# Image Analysis
# Week 1

## Objectives of the lab:
The goal this week is to introduce you to one method for quantifying the kind of visual data you might collect during the course of lab this semester:
1) Use image analysis software, ImageJ/FIJI, to quantify the distribution of colocalization of proteins in transgenic mammalian NIH3T3 cells
2) Identify the potential strengths and weaknesses of using this type of analysis in this system

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### In order to open your images and add scale bars you will need to download the following program: ImageJ/FIJI. (https://imagej.net/Fiji#Downloads)

*Please download the FIJI software as it comes preloaded with plugins necessary for opening your images).*

You will also need to download the test files from the BI227 google drive, ER-RFP test.czi and Emerals-ELP1 test.czi found on the course Google Drive.

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**To help you to get a bit more familiar with the software,**
### In FIJI: Start by opening your image file.

**Then,**
1) To zoom in (or out) just hit the “+” (or “-“) key.
2) You can select the “Hand” (selected in the screenshot below) from the FIJI controls to move around in your image to find the cell you are interested in.


*NOTE: Your image is in Black and White (Greyscale).*


**Let’s make the RFP “Red”:**

To pseudocolor your image you will need to select a new Lookup Table:

1) Select “Image” --> “Lookup Tables” --> “Red”


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### ***Open your second test image (Emerald-ELP1 test.czi) and make the Emerald-ELP1 signal Green.***


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### ***Now save your images as Tiff files:***

1) File --> Save As --> TIFF, add to name

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### ***Let’s merge the images:***
To Merge the images:

1) Image --> Color --> Merge Channels
2) Select ER-RFP test.czi from the dropdown for C1 (red).
3) Select Emerald-ELP1 test.czi from the dropdown for C2 (green).
4) Make sure that “Create Composite” is checked as well as “Keep Source Images”


*Your Composite image should look like this:*


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## • What is Green in the image? _______________

## • What is Red in the image? _________________

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### ***Let’s add a scale bar to the image:***

*When you add the scale bar to your image it will be added to the image at the original scale (so you may not be able to see it if you are zoomed in).*

**To add scale bars to your images:**
1) Open your image file.
2) On the menu bar go to: Analyze --> Tools --> Scale bar
3) Set the width in microns (µm) or pixels of your scale bar to: 500
(*Image scale data is embedded in the metadata of image files. You will only be given the option to set the scale in microns by the program if that information is available in the metadata of the file.*)

4) Set the location of your scale bar to “upper right”.
5) Set the color to “white”.
6) Be sure that you have selected the “Overlay”.


7) Save the image in whatever file format is required (.jpg or .tiff are recommended for most applications).


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## ***Let’s crop the image:***

To crop the image to the region you are interested in:

1) zoom in on you cell of interest
2) select the “square” tool (selected in the screenshot below)


3) click on your image and drag to select your rectangular region of interest (yellow box)
4) Go to: Image --> Crop (The program will crop the image to the selected region)


5) Save the image in whatever file format is required (.jpg or .tiff are recommended for most applications).

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Other features of FIJI:

Image --> Color --> Channels Tool
Color: turn on and off individual colors
Composite: combine colors (turn on and off individual colors)
Can go back to Colors and alter color etc. (Brightness/Contrast/Look Up Table)

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ASSIGNMENT:

1) Take a picture of NIH3T3 Cells co-transfected with ER-RFP and Emerald-ELP1-25.
Take a picture of one field of the control cells (DMEM only) and one field of the cells treated with Brefeldin A at 20X.
Save the images with the name of the plasmid as well as your initials and the “Control” or “Experimental”
Using FIJI, pseudo color your images RFP--> Red and Emerald --> Green
Make a merge image with a scale bar
2) Make a figure:
Your figure should have 6 panels: one for ER-RFP, one for Emerald-ELP1-25 and one merged panel for your control and your experimental (Brefeldin A-treated) cells. All panels should be labeled.
The merged image should have a scale bar
Each panel should have an inset of a single cell cropped from the larger image.
Include a figure legend
3) Results: Answer the following:
Describe what you see in your images.
Based on your hypothesis from last week, do your results match your predictions? Support your hypothesis? Why or why not?
Each student should turn in a .docx or .pdf file containing your figure, figure legend and answers before the next lab session.

*Data collection may be completed in groups. However, data analysis and interpretation should be completed individually. Thus all graphs and interpretations of results should be your own. Failure to abide by this will be treated as a violation of college academic integrity policies.*