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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. 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.

 

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 Thursday, August 24.

2.  In groups, image 1um green fluorescent beads (x10):

  • Using different step sizes/camera bins (x3, including Nyquist sampling for the A1R)
  • Using different objectives (x2)
  • Do at least one high-resolution z-stack

3.  Repeat above using 100nm beads (green or red fluorescence) (x10)

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

4.  Using Matlab (please try 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

5.  In groups, summarize your results and discuss the implications of your findings (no more than a couple of pages including the figures).  There is no need to write up your methods as we are fully aware of how this was done.  The focus for this write up will be the results and discussion.

  • Fully annotated (legend; complete caption) figures that represent the raw data and typical curve fit of the 100nm and 1um beads.  Please include in the plot your FWHM line as well.
  • A fully annotated table summarizing your results (or more than one if you think it is necessary): Average and Standard Deviation of your fits as a function of the above variables.
  • 1 - 2 pages discussing your results and their implications within light microscopy.  The main discussion should be around how the psf shape changes as a function of wavelength, objective, and camera bin, and what this implies when imaging samples like microtubules, actin filaments, nuclei, cell membrane, bacteria, ...

6.  Please deposit your reports and your fully annotated mfile into the network drive.

 

 

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