Summary: Designing a Fluorescence Microscopy Experiment

Talk Overview

With all the microscopy techniques now available, which one should you use for your experiment? Here we describe some considerations to take into account when designing a microscopy experiment including: choice of microscope, choice of dye or fluorescent protein, and using confocal, TIRF, or widefield.

Questions

You would like to obtain a high-resolution image on a thin, 10 µm sample. Which of the following statement(s) is true? (Select all that apply)
1. Using a confocal over a widefield microscope will increase the resolution of the image
2. Using a high NA over a low NA will increase the image resolution
3. Using a spinning disk confocal over a laser-scanning confocal will be preferable if this is a live sample
4. None of the above
In his talk, Dr. Thorn explains that the resolution of an image can be calculated based on the following mathematical formula: dmin = 0.61 λ/ N.A. Would you agree or disagree with the following statement: “The higher the NA, the higher the image resolution.” Explain why.
You are in charge of the microscopy facility at your institution. An investigator comes to you and explains that, after several months of research, she finally identified a lab which has a transgenic mouse colony expressing mCherry fusion proteins for the gene she is researching. mCherry is a monomeric red fluorescent protein (RFP) that is excited with a 561 nm yellow-green laser. However, you do not have this laser line at your facility. Should you recommend that this investigator not move forward with her study until she finds transgenic mice that match the laser lines available at your facility? Explain your answer.
You are in the process of designing an experiment which will involve long-term, high-resolution 3D live cell imaging on a 10 µm sample, and involving fusion proteins with mCherry. List 3 pieces of equipment that you will need to check for before you begin your study.
In the list below, choose the steps that could improve data analysis in the long run:
1. Using fewer than 4 dyes at onc
2. Determining whether protein localization is impacted by fixing, mounting or labeling prior to beginning your study
3. Reducing the amount of light to which you are exposing your samples
4. Including controls that will correct for variations during data collection
5. None of the above

Answers

View Answers

B, C
1. Incorrect – Using a confocal microscope will NOT increase the resolution of the image.
2. Correct – Using a high NA over a low NA will increase the XY resolution.
3. Correct – Using a spinning disk confocal over a laser-scanning confocal will be preferable if this is a live sample because
Agree. dmin stands for the smallest distance separating two distinct objects. A high resolution image allows one to distinguish between two objects that are very close together, and therefore a high resolution image will have a low dmin. Therefore, the higher the NA, the lower the dmin, and therefore the higher the resolution.
In his talk, Dr. Thorn explains that he recommends checking that the dyes used when labeling samples match the laser lines available at the design stage, before launching an experiment. However, he does explain that in certain conditions, such as with fusion protein constructs in transgenic mice, the availability of laser lines cannot be taken into account. In this case, the investigator should probably look for the right microscope or look into buying a laser line for an existing microscope at her or other institutions so she can complete her study.
i) A spinning disk confocal to rapidly image a thin, live specimen;
ii) An environmental chamber, which is used for live cell work. It allows the investigator to control for temperature, CO2 levels, and humidity to keep the specimen alive during image recording;

iii) The appropriate laser line for mCherry (in this case, 561 nm yellow-green laser)
A, B, C, D
1. Correct: Using more than 4 dyes at once may result in crossover in light detection.
2. Correct: Protein localization can occasionally be impacted by fixing, mounting or labeling, so it is important to determine if they do prior to beginning your study.
3. Correct: Reducing the amount of light to which you are exposing your samples will avoid (or lessen) photobleaching and phototoxicity issues.
4. Correct: Including the right controls will allow to correct for day-to-day variations during data collection (due to lamp power and alignment, for example)

Speaker Bio

Kurt Thorn

Kurt Thorn is an Assistant Professor of Biochemistry and Biophysics at UCSF and Director of the Nikon Imaging Center – a facility that provides cutting edge light microscopy equipment to UCSF researchers. Kurt can be followed on his blog at http://nic.ucsf.edu/blog/. Continue Reading