# Recursive Image Alignment and Statistics

If it is awake, it will move.  And if it moves, your images are likely to do it too.  This means that, almost surely, the first step in processing your data will be to align (or register) all the images in the sequence. This is such a basic problem that there are probably as many solutions as readers of this blog, and I invite you to share yours  in the comments below.

Below is our approach, which seems to work reasonably well.  (At some later time I will discuss the possibility of doing this alignment in real-time as images are being acquired.)

Suppose we have a stack of images indexed $x_i$ to align.  We will write a function that takes as input this set of images and returns a corresponding set of transformations  $t_i$ that, when applied to each of the images, results in their alignment.  The function also returns the mean image after the alignment $\mu$.

The general strategy consists in splitting the sequence in two sets, A and B.  The set A  holds the first half of the sequence and B the second half.  Then, we recursively call our alignment function to register the set in A, which returns $(\mu_A, {t_i^A})$, and then the one in B, which returns $(\mu_B, {t_i^B})$.

The final step is to find the transformation required to align the mean image of A with the mean image of B, $t_{AB}$, and return the mean after the alignment $\mu = (|A|t_{AB}\mu_A + |B| \mu_B )/(|A| + |B|)$ and the transformations $(t_{AB} \odot t_i^A, t_j^B)$.  Here, $\odot$ means the composition of the transformations.  That is, the transformation $t_{AB}$  is applied to all the transformations of the set A.  The recursion ends when the function is called with a single image, in which case it returns the image itself as the mean and the identity transformation.

It turns that you can not only compute the mean recursively, but all the other moments as well. Thus, in a single pass through the data, the algorithm can align the images, and compute the mean, second, third and fourth moments of the aligned sequence.  From there it is easy to compute the images corresponding to the variance, skewness and kurtosis of the distributions of the signal at each location.

Note the algorithm works for any class of transformations, but it helps to keep it simple.  If you are using a resonant, two-photon microscope then, because of the relatively high rate of imaging the distortions from one frame to the next are well approximated by a simple translation.  Feel free to try other transformations, but beware of the fact that you will pay a heavy prize in processing time.

The resulting code is only a few lines long:

function r = sbxalign(fname,idx)

if(length(idx)==1)

S = sparseint; % sparse interpolant for...
A = squeeze(A(1,:,:))*S; % spatial correction of green channel

r.m{1} = A; % mean
r.m{2} = zeros(size(A)); % 2nd moment
r.m{3} = zeros(size(A)); % 3rd moment
r.m{4} = zeros(size(A)); % 4th moment

r.T = [0 0]; % no translation (identity)
r.n = 1; % # of frames

else

idx0 = idx(1:floor(end/2)); % split into two groups
idx1 = idx(floor(end/2)+1 : end);

r0 = sbxalign(fname,idx0); % align each group
r1 = sbxalign(fname,idx1);

[u v] = fftalign(r0.m{1},r1.m{1}); % align their means

for(i=1:4) % shift mean image and moments
r0.m{i} = circshift(r0.m{i},[u v]);
end

delta = r1.m{1}-r0.m{1}; % online update of the moments (read the Pebay paper)
na = r0.n;
nb = r1.n;
nx = na + nb;

r.m{1} = r0.m{1}+delta*nb/nx;
r.m{2} = r0.m{2} + r1.m{2} + delta.^2 * na * nb / nx;
r.m{3} = r0.m{3} + r1.m{3} + ...
delta.^3 * na * nb * (na-nb)/nx^2 + ...
3 * delta / nx .* (na * r1.m{2} - nb * r0.m{2});
r.m{4} = r0.m{4} + r1.m{4} + delta.^4 * na * nb * (na^2 - na * nb + nb^2) / nx^3 + ...
6 * delta.^2 .* (na^2 * r1.m{2} + nb^2 * r1.m{2}) / nx^2 + ...
4 * delta .* (na * r1.m{3} - nb * r0.m{3}) / nx;

r.T = [(ones(size(r0.T,1),1)*[u v] + r0.T) ; r1.T]; % transformations
r.n = nx; % number of images in A+B

end


The algorithm lends itself to parallel processing by enclosing the recursive calls (lines 22 and 23) in a parfor loop. However, the gains (or not) will depend largely on the disk array you have.

The optimal translation (with single pixel resolution) is found using FFTs:

function [u,v] = fftalign(A,B)

N = min(size(A));

yidx = round(size(A,1)/2)-N/2 + 1 : round(size(A,1)/2)+ N/2;
xidx = round(size(A,2)/2)-N/2 + 1 : round(size(A,2)/2)+ N/2;

A = A(yidx,xidx);
B = B(yidx,xidx);

C = fftshift(real(ifft2(fft2(A).*fft2(rot90(B,2)))));
[~,i] = max(C(:));
[ii jj] = ind2sub(size(C),i);

u = N/2-ii;
v = N/2-jj;



You can allow for more complex transformations by using Matlab’s  imregtform but, I warn you once more, you will have to wait substantially longer. Remember that in a typical experiment you will be aligning tens of thousands of images (18,000 in the example below.)

So here is an example of the estimated translations along with the movement of the ball, which is obtained via a camera synchronized to the frames of the microscope. Not surprisingly, when the mouse runs there is relative movement detected in the estimated translation of the alignment.

Also note that the movement in at this magnification has an extent of about 5-6 pixels, this is comparable to the radius of a cell body. A typical result of aligning the images using this method is shown below, where you can still discern individual processes after a 20 min imaging session.

The outcome of the alignment is the mean image, along with the moments and the sequence of transformations.  We do not store a new set of aligned images, as doing so would require much space. Instead, next time we read the image from the raw data file, if the alignment data is present,  we can apply the alignment transformation so we can read the aligned stack.

What do you do?  Do you have a different method to share?