Skip to content. | Skip to navigation

Personal tools

Navigation

You are here: Home / Imaging / IQBio / Lab 4: Live Cell Imaging of Tetrahymena

Lab 4: Live Cell Imaging of Tetrahymena

Lab 4: Live Cell Imaging of Tetrahymena

Welcome to the last wet lab of this course. The final wet lab aims encompass much that you have learned over the previous three labs: how to operate a microscope; how to minimize the effects of light on a sample; and how to track moving objects. This weeks lab will include the observation of live tetrahymena. Tetrahymena are a single-celled organism that are commonly found in pond water. What makes them so fun, for us at least, is that they can propel themselves at a very rapid rate using their significant number of cilia. This should provide a challenge to you with respect to the tracking portion of your lab as they will bump into each other and swim over each other. You will be monitoring two variants of the tetrahymena: a wild-type strain and a mutant variant. The cilia of the mutant variant should be much stiffer than the wild type, reducing their ability to move.

Useful Reading and Watching

Tetrahymena are a common model organism for biology so there is a lot out there to read and watch. For our purposes not too much information is needed. The links below will provide you with some background information:

  • As always, a decent place to start is wikipedia (click here)
  • Youtube also has some fun videos (click here)

To Do:

Like the previous lab, the data acquisition for this lab should not take long.  You only need to acquire a couple of time lapses.

I will prepare for you slides containing

  1. Live wild-type tetrahymena
  2. Live mutant tetrahymena

You will acquire two time lapse videos, one for each live variant of the tetrahymena. I would recommend recording at a reasonable frame rate, around 30 fps, for 1000 frames. The idea is to have a video with with enough moving tetrahymena in it that your tracking code will be challenged by interacting and overlapping tetrahymena. You can use ImageJ/Fiji to crop the video (in x,y and/or time) to reduce the file size necessary for Matlab. You should use the 10x objective for this part. The tetrahymena will not be fluorescently labeled, so you will need to acquire the images using brightfield illumination.

To acquire clean brightfield images you need to make sure that he condenser head is appropriately positioned and that the microscope is Köhler aligned (click here and here for a nice overview. The second one has an awesome comb over in it.). For the sake of time, I will do this for you on each microscope, but it is nice to know what Köhler alignment is and what it is used for (click here for some information about DIC imaging).

A1R

Remember, laser scanning confocal microscopy is inherently slow so you will want to use the resonant scanners (click here for a nice overview). To enable the resonant scanner, select Resonant in the upper right hand corner of the Nikon Elements window. You will hear a high-pitch vibrating sound (it sounds like a mosquito - please do not hit the microscope). To enable the DIC imaging, make sure the TD option of the 4Ch + DIC optical configuration is selected. You need to use at least one laser line for this to work, so select either the 488 or the 561 laser as well. The collected data will have two channels: the laser and the DIC. You can extract the DIC channel to reduce your file size by right-clicking on the channel tab (below the image) and select extract the selected channel.

NSTORM

The NSTORM has two camera options: An Andor 897 Ultra EMCCD and a Hamamatsu Flash4.0 sCMOS. The sCMOS has a much larger chip so the field of view is greater. Also, its pixels are much smaller (6.5 microns vs 16 microns). I will let you decide which camera to use. To choose, launch Elements using the appropriate startup icon (labeled Andor and Hamamatsu, respectively). If you go with the Hamamatsu, I highly recommend a 4x4 bin size, otherwise your file sizes will be enormous. Once you have selected a camera, set your exposure time to 30ms. This will allow you to acquire images at approximately 30 frames per second. Remember, the Andor is on the Left camera port and the Hamamatsu is on the Right camera port be sure that your light path is in the correct orientation. To image the cells you will want to select the Brightfield preset. This will use the diascopic lamp (DIA) to illuminate your sample. Be sure to adjust the intensity of the lamp to improve your image quality.

Spinning Disc

Once the software has started, set the exposure time to 30ms from the drop down menu. Adjust your DIA Intensity and gain accordingly. The Frame rate will be nearly 30 frames per second.

All

  1. Trace the movement of your cells.
  2. Since you know the frame rate and the size of the image, calculate the average linear velocity of each tetrahymena (report as average and standard deviation). Compare the linear velocity of each variant. Is there a difference? Here are a few links that may be of help (click here and here for two examples in Matlab. The second may be more relevant).
  3. For those of you who are interested in a challenge, try to image the live tetrahymena using the 100x Oil objective under DIC. If you crop your field of view down using either the Camera ROI (NSTORM, Spinning Disc) or a very small scan area and few steps (A1R), you should be able to approach 400 frames per second. If you can find a tetrahymena that is stationary (try looking near an air bubble or the edges), you might be able to observe the cilia moving.
This is Pacific Theme