
tm[2, 2] = 1
transformation_matrixC = np.linalg.inv(tm)
else:
#print('çalculating automatic map')
# Open image
if type(file_tetra) == str:
image_tetra = tiff.imread(file_tetra)
else: #assume you are passing an image
image_tetra = file_tetra
# default for 16 bits 50000, for 8 bits 200 (=256*50000/64000)
if f == None:
if np.max(image_tetra) > 256:
f = 50000
else:
f = 200
if bg == None:
# take two different backgrounds, one for donor, one for acceptor channel
sh = np.shape(image_tetra)
thr_donor = get_threshold(image_tetra[:, 1:sh[0] // 2])
thr_acceptor = get_threshold(image_tetra[:, sh[0] // 2:])
bg = np.zeros(sh)
bg[:, 1:sh[0] // 2] = thr_donor
bg[:, sh[0] // 2:] = thr_acceptor
image_tetra = remove_background(image_tetra.astype(float), bg)
# image_tetra=image_tetra.astype(float)-bg
# image_tetra[image_tetra<0]=0
image_tetra = image_tetra.astype(np.uint16)
position1 = []
position2 = []
# left, right, enhanced left and enhanced right image for keypoint detection, adapt f
while np.shape(position1)[0] < 50 or np.shape(position2)[0] < 50:
# while loop to lower f and increase the number of spots found
l, r, l_enh, r_enh = enhance_blobies(image_tetra, f, tol)
gray1 = l_enh
gray2 = r_enh
# initialize the AKAZE descriptor, then detect keypoints and extract
# local invariant descriptors from the image
detector = cv2.AKAZE_create()
(kps1, descs1) = detector.detectAndCompute(gray1, None)
(kps2, descs2) = detector.detectAndCompute(gray2, None)
position1 = cv2.KeyPoint_convert(kps1)
position2 = cv2.KeyPoint_convert(kps2)
f = f * 0.9
gray1 = l_enh
gray2 = r_enh
# initialize the AKAZE descriptor, then detect keypoints and extract
# local invariant descriptors from the image
detector = cv2.AKAZE_create()
(kps1, descs1) = detector.detectAndCompute(gray1, None)
(kps2, descs2) = detector.detectAndCompute(gray2, None)
position1 = cv2.KeyPoint_convert(kps1)
position2 = cv2.KeyPoint_convert(kps2)
if 0: #automatic mapping based on matching features
print("keypoints: {}, descriptors: {}".format(
len(kps1), descs1.shape))
print("keypoints: {}, descriptors: {}".format(
len(kps2), descs2.shape))
# Match the features
#this part is working properly according to the overlayed images
bf = cv2.BFMatcher(cv2.NORM_HAMMING)
matches = bf.knnMatch(descs1, descs2, k=2) # typo fixed
# Apply ratio test
pts1, pts2 = [], []
size_im = np.shape(gray1)
heigh, widt = np.shape(gray1)
for m in matches: # BF Matcher already found the matched pairs, no need to double check them
pts1.append(kps1[m[0].queryIdx].pt)
pts2.append(kps2[m[0].trainIdx].pt)
pts1 = np.array(pts1).astype(np.float32)
pts2 = np.array(pts2).astype(np.float32)
transformation_matrixC, mask = cv2.findHomography(
pts2, pts1, cv2.RANSAC, 20)
A = pts1[0:len(matches):int(len(matches) / 15)]
im3 = cv2.drawMatchesKnn(gray1,
kps1,
gray2,
kps2,
matches[1:100],
None,
flags=2)
if show:
plt.figure(1)
plt.imshow(im3)
plt.title('mapping matching keypoints')
plt.show
else:
# find matching points (no other features) based on the manual mapping
dst = cv2.perspectiveTransform(
position1.reshape(-1, 1, 2),
np.linalg.inv(tf1_matrix)) #reshape needed for persp.transform
dst = dst.reshape(-1, 2)
dist = np.zeros((len(position2), len(dst)))
for ii in range(0, len(position2)):
for jj in range(0, len(dst)):
dist[ii, jj] = np.sqrt((position2[ii, 0] - dst[jj, 0])**2 +
