# A virtual laboratory on cell division
This week in lab you will conduct an analysis of the stages of cell division using real data provided by the Allen Institute for Cell Science in Seattle, WA and freely available on their website https://www.allencell.org. Mitosis is a dynamic process; however, you will analyze still images which will mimic the kind of data collection and analysis performed by both basic science (https://bmcvetres.biomedcentral.com/articles/10.1186/s12917-017-1030-7) and commercial (https://patents.google.com/patent/US20180095070A1/en) laboratories.

**The experimental logic can be thought of as follows:**

Suppose you are interested in the behavior of an organism; let’s say zebras. You are interested in how much time they spend eating, sleeping, running, conducting “zebra” play and so on. You could make a movie of individual animals, but this might be very inefficient. Getting one zebra’s worth of data for one day would take you a whole day of filming. Instead, you could take random images of a herd of many zebras and then count the zebras in each image doing specific things. This might be more efficient than filming one zebra at a time, but you’d have to think a bit about how to interpret the data. For one thing, if an event was very short (say only a minute or two), you might miss those events by taking random pictures, unless you took a lot of images and looked at a lot of zebra pictures. Now imagine that to gather more data you hire someone to take images for you, but when you analyze the images you discover that there were no sleeping zebras in the images. You might conclude that zebras do not sleep, or that they sleep for very short periods of time. On the other hand, you might conclude that your assistant was sleeping instead of taking pictures while the zebras were asleep. So, there are lot of things to consider when designing the experiment and interpreting the data.
### Learning Goals:
* Students will understand the events of mitosis and the cellular processes that occur during different stages of cell division.
* Students will understand how data on dynamic cellular events are collected and analyzed.
* Students will appreciate how cell biologists address experimental questions.

### Background

Cell division is the process by which the contents of a cell are duplicated (DNA) and segregated (DNA, proteins, organelles, etc.) to generate two daughter cells. Each cell cycle has two major phases: Interphase and M phase. Interphase is the period of the cell cycle during which cells grow and replicate their DNA. This is the longest phase of the cell cycle (the phases of the cell cycle in Figure 18-1 from your textbook and the panel included below are NOT drawn to scale) and can be further broken down into 3 stages: G1, S and G2. G1 is the gap between M phase and S phase, DNA replication is confined to S phase and G2 is the gap between S phase and M phase. During M phase, the nucleus divides in a process called mitosis; then the cytoplasm divides, in a process called cytokinesis. Like Interphase, Mitosis can be broken down into several stages: prophase, prometaphase, metaphase, anaphase and telophase.

###### Cell Division and the Cell Cycle (Panel 18–1, part 1; Alberts, Bruce, et al. Essential Cell Biology. 5th ed., Garland Publishing, 2018.)

During M phase, the nucleus divides in a process called mitosis; then the cytoplasm divides, in a process called cytokinesis. Like Interphase, Mitosis can be broken down into several stages: prophase, prometaphase, metaphase, anaphase and telophase.

For your reference, included below is a description of the chromatin and microtubule dynamics associated with each of these stages and cytokinesis from your textbook:

###### The principal stages of m phase in an animal cell (Panel 18–1, part 3-7; Alberts, Bruce, et al. Essential Cell Biology. 5th ed., Garland Publishing, 2018.)

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## Assignment
For lab this week, you will be analyzing a number of images containing pictures of cells stained to reveal their DNA and mitotic spindles, and you will use your knowledge of the events of mitosis to count the cells at each stage of the cell cycle: Interphase, Prophase, Prometaphase, Metaphase, Anaphase and Telophase.

You have been provided with a [link](https://drive.google.com/drive/folders/1q_m4zIdDprkd2huBMJxtOMYa20w5V9X7?usp=sharing) to a folder with an Excel spreadsheet containing images with examples of cells at each stage of the cell cycle. The first column in your Excel file will contain links to fields of cells on the Allen Institute for Cell Science web site. The links are labeled “Image 1”, “Image 2” and so on. Click on each link and an image should open in your default web browser.


Working alone, you will count all the cells having enough of the nucleus in the image to identify its stage, enter the numbers and then tabulate the results from the images. You will then generate a graph, table or chart that shows your results.

# **Before you begin collecting data:**
**1. Create and discuss a hypothesis regarding what you expect to see in terms of the *mitotic stages* of the cell cycle (Prophase, Prometaphase, Metaphase, Anaphase and Telophase).**
* Consider what you know about what happens at each stage of cell division, and think about how long completing those actions might that require?

***Do you think all stages will have equal counts? Why or why not?*** If not, which stage of mitosis do you think will be most, least, etc. common in your data and why?

*Note: Most of the cells in your images will be in interphase because this phase makes up the largest part of the cell cycle. The assignment is to discuss the stages of mitosis. Please do not tell me your hypothesis is that most cells will be in interphase.*

### **Your initial hypothesis/rationale should be turned in to me along with the remainder of the assignment before lab next week.**

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### Counting cells:

**2. Working alone, from the images provided, select (at least) 20 images from which you will count the cells.**
* Try to select images in which there is at least one mitotic cell so that you have the best chance of seeing what the distribution of cells in various stages of mitosis looks like.

**3. Use the 3D cell viewer (instructions provided below) to identify the stages of the cells in your images.**

**4. For each image, once you have counted the cells, enter the total number of cells at each stage of the cell cycle in the table in the row for that image.**
* You should count all of the cells in the images for which enough of the nucleus is visible in the image to identify the cell's stage. This includes all cells in interphase as well as the stages of mitosis.

