New user project questions

Thanks for contacting us about using the imaging facilities we offer. Before going to a microscope system, we require a face to face meeting with you:

Since you are a new user and/or this is a new imaging experimental project, we will need to work with you in order to help you:

• Carefully define the imaging experiment with a testable hypothesis.
  • "I just want to see what it looks like" is not an experiment - you will be asked to go and think hard about that.
  • It's very likely that you will need control samples and images of them.
• Select appropriate microscope systems able to get the info you need from the samples to test the hypothesis
  • We have many tools at hand. The hammer (point scanning confocal) is a great general tool, but its not always the most suitable.
• Figure out sample preparation/mounting/environmental control
  • You need to know the spectral details of the dyes and understand the biochemistry you are using for your staining/preparation.
  • High resolution images can only be gotten from well prepared and properly mounted samples
  • Environmental control can be tricky to set up and optimise - dont underestimate the complexity.
• Determine optimal imaging hardware settings to get the best image quality / speed tradeoff.
  • Your first images of a sample will hardly ever be THE final images
  • Expect it to take some training, patience, and effort to become able to use a new microscope system properly. These are not point and shoot cameras.
  • You are going to need to spend considerable time fine tuning the setup to get optimal image data.
  • DON'T RUSH - make an estimate of how long the imaging experiment will take, then multiply it by at least 3x.
    • "But I need the images for my TAC on monday!" is a bad way to attempt an imaging experiment - in our experience this rushed approach will be ineffective and traumatic.

Together we will find the best and/or most sensible way to get the image Information you need to test your hypothesis. During the new user project meeting the answers you give to the below questions will be the basis of the discussion.

Please come to our upcoming meeting having already replied by email to LMF@mpi-cbg.de with answers to the following questions. You probably should discuss these specific points with your adviser / supervisor, who might also want to come to the new user project meeting. (This is not supposed to be yet another form to fill in, rather, an exercise to get you thinking about what you really want to do, and get it done faster and better!)

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First we have to start with your "Experimental Hypothesis": Please try to explain to us the following:

1) What is your experimental hypothesis?

2) How will you test it - what info do you need?

3) How will you get that info - what will you measure ?

4) ...and how will you analyze the info you get in order to prove your hypothesis right or wrong?

5) What controls will you need?

6) What statistical test will you use to test your hypothesis?

7) Are you interested in colocalization analysis?

All these affect the way the images are best collected: on what kind of microscope and with what hardware settings / objective lens etc.

The laser scanning confocal microscope is very useful, but its not the only tool we have! Don't be afraid to open the toolbox and see what microscopes are inside...

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Second, we can talk about "Sample Preparation, Dyes and Mounting" The answers to the questions in the Experimental Hypothesis" section will help determine some of the answers here:

1) What is the sample? Organism? Tissue? Cell Type? Living? Fixed?

2) How is it prepared? Grown on glass? Tissue section? Living Embryo?

3) How is it mounted? Under a (proper exactly 0.17 mm) coverglass? Underwater in an agarose well? In an agarose column? On a filter/carrier/special chamber?

4) What Staining / Dyes / contrast agents will you use? Fluorescence and/or transmitted light? Electron beams?

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The third task is to use the answers from parts 1 and 2 to select appropriate microscope systems which will be able to get the information you need out of your samples in a sensible and efficient way.

If the only tool you have is a hammer - every problem begins to resemble a nail.

We have a large toolbox of different kinds of microscopes ... lets take a look inside!

1) Do you need 2D (single focal plane) or 3D images (z stacks)?

2) Do you need time series? If so, what time resolution / frames per seconds do you need?

3) Do you need high Signal : Noise (see the difference in intensity accurately), or will somewhat noisy images work for you (just see where stuff probably is)

4) Do you need high dynamic range (see very dim and very bright things in the same image at the same time)?

5) What is the size of the field of view that you need?

6) How big are the objects you are interested in? What (optical) resolution do you need (what should the pixel spacing at the sample be)? 5 nm? 200 nm? 1 micro meter? Bigger? (Hint: Theory says you need no more than 2.3-3 pixels across the object of interest in order to image it properly)

cheers your imaging facility team