(position2[ii, 1] - dst[jj, 1])**2)
pts1, pts2 = [], []
for ii in range(0, len(position2)):
jj = np.where(dist[ii, :] == min(dist[ii, :]))
if dist[ii, jj] < 4:
pts1.append(position1[jj])
pts2.append(position2[ii])
pts1 = np.array(pts1).astype(np.float32)
pts2 = np.array(pts2).astype(np.float32)
transformation_matrixC, mask = cv2.findHomography(
pts2, pts1, cv2.RANSAC, 20)
# produce an image in which the overlay between two channels is shown
array_size = np.shape(gray2)
imC = cv2.warpPerspective(gray2, transformation_matrixC,
array_size[::-1])
#cv2.imshow("transformed ", im4)
if show:
plt.figure(11, figsize=(18, 9))
plt.subplot(1, 6, 6),
AA = (gray1 > 0) + 2 * (imC > 0)
plt.imshow((gray1 > 0) + 2 * (imC > 0),
extent=[0, array_size[1], 0, array_size[0]],
aspect=1)
#plt.colorbar()
plt.title('automatic align \n#spots overlap {:d}'.format(
np.sum(AA == 3)))
plt.show()
plt.pause(0.05)
#saving to .map file
P = np.zeros((4, 4))
tm = np.linalg.inv(transformation_matrixC)
P[0, 0] = tm[0, 2]
P[0, 1] = tm[0, 1]
P[1, 0] = tm[0, 0]
Q = np.zeros((4, 4))
Q[0, 0] = tm[1, 2]
Q[0, 1] = tm[1, 1]
Q[1, 0] = tm[1, 0]
with open(save_fn, 'w') as outfile:
for ii in range(P.size):
outfile.write('{0:4.10e}\n'.format(np.hstack(P)[ii]))
for ii in range(Q.size):
outfile.write('{0:4.10e}\n'.format(np.hstack(Q)[ii]))
#print('done with automatic align')
return transformation_matrixC
def set_background_and_transformation(self):
"""
sum 20 image, find spots& background. Then loop over all image, do background subtract+ extract traces
:return:
"""
_, hdim, vdim, n_images = read_one_page(self.image_fn, pageNb=0)
im_array = np.dstack([(read_one_page(self.image_fn,
pageNb=ii)[0]).astype(float)
for ii in range(20)])
im_mean20 = np.mean(im_array, axis=2).astype(int)
bg = rollingball(im_mean20)[1]
im_mean20_correct = im_mean20 - bg
im_mean20_correct[im_mean20_correct < 0] = 0
threshold = get_threshold(im_mean20_correct)
im_mean20_correct = remove_background(im_mean20_correct, threshold)
#note: optionally a fixed threshold can be set, like with IDL
# note 2: do we need a different threshold for donor and acceptor?
root, name = os.path.split(self.image_fn)
pks_fn = os.path.join(root, name[:-4] + '-P.pks')
if os.path.isfile(pks_fn):
ptsG = []
dstG = []
with open(pks_fn, 'r') as infile:
for jj in range(0, 10000):
A = infile.readline()
if A == '':
break
ptsG.append([float(A.split()[1]), float(A.split()[2])])
A = infile.readline()
dstG.append([float(A.split()[1]), float(A.split()[2])])
ptsG = np.array(ptsG)
dstG = np.array(dstG)
pts_number = len(ptsG)
# load
else:
pts_number, label_size, ptsG = analyze_label.analyze(
im_mean20_correct[:, 0:int(vdim / 2)])
# there should be different options:
# donor: im_mean20_correct[:,0:vdim//2]
# acceptor: im_mean20_correct[:,vdim//2:]
# donor+acceptor
dstG = cv2.perspectiveTransform(
ptsG.reshape(-1, 1, 2),
np.linalg.inv(self.mapping._tf2_matrix)) #transform_matrix))
dstG = dstG.reshape(-1, 2)
dstG = np.array([[ii[0] + 256, ii[1]] for ii in dstG])
#saving to pks file
with open(pks_fn, 'w') as outfile:
for jj in range(0, pts_number):
pix0 = ptsG[jj][0]
pix1 = ptsG[jj][1]
outfile.write(
' {0:4.0f} {1:4.4f} {2:4.4f} {3:4.4f} {4:4.4f} \n'.
format((jj * 2) + 1,
pix0,
pix1,
0,
0,
width4=4,
width6=6))
pix0 = dstG[jj][0]
pix1 = dstG[jj][1]
outfile.write(
' {0:4.0f} {1:4.4f} {2:4.4f} {3:4.4f} {4:4.4f} \n'.
