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IQBio 2016 Syllabus

Quantitative Optical Microscopy Lab for IQ Bio 2016

  • 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
  • Guest Lecture Location (week 3): JSCBB B331
  • Wet Lab and Matlab Discussion hours: Thursdays, 1:00 - 4:00pm
  • Office Hours: Thursdays, 1:00 - 4:00pm
  • Office Hours Location: JSCBB C315
  • Matlab Assistant: Jian Wei Tay

 

Course Description:

There are 5 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 B224).

Week 3: Reports due and guest lecture that will reinforce the lab (JSCBB B331).

Week 1 will involve three groups of 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 is 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.

During Week 3 each group will turn in a report summarizing your results and their implications. I will do my best to provide clear guidance as to what I am looking for each lab.  Week 3 will further be accompanied by a guest lecture that will reinforce the lab and the lectures to date.

 

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. During 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. Each microscope uses the same operating software with slight variations and their configurations are a bit different. The lab itself will take place at the end of the training.  Here, you will acquire images of sub-resolution multispectral beads using multiple objectives, step sizes (for the A1R), bin factors (for the NSTORM and Spinning Disc Confocal), and different colors (ex, blue, green, and red). For each condition you will do a high-resolution z-stack. For the image analysis you will determine the shape and size (full-width half-maximum) for at least 10 beads and three colors at a central focal plane.  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. Physiological Effects of Light

Lab 2 will examine the effects of light on living samples. 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 cyanobacteria 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. Fluorophores (in live or dead cells) can also photobleach, which is the phenomenon whereby a fluorophore is fundamentally destroyed when exposed to an overabundance of light. In lab 2, you will expose fluorescent cyanobacteria to various light intensities through multiple objectives. These bacteria have the interesting property that their fluorescence will increase with light exposure. This increase will be used to gauge the physiological impact of light onto the sample. 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.

Coming Soon

Lab 3. Particle Tracking

Lab 4. Live Cell Tracking

Lab 5. Analysis of Large Data Sets

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