Carnegie Mellon University Comparing Images of Protein Distributions Excel Worksheet

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I need a screenshot for every step and explain every step and for question 2 please use excel

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Homework 11 Comparing Images of Protein Distributions Assigned April 17, 2020, Due April 24, 2020 (revision 2) Download the following files from the Human Protein Atlas site into the same folder: https://images.proteinatlas.org/7244/18_H7_1_blue_red_green.jpg https://images.proteinatlas.org/7244/18_H7_2_blue_red_green.jpg https://images.proteinatlas.org/40802/509_F7_1_blue_red_green.jpg https://images.proteinatlas.org/40802/509_F7_2_blue_red_green.jpg https://images.proteinatlas.org/71138/1433_G4_1_blue_red_green.jpg https://images.proteinatlas.org/71138/1433_G4_2_blue_red_green.jpg The first two files are for protein “ACAA1”, the second two are for “VPS35”, and the third two are for protein “CEP19”. 1. Do the following for each image and give the commands that you used (see note after question 3). a. Open the image in ImageJ, split the colors and select the blue window (DNA image). Set the threshold to the default value chosen by ImageJ. b. Apply the threshold to generate a binary image (containing just the nuclei). c. Fill Holes. d. Duplicate the resulting blue (binary) image and invert it. e. Use the Image Calculator to mask the green image with the binary blue image (use AND). This will set pixels that are not in the nuclei to zero. f. Select the resulting image and set the threshold to 50 (but to not apply it, which will convert it to binary). g. Use Analyze Particles with a minimum size of 20 (be sure to Clear the results before each run). h. Save the Results to a CSV file (choose a useful name, like “18_H7_1_nuclear.csv”). i. Repeat steps e-h but use the inverted mask (this will find particles that are NOT in the nuclei; change the name to something like “18_H7_1_cyto.csv”) 2. Analysis and Comparison Open the CSV files in a plotting program (such as Excel or Python). Submit the following. a. Calculate the following ratios for each file: i. the number of objects in the nucleus and the cytoplasm ii. the average size of objects in the nucleus and the cytoplasm. iii. the average of the mean intensity per object in the nucleus and cytoplasm b. b. Calculate the following ratios for the pair of images for each protein (_1_ c. and _2_): i. the number of objects in the nucleus ii. the average size of objects in the nucleus iii. the average of the mean intensity of objects in the nucleus 3. Interpretation Describe and justify your tentative conclusions about: d. a. the general reproducibility of results for the two images of each protein. e. b. the size, mean intensity and spatial distribution of the objects for the three proteins, and how your conclusions are influenced by the reproducibility. f. c. whether the results agree with existing information (e.g., from UniProt or ENSEMBL) about the organelle/subcellular location of these three proteins (include a copy of the information you use and its source). g. Extra credit (15 points): In place of the written description for question 1, you may submit an ImageJ Macro (as a plain text file) that does some or all of the steps. In order to receive any extra credit, the macro must 1. run without errors on our grading system (after adjusting locations for files) (it is better to submit a working macro for a subset of the steps than to submit a nonworking macro for all of the steps), and h. 2. contain comments (lines beginning with //) for at least one-third of the commands in the macro.
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1

Homework 11
Comparing Images of Protein Distributions
Assigned April 17, 2020, Due April 24, 2020 (revision 2)
Download the following files from the Human Protein Atlas site into the same folder:
https://images.proteinatlas.org/7244/18_H7_1_blue_red_green.jpg
https://images.proteinatlas.org/7244/18_H7_2_blue_red_green.jpg
https://images.proteinatlas.org/40802/509_F7_1_blue_red_green.jpg
https://images.proteinatlas.org/40802/509_F7_2_blue_red_green.jpg
https://images.proteinatlas.org/71138/1433_G4_1_blue_red_green.jpg
https://images.proteinatlas.org/71138/1433_G4_2_blue_red_green.jpg
The first two files are for protein “ACAA1”, the second two are for “VPS35”, and the
third two are for protein “CEP19”.
1. Do the following for each image and give the commands that you used (see note after
question 3).
a. Open the image in ImageJ, split the colors and select the blue window (DNA
image). Set the threshold to the default value chosen by ImageJ.

2

b. Apply the threshold to generate a binary image (containing just the nuclei).

3

4

c. Fill Holes.

5

d. Duplicate the ...


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I was struggling with this subject, and this helped me a ton!

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