format((jj * 2) + 2,
pix0,
pix1,
0,
0,
width4=4,
width6=6))
# ALL_Gaussians_ptsG=np.zeros((11,11,pts_number))
# ALL_Gaussians_dstG=np.zeros((11,11,pts_number))
ALL_GAUSS = makeGaussian(11, fwhm=3, center=(5, 5))
# for jj in range(0,pts_number):
# xpix = ptsG[jj][1]
# ypix = ptsG[jj][0]
#
# xpix_int = int(xpix)
# ypix_int = int(ypix)
# ALL_Gaussians_ptsG[:,:,jj]=makeGaussian(11, fwhm=3, center=(ypix - ypix_int + 5, xpix - xpix_int + 5))
#
# xf2 = dstG[jj][1] # approach3
# yf2 = dstG[jj][0] # approach3
# xf2_int = int(xf2) # approach3
# yf2_int = int(yf2) # approach3
# ALL_Gaussians_dstG[:,:,jj]= makeGaussian(11, fwhm=3, center=(yf2 - yf2_int + 5, xf2 - xf2_int + 5)) # approach3
#
return bg, threshold, pts_number, dstG, ptsG, im_mean20_correct, n_images, hdim, ALL_GAUSS
def mapping_manual(file_tetra,
show=0,
f=None,
bg=None,
tol=1): #'E:\CMJ trace analysis\\autopick\\tetraspeck.tif'
root, name = os.path.split(file_tetra)
save_fn = os.path.join(root, name[:-4] + '-P.coeff')
transformation_matrixC = np.zeros((3, 3))
transformation_matrixC[2, 2] = 1
if os.path.isfile(save_fn):
#print('loading manual transformation coeff')
#import from .coeff file:
with open(save_fn, 'r') as infile:
transformation_matrixC[0, 2] = float(infile.readline()) - 256
transformation_matrixC[0, 0] = float(infile.readline())
transformation_matrixC[0, 1] = float(infile.readline())
transformation_matrixC[1, 2] = float(infile.readline())
transformation_matrixC[1, 0] = float(infile.readline())
transformation_matrixC[1, 1] = float(infile.readline())
return transformation_matrixC
else:
global points_right # globals in nested function, need to be defined here as well
global ii
# Open image
if type(file_tetra) == str:
image_tetra = tiff.imread(file_tetra)
else: #assume you are passing an image
image_tetra = file_tetra
# default for 16 bits 50000, for 8 bits 200 (=256*50000/64000)
if f == None:
f = 50000
#print(f)
if type(bg) == type(None):
# take two different backgrounds, one for donor, one for acceptor channel
sh = np.shape(image_tetra)
thr_donor = get_threshold(image_tetra[:, 1:sh[0] // 2])
thr_acceptor = get_threshold(image_tetra[:, sh[0] // 2:])
bg = np.zeros(sh)
bg[:, 1:sh[0] // 2] = thr_donor
bg[:, sh[0] // 2:] = thr_acceptor
image_tetra = remove_background(image_tetra.astype(float), bg)
# image_tetra=image_tetra.astype(float)-bg
# image_tetra[image_tetra<0]=0
image_tetra = image_tetra.astype(np.uint16)
position1 = []
position2 = []
# left, right, enhanced left and enhanced right image for keypoint detection, adapt f
while np.shape(position1)[0] < 50 or np.shape(position2)[0] < 50:
# while loop to lower f and increase the number of spots found
l, r, l_enh, r_enh = enhance_blobies(image_tetra, f, tol)
gray1 = l_enh
gray2 = r_enh
# initialize the AKAZE descriptor, then detect keypoints and extract
# local invariant descriptors from the image
detector = cv2.AKAZE_create()
(kps1, descs1) = detector.detectAndCompute(gray1, None)
(kps2, descs2) = detector.detectAndCompute(gray2, None)
position1 = cv2.KeyPoint_convert(kps1)
position2 = cv2.KeyPoint_convert(kps2)
f = f * 0.9
# now we start to move the markers in the right part, first an overall move
from pynput.keyboard import Key, Listener
import matplotlib.pyplot as plt
import time
# points_left=[[ii, ii+10] for ii in range(11)]
# points_right=points_left.copy()
fig = plt.figure(10, figsize=(18, 9))
ax1 = fig.add_subplot(1, 2, 1)
ax1.imshow(gray1)
ax1.set_title('click on bright spots in the left image')
ax2 = fig.add_subplot(1, 2, 2, sharex=ax1, sharey=ax1)
ax2.imshow(gray2)
points_left = plt.ginput(
4
) # adapted it to be 4, I expected 3 to work, to match with IDL code
points_right = points_left.copy()
[
ax1.plot(xx,
yy,
markersize=10,
c='w',
marker='o',
fillstyle='none') for xx, yy in points_left
]
xp = [xx for xx, yy in points_right]
yp = [yy for xx, yy in points_right]
line2, = ax2.plot(xp,
yp,
markersize=10,
c='w',
marker='o',
fillstyle='none',
linestyle='none')
fig.canvas.draw()
