Interpretation and Image Processing

The next stage of the process asks whether information about the experimental conditions

(proteins and their concentrations) should be automatically interpreted from the name of

the directory ’Image Conditions’. If so it expects the format of the directory name ’Image

Conditions’ to be in the form:

‘imaging date protein 1 concentration protein 1 name protein 2 concentration ....

temperature chamber imaging number’

i.e.’140618 24uM 12percTub488 100nM EB1mCherry T25C 005’.

Due to the above-prescribed format of the image files, the user was asked if they would

like the directory names to be automatically deciphered. If yes, then the directory name

was separated into parts delimited by an underscore (‘ ’). The first and the last two

entries in the directory name are automatically assigned. The remainder are paired up

in the format concentration-name. There needs to be at least the same number of pairs

as the number of channels selected. The user will be asked to assign each channel to a

concentration-name pair. If the file name was not right for automatic deciphering, or you

decide not to automatically decipher the file names, the fields are still generated but are

left blank. If these settings are not the same for all samples, there was an option later on

to manually edit each field.

The only image processing required at this point was that which needs to be applied

to the original microscope images. The imaging system was checked for chromatic

aberration/shift between excitation in the 488 nm and 561 nm channels. Fluorescently

labelled beads were imaged and the corresponding images analysed. A 2-Dimensional

3.3. IDENTIFICATION OF LABELLED MICROTUBULES

channel, a 2-dimensional Gaussian was fitted to the neighbourhood of each identified

point from the 488 nm channel. The offset (distance and direction) was calculated

between corresponding Gaussians. There was no discernible pattern detected in the

measured offset between the fitted centres of the Gaussians between the two channels.

As such it was concluded that the microscope set-up did not suffer from any significant

chromatic shift or aberration requiring adjustment. There was no testing done between

the 488 nm, or 561 nm channels, and the 640 nm channel. This was because the 640 nm

channel is reserved for stabilised seeds and was only used as a generalised reference

point. As such a couple of pixels deviation between the imaged position in the 640 nm

channel and the observed position with relation to the 488 nm and 561 nm channels was

not considered a problem.

Spatial drift through time is occasionally observable in obtained images, especially in the

first few minutes a chamber is on the microscope. The main method normally used to

correct this type of error is stack registration. Stack registration normally involves whole

pixel shifting to minimise the lateral displacement between time points in a temporal stack.

More elaborate routines are also capable of dealing with a slight rotational displacement,

and sub pixel shifts to better align images. The accuracy of these methods is determined

by how well the algorithm can identify key features in each image slice. Due to the lack of

fixed features in these images, standard stack registration routines were ignored. Instead

each microtubule has it’s own fixed control point in the ‘dark’ seed join. This junction

between the GMP-CPP seed and the dynamic microtubule can be used as a fixed point

that is independent of temporal drift. By fitting a dual ended GEF (Section 3.4.1, Equation

3.4) it was possible to calculate microtubule length independent of drift.

Obtained microscope images do contain a distinctive background pattern due to uneven

illumination. By taking a reference image using a coloured plastic slide, we get

a representative image of our background illumination. The raw images are then

normalised by the representative background image as follows.

To background correct, the reference image was binned by averaging 16x16 pixel blocks,

smoothed and then re-expanded to be 1024x1024 by/using bi-cubic interpolation. As the

3.3. IDENTIFICATION OF LABELLED MICROTUBULES

manipulation, background correction was done in MATLAB. For every image stack,I, the

following transformation was applied:

I(t) = ((I(t)−3000)1/S)∗max(S) ∀t

whereis the hadamard (or element-wise, point-wise) product, andtis the time slice of

the image stack, andSis our reference image. The values ofSare sufficiently large that

no special case was considered for values ofSclose to zero.

In situations where the reference image was not obtained, it is constructed from the

images available from the experiment. To do this, 10 uniformly spaced time slices are

extracted from each image stack. The intensities for each extracted image are normalised

to have a mean of 2000 after subtracting the camera offset of 3000. The median is then

calculated from all of the normalised images to create the reference imageS.

Each transformed image is saved in the same directory as it was loaded from, but it

has ’ back’ appended to its file name. The user inputted information and information for

automatic deciphering the image file names are stored in a structure called Image. The

structure is saved to ’Parent Directory/Results/Image.mat’.