**5. Look at your results. You may wish to/need to determine the average, mean or total cell counts as well as the standard deviation or standard error in each of your categories inorder to better appreciate your results.**
* *Do your results support or refute your initial hypothesis?*

**6. Consider the best way of presenting your results (graph, table or chart) to your audience (me!).**
* What method of presenting your data will make your results easiest for your audience to interpret?
* Will any of these means of presenting your results do a particularly good job of highlighting the data that is most supportive of your conclusions?

**7. Once you have chosen the best way of presenting your results (graph, table or chart); create a document that includes:**

1) The initial hypothesis and rationale/discussion supporting this hypothesis you generated *before* counting your cell.
2) Your graph, table or chart that shows your results.
3) A discussion of your results that addresses whether or not the data you collected support your original hypothesis and why or why not you think that happened.

**Please submit your final results document via the "Lab 6 Assignment" on the course Moodle site before coming to lab next week.**
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### Instructions for using the 3D cell viewer:

The 3D Cell Viewer is a powerful web-based tool for accessing and visualizing cell data hosted at allencell.org. The viewer has many features and you are welcome to explore them at your leisure. However, completion of the exercise will only require you to know how to use 4 parts of the viewer and what they do.

### Overview of the Cell Viewer:

* The **panel on the left** is used to access different image sets. You will **NOT** need it for this exercise.


* The **main view pane**l is where you will see the fields of cells for analysis. The Z slider allows you to change the focus of the virtual microscope and increase or decrease the depth of focus (the amount of each cell that is seen). I recommend increasing the depth of focus, as described below.


* The **right-hand panel** controls how each field of cells appears. There are many options, but I recommend just using the Alpha-tubulin checkbox to show and hide microtubules. Some images may also look better if you adjust the brightness using the left-hand part of the brightness slider.


## Following are a series of steps that I suggest using to make the view of the cells easier to interpret.

* The first thing I do is open the space between the limiting arrows to show a bit more of the cells. (Red circle)

*From this...*
*To this...*

* It may also be helpful to grab the leftmost handle on the intensity slider and move it slightly to the right (green circle).


* It may be helpful to toggle viewing of the microtubules, using the alpha-tubulin checkbox

*Like this…*


***With microtubules shown, you can see this is a mitotic cell – but which stage?***


* Sometimes, you can page through the images with the “play arrow” (blue arrow) and get a better idea of what the cell looks like:


* Most cells can be identified using the XY images that appear in the viewer by default. Occasionally, you may find a cell whose stage can’t be identified easily using the XY view. You can choose to ignore them, but you might want to give looking at the cell from the side a try. To do this, click on “XZ” at the top right of the screen, instead of “XY”. Then click on the play button to move through the layer of cells.



*There is it on the right! This cell is in metaphase!*

## Counting and analysis

It is recommended that you look at the DNA staining and microtubules separately to begin analyzing each image by checking and unchecking the appropriate checkboxes on the right-hand side of the 3D Cell Viewer. This will help you rapidly identify mitotic cells, since their microtubules and DNA are different than that of interphase cells. There are also relatively few cells undergoing cell division in each field, so they are easy to count. However, interphase cells are numerous. In some images you will encounter only a few cells, and it will be easy to count all the interphase cells by just looking at the image. However, other images may have a lot of cells, and it can be hard to keep track of them all. Below are some solutions:

* **A low tech solution** is to put a piece of transparent plastic over your computer screen and then use a marker to check off each interphase cell as you count it. Believe it or not, this used to be the main way that scientists counted things in digital images in the past.

* **Snipping tool for Windows.** If you are using your own computer and have a Windows PC, you can use the built in “Snipping Tool” application. See here (https://support.microsoft.com/en-us/help/4027213/windows-10-open-snipping-tool-and-take-a-screenshot) for instruction on how to access the snipping tool if you are not familiar with it. It is my understanding that the tool is available for Windows 7 through the current version.


* * To use the snipping tool, start the application and then chose “New Snip” (Cntrl-N). Then draw a box around the image of the cells. Use the marking tools and options to set up a thick pen in a color that’s easy to see. Click (mark) each cell as you count it and then write the total number of counted cells down in the appropriate column of your Excel file.

* **Preview for Macintosh Computers.** On a Mac computer (the computers available in the lab), you can do something similar using the screen shot commands and the built in Preview application.


* * First, press Command-Shift-4. A cross-hair cursor will appear, and you can click and drag to select the area of your screen you wish to capture. The function will save an image on your desktop or other location that you designate. Double clicking on the image file will open it in Preview. You can make marks on the image to keep track of the cells you count using the pen tool, and you can change the thickness and color of the marks using the tool options shown by the green arrows in the image below.

* **ImageJ/FIJI.** Finally, if you are interested in using a different image analysis program, consider using FIJI. (If you have not yet downloaded the program it is available here: [https://imagej.nih.gov/ij/](https://imagej.nih.gov/ij/)). You can find a discussion of how to use the “point tool” for marking and counting image features here ([https://imagej.nih.gov/ij/docs/tools.html](https://imagej.nih.gov/ij/docs/tools.html))

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### References:

Alberts, Bruce, et al. Essential Cell Biology. 5th ed., Garland Publishing, 2018.

E. Shelden, E. Offerdahl, G. Johnson. 2019 A Virtual Laboratory on Cell Division Using a Publicly-Available Image Database. [https://qubeshub.org/community/groups/coursesource/publications?id=2631&v=1CourseSource](https://qubeshub.org/community/groups/coursesource/publications?id=2631&v=1CourseSource). Accessed October 2019.

Various. 2018. 3D Cell Viewer, on Allen Institute for Cell Science. https://www.allencell.org/3d-cell-viewer.html. Accessed October 2019.