Output

Saved in the destination folder was a large number of files in .tif, .txt and .xls format displaying all the data extracted from the images.

The .tif files showed the segmentation that had been carried out in a visual format; a representative of these is shown in Figure 4.22 below.

Figure 4.21. Shows the binary image files of the Border cell cluster generated by the pre-processing steps. (A) Shows the same frame before and after part of the epithelium has been masked, indicated by the red arrows. (B) Shows the same frame before and after part of the protrusion has been filled in, indicated by the red arrows. Note only one object can be present in each frame for the macro to function correctly.

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The excel files in the output contained most of the useful information about the cluster migration and the protrusions. Each of the files was separated based on specific information about the cluster, for example data such speed, cluster shape, duration etc. This was

important when initially looking at differences between genotypes, to see if there was an

Figure 4.22. Shows representative visual output of the extension analysis at four consecutive time points

(2mins apart). Here, the cluster is travelling from left to right. (A) The cell body is labelled in blue and the protrusions in red; the .tif file was used to determine if the image segmentation has taken place correctly. The small circle indicates the centre of the cluster and is used to calculate migration distance. (B) Shows each of the protrusions measured in each time frame. Each protrusion was measured from the centre of the cluster. All protrusions were counted, resulting in data on the actual number of protrusions; it did not distinguish new from old protrusions. (C) Shows the number of protrusions counted as single protrusions. The cell body was not present, only the protrusions. These were counted and identified as persistent or new and labelled singularly or in multiple frames.

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impairment.

Other outputs gave information with regards to the extensions such as the speed of the cluster, extension area and length, at each of the separate sections, front, back and sides. It also described the persistence, angle of each of the individual extensions. The output also contained information on a frame-by-frame basis, including the direction of the extensions at each time point. These were represented as absolute and binary numbers for each direction in each frame, allowing direct frame-by-frame analysis to be carried out if necessary. Additional spreadsheets were also given with more detailed data about the maximum length and area of individual protrusions.

Using all the data combined enabled a clear accurate picture of the migration process, which could be used to compare the behaviour in different genotypes.

Additional Macro Uses

In addition to extension analysis, information about the effects of the protrusions on the migration process could also be generated using the X-velocity and Max/Sum macros. This used the pre-processed movies generated to determine what effect the extension size, area and persistence etc. related to the cluster velocity in the X axis. It was found to give a more informative view on the role protrusions play in the migration process, especially if no differences were seen in protrusion number or direction. It used the velocities that the centre of mass travelled, to determine if any protrusions were productive in forwards movement. The macro essentially reuses data generated by the extension analysis and displays it in a different way.

Another operation of the macro was to collate all of the data files together. This was especially useful when more than one image file was being used to represent movement

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within a single egg chamber, e.g., when adjustments were made in the focus during imaging. It allowed the results files for all movie parts and genotypes to be collated into one excel spreadsheet, making analysis easier.

Results

This results section is going to focus on the tracking aspects of the macro, and not the protrusion analysis, which has been briefly described earlier and will be discussed in more detail in Chapter 5, Section 5.3.

border cell clusters from wild type egg chambers (c306 GAL4, UAS Lifeact-GFP) were imaged and tracked for the first half of migration. The results from the automated macro show that the border cell cluster travelled on average 1.42 µm/minute, based on the average velocity from one frame to the next, using XY values only. These values are much higher than seen previously in XY (Section 4.3.2)with slbo GAL4, UAS mCD8-GFP in the first half of migration (0.61µm/min). As all imaging techniques were the same, the differences in speeds are either a result of the genotype or the tracking method used. To determine the cause of the

discrepancy, c306 GAL4, UAS Lifeact-GFP border cell clusters were tracked in XY and XYZ using the MTrackJ plugin to compare the migration rates using the same genotype. In XY, the tracked migration rate was very similar to the macro at 1.39 µm/minute. The rate in XYZ was higher (1.68 µm/minute), again most likely because of drift in the Z direction.

Therefore the differences seen in migration rate can be attributed to the genotype of the reporter strains and not due to discrepancies in the tracking methods.

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Figure 4.23. Shows graphs comparing the migration speed calculated by the automated macro and MtrackJ. (A) Shows the migration speed during the first half of migration in two different genotypes, analysed by two different methods, automated and MtrackJ.

Genotypes are as follows: c306 GAL4, UAS-LifeactGFP, an actin based reporter under the control of c306 which enables expression in the follicle and polar cells. slbo GAL4, UAS-mCD8-GFP a membrane based reporter under the control of Slbo expressing in the follicle cells but not the polar cells. (B)

Shows the migration speeds of the same genotype (c306 GAL4, UAS-LifeactGFP) using three different methods. Error bars represent the standard deviation of the mean.

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