fig.canvas.flush_events()
plt.pause(1)
ax1.set_title('')
ax2.set_title(
'move point with arrows, press esc when it matches the location in other channel'
)
def on_release(key):
global points_right
global ii
#print(points_right)
if key == Key.up:
points_right[ii] = [
points_right[ii][0], points_right[ii][1] + 1
]
# points_right= [ [xx,yy+1] for xx,yy in points_right]
elif key == Key.down:
points_right[ii] = [
points_right[ii][0], points_right[ii][1] - 1
]
# points_right= [ [xx,yy-1] for xx,yy in points_right]
elif key == Key.right:
points_right[ii] = [
points_right[ii][0] + 1, points_right[ii][1]
]
# points_right= [ [xx+1,yy] for xx,yy in points_right]
elif key == Key.left:
points_right[ii] = [
points_right[ii][0] - 1, points_right[ii][1]
]
# points_right= [ [xx-1,yy] for xx,yy in points_right]
if key == Key.esc or (key == Key.up) or (key == Key.down) or (
key == Key.right) or (key == Key.left):
# Stop listener
return False
matches = len(points_left)
for ii in range(matches):
xp_new = 0 * xp
yp_new = 0 * yp
ax1.set_xlim(points_left[ii][0] - 50, points_left[ii][0] + 50)
ax1.set_ylim(points_left[ii][1] - 50, points_left[ii][1] + 50)
line1 = ax1.plot(points_left[ii][0],
points_left[ii][1],
markersize=10,
c='y',
marker='+',
fillstyle='none')
line2b = ax2.plot(points_right[ii][0],
points_right[ii][1],
markersize=10,
c='y',
marker='+',
fillstyle='none')
fig.canvas.draw()
fig.canvas.flush_events()
plt.pause(0.1)
while xp != xp_new or yp != yp_new:
xp = [xx for xx, yy in points_right]
yp = [yy for xx, yy in points_right]
line2.set_xdata(xp)
line2.set_ydata(yp)
fig.canvas.draw()
fig.canvas.flush_events()
plt.pause(0.1)
# Collect events until released
with Listener(on_release=on_release) as listener:
listener.join()
xp_new = [xx for xx, yy in points_right]
yp_new = [yy for xx, yy in points_right]
# print(np.int(xp_new[ii]),np.int(yp_new[ii]))
line1[-1].remove()
line2b[-1].remove()
fig.canvas.draw()
fig.canvas.flush_events()
plt.pause(0.1)
# now loop over each spot to update its position in a zoomed in window
# get the points_right to match syntax pts1, pts2
points_right = np.array(points_right)
points_left = np.array(points_left)
transformation_matrixC, mask = cv2.findHomography(
points_right, points_left, cv2.RANSAC, 20)
# produce an image in which the overlay between two channels is shown
array_size = np.shape(gray2)
imC = cv2.warpPerspective(gray2, transformation_matrixC,
array_size[::-1])
#cv2.imshow("transformed ", im4)
if show:
plt.figure(11, figsize=(18, 9))
plt.subplot(1, 1, 1)
plt.subplot(1, 6, 1),
plt.imshow(gray1,
extent=[0, array_size[1], 0, array_size[0]],
aspect=1)
plt.title('green channel')
plt.subplot(1, 6, 2),
plt.imshow(gray2,
extent=[0, array_size[1], 0, array_size[0]],
aspect=1)
plt.title('red channel')
plt.subplot(1, 6, 3),
plt.imshow(imC,
extent=[0, array_size[1], 0, array_size[0]],
aspect=1)
plt.title('red transformed')
plt.show()
plt.subplot(1, 6, 4),
A = (gray1 > 0) + 2 * (gray2 > 0)
plt.imshow(A,
extent=[0, array_size[1], 0, array_size[0]],
aspect=1)
# plt.colorbar()
plt.title('unaligned #(yellow) \nspots overlap {:d}'.format(
np.sum(A == 3)))
plt.subplot(1, 6, 5),
AA = (gray1 > 0) + 2 * (imC > 0)
plt.imshow((gray1 > 0) + 2 * (imC > 0),
extent=[0, array_size[1], 0, array_size[0]],
aspect=1)
#plt.colorbar()
plt.title('manual align \n#spots overlap y{:d}'.format(
np.sum(AA == 3)))
plt.show()
plt.pause(0.05)
plt.figure(10), plt.close()
#saving to .coeff file:
with open(save_fn, 'w') as outfile:
outfile.write('{0:4.10e}\n'.format(transformation_matrixC[0, 2] +
256))
outfile.write('{0:4.10e}\n'.format(transformation_matrixC[0, 0]))
outfile.write('{0:4.10e}\n'.format(transformation_matrixC[0, 1]))
outfile.write('{0:4.10e}\n'.format(transformation_matrixC[1, 2]))
outfile.write('{0:4.10e}\n'.format(transformation_matrixC[1, 0]))
outfile.write('{0:4.10e}\n'.format(transformation_matrixC[1, 1]))
# print(transformation_matrixC)
# print('done with manual align')
return transformation_matrixC