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Lab 1: Point Spread Function Determination

Lab 1.  Microscope training and Point Spread Function Determination

Lab 1 will most likely be the most intensive lab out of the four. If necessary, I will finish the microscope training at the beginning of Lab 1. Fortunately each microscope uses the same operating software with slight variations and their configurations are a bit different. This lab will have a pretty clear protocol (hopefully) for you to follow, to help facilitate your microscope use. In this lab, you will acquire images of sub-resolution green beads using a 100x objective under multiple step sizes (for the A1R) or bin factors (for the NSTORM and Spinning Disc Confocal). 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 as a function of z. 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.

 

Pre-lab Reading and Websites of Interest:

Lab 1 will be largely based off of the 2011 Nature Protocols article written by Richard Cole, Tushare Jinadasa, and Claire Brown. The paper explains in detail the basics of a PSF, its uses, and the overall implications of its shape. Please click here to download it.

Zeiss On Your Campus is a web series that describes light microscopy in significant detail, two of which are highly relevant to this week's lab. The first discusses the Numerical Aperture of an objective and its affect on image quality (click here), and the second demonstrates the change in PSF shape and its implications (click here).

Finally, Claire Brown also describes in detail the effects of over and under sampling and other pitfalls of light microscopy. This is a cool poster (click here).

 

To do:

1.  Learn how to operate the microscope(s).  Please read the A1R manual before the start of Lab 1.

2.  Image 100nm beads (green fluorescence, x10 beads)

  • Using different step sizes/camera bins (x3, including Nyquist sampling for the A1R)
  • Using 100x Objective
  • Do at least one high-resolution z-stack

3.  Using Matlab (please work on this independently at first, then work with your groups and others):

  • Fit the intensity profile of each bead
  • Calculate the average full-width at half-maximum intensity and the standard deviation for the beads
  • For the adventurous: Plot the z-axis profile of the z-stack

4.  Your mFile should produce all of the fully annotated (axis labels, other things) graphs and plots.

5.  Please deposit your fully annotated mFile into the network drive. Three of you will present your mFiles.

The protocols for the lab can be found below:

 

 

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