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IQBio 2017 Syllabus

Quantitative Optical Microscopy Lab for IQ Bio 2017

  • Instructor: Joe Dragavon
  • Office: JSCBB C315
  • Email: joseph.dragavon@colorado.edu
  • Phone: 303.345.6988
  • Cell: 720.934.2933
  • Wet Lab Location (week 1): JSCBB C350, BioFrontiers Advanced Light Microscopy Core
  • Matlab Discussion Location (week 2): JSCBB B331
  • Presentation Location (week 3): JSCBB B331
  • Wet Lab and Matlab Discussion hours: Thursdays, 1:00 - 4:00pm
  • General Office Hours: Thursdays, 1:00 - 4:00pm
  • General Office Hours Location: JSCBB C315
  • Matlab Assistant: Jian Tay

 

Course Description:

There are 4 labs that will accompany the course Quantitative Optical Microscopy taught by Professor Joel Kralj. The labs will be used to reinforce the lectures of the class and to provide significant hands on imaging experience. Further, the labs will be used to greatly enhance each student’s capabilities and understanding of Matlab.

The labs will follow the following pattern:

Week 1: Image/data acquisition on a microscope (JSCBB C350).

Week 2: Office hours to go over the image analysis using Matlab (JSCBB B331).

Week 3: mFiles due and individual presentations about the lab (JSCBB B331).

Week 1 will involve three groups of 2 or 3. Ideally the members of each group will take turns piloting the microscope but this is not necessary. After Lab 1 (more later), the hours of Week 1 will be flexible and fit the needs of the group. My goal is for each wet lab to take less than the planned 3 hours, but this is not certain. After the data are acquired I would like each person to spend the next several days (until office hours) working on analyzing the data in Matlab individually. The ability to use and create analysis scripts in Matlab will greatly set each of you apart from others in your field, so it is important that each of you take the time to learn the program now. This will also be beneficial for your independent research careers.

The Week 2 office hours will be used to ensure each person has functioning image analysis code for the given lab. During these hours I will ask that each of you be present. This will be the time where those of you that were able to create a functioning script will help those that were not able. In science, especially interdisciplinary programs, it is important to be able to educate others on how you approached a problem and how you acquired your results. Interdisciplinary research also requires that you often work in teams. In such a setting the ability to communicate across domains is essential. Ideally Week 2 will promote such an environment.

Each person will turn in their own mFiles the day before the Week 3 session (so Wednesday). Three to four people will present their mFiles to the group. I will provide guidance as to what kind of plots/figures/movies I would like your mFile to generate. The presentations will be around 20 minutes in length. The purpose of presenting the mFiles is to give you practice explaining what your code does both in lay and advanced terms, since you will most likely experience this throughout your careers. Overall, I am not too concerned on whether or not you are able to have a complete mFile, but significant effort should be given. Further, you can utilize the group to learn better ways to proceed with your code and/or to discuss issues and difficulties that you had. My goal is to have each of you present at least twice over the four labs.

 

Grading:

Participation: 70%

Reports: 30%

Graduate school is not the same as your undergraduate studies. The idea of the class is to provide you with tools that will aid you in your future as well as expose you to a variety of topics that may assist you down the road. 

 

Brief outlines of the labs:

Lab 1.  Microscope training and Point Spread Function Determination

Lab 1 will most likely be the most intensive lab out of the four. Prior to Lab 1 I will complete the training for each of you on three different microscopes: the Nikon A1R laser scanning confocal; the Nikon NSTORM (for widefield use only); and the Nikon Spinning Disc Confocal. If necessary, we can refine the training during the first lab. Each microscope uses the same operating software with slight variations and their configurations are a bit different. Here, you will acquire 3D images of sub-resolution multispectral beads suspended in a gel using multiple objectives and different fluorescence intensities. For the image analysis you will determine the shape and size (full-width half-maximum) for at least 10 beads and at multiple different focal planes. You can report the average and standard deviation for each. For those more advanced in their Matlab, you will plot the z-profile of the PSF under each condition, and describe what this shape implies.

Lab 2. Photobleaching

Lab 2 will examine the ability of light to permanently destroy fluorophores (photobleaching). Light sources are very powerful, with the capability of inducing blindness if one is not careful. However, in fluorescence microscopy, one must use a light source in order to induce a fluorescent response. In Lab 2, the effect of light exposure onto fluorescent samples will be investigated, and the resulting photoresponse determined. In live cell imaging, too much light exposure can lead to phototoxicity, which often results in cell death. In lab 2, you will expose fluorescent samples to various light intensities through multiple objectives. The role of oxygen in photobleaching will also be investigated. Further, by acquiring a large image around your central illuminated field of view, you will be able to calculate the actual exposure area and compare this to your field of view. Using time-lapse imaging, you will record the fluorescence intensity as a function of time, and relate that to illumination intensity and the objective. Using Matlab you will plot out and fit each intensity curve.

Lab 3. Complex Particle Tracking

Light microscopy is used to observe a vast array of moving particles, including endosomes, organelles, bacteria, viruses, lipid nanoparticles, micelles, proteins, ... The localization of these minute features with respect to cellular structure is very informative. More powerful is the ability to track the position and velocity of each over time. By doing so, one can begin to infer cellular transportation mechanisms, diffusion characteristics, and protein interactions, amongst others. Beyond tracking minute particles, the same technical mechanisms can be used to track macroscopic entities such as individual cells or even the movement of whole animals. Needless to say, the ability to track multiple fluorescent entities over time is an essential tool for today's researchers.

While it would be fun and interesting to perform particle tracking on the translocation of membrane proteins to the nucleus, or injection of virulence factors from infectious bacteria, in Lab 3 your are going to start with something more simple. You will perform two observations: 1) the natural Brownian movement of fluorescent beads in suspension and 2) track the movement of fluorescent beads within a simple flow chamber. While this may appear to not relate, many of the same techniques will be developed. You will need to identify the initial location of each particle within your field of view, track that particle over time, determine its trajectory and its velocity.

Lab 4. Dynamic Live Cell Imaging

The final lab in the series will attempt to unify many of the Matlab and imaging skills that you have developed so far. Within Lab 4, you will image the dynamic movement of cells over time. More specifically, you will monitor the contraction and relaxation of adult cardiomyocites or the calcium activity within HeLa cells (think line profiles). Both of these processes are rapid and dynamic, but can be easily altered through intense illumination (phototoxicity). Over time the cells themselves will move, so you will need to update your region of interests on a regular basis (think particle tracking). Here you will report out various physiological values as a function of time (i.e., cells length, fluorescence intensity, pulse frequency, pulse duration,...). Basically this is a fun lab that you will allow you to pursue multiple routes of analysis.

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