How would you set up the SP8 to capture all five fluorescent proteins, using 2 sequential scan settings? How would you improve on this with 3 sequential scan settings?
BSC 411. Confocal take-home Final Exam. Due 12-8-20.
The Written Final is in 2 parts. For the first, answer the questions here. For the second, submit the Photoshop Homework (more to be posted).
Answer each using a concise but specific and suitably detailed essay. Type your answer below each question (you will submit the Word file containing both the question and answer). Use complete sentences. You may discuss the material with other 411 students, but make sure your writing is entirely your own, or it will not be credited. Email it to kaedwar@ilstu.edu with 411-confocal Final in the Subject line.
Answers will be graded on accuracy, clarity, completeness, use of specifics and examples to support your assertions, and originality.
Discouraged: using brief general statements, lack of specifics, use of undefined terms, and copying phrases from elsewhere.
- Consider the worm Brainbow line described in Lec.6 PPT. How would you set up the SP8 to capture all five fluorescent proteins, using 2 sequential scan settings? How would you improve on this with 3 sequential scan settings?
- Valid/invalid image manipulation. You are preparing your images for publication using Photoshop. Describe examples that fit these three scenarios, giving the specific Photoshop commands used and why they fit the description:
(A) you make an image adjustment that is useful and for which there should be no objection.
(B) you make an adjustment, and have to consider carefully whether it is acceptable by the journal.
(C) you make an adjustment that would help you “sell” your conclusions, but would be considered absolutely unacceptable.
Answer 4 of the next 7 questions; you may omit three.
- You wish to image live cells, and want to be able to clearly see a dim signal from GFP. What are the two best ways to increase the signal? How is your answer more relevant to live, rather than fixed, cells?
- For antibody staining experiments, (a) what is the experimental advantage of having the ability to completely remove the antigen of interest from your sample? (b) What is the experimental use of performing a “no primary” control staining?
- Why was the discovery of GFP technology so important? What problem did it solve, and why was it so rapidly and fully adopted (when antibody staining works “just fine” for seeing individual proteins?)
- Discuss at least 5 major parameters that one should consider when selecting a marine fluorescent protein for use as a protein tag in live microscopy.
- Explain the basic mechanism and concept of ‘fluorescence lifetime‘ and how this is used for FLIM on our SP8 system. Why is a pulsed laser required? How is it that a FLIM-based image can distinguish two dyes that excite and emit at essentially the same wavelength?
- Viral detection in saliva samples with the confocal. Suppose all the world’s Taq supply was gone, making PCR testing impossible, but you need to continue testing students on campus for the presence of SARS-CoV-2. How would you do this on the SP8 system? You do not need to give very specific details like scan settings or reagent names, but the general plan should make sense, and the controls should be spelled out.
- For one of the assigned iBio seminars listed in Lec. 1/slide 4, provide a clear succinct outline. Use one you did not use on the Midterm.
——–
PHOTOSHOP HOMEWORK 1. This is due by end of Finals Week.
Find any image with some obvious reddish, greenish, and bluish regions.
Open in Photoshop.
Make it JPG format.
Reduce size if needed so it is no more than 400KB.
Save each of the color channels as its own grayscale JPG image, with ‘-red’, ‘-green’, and ‘-blue’ added to the filename as appropriate.
Recombine these into a new RGB image with the red and green channels switched with each other, save as ‘-switched’.
Email all 5 files to kaedwar@ilstu.edu.
