University of Arizona
spacer

Superresolution Optical Microscope
NIH Shared Instrumentation Grant (S10) proposal

Overview | SIM information | Benefits | Limitations | Example Image | Comparing Superresolution techniques | Sample Prep

 

Contacts:

PI: David Elliott, Ph.D., 626-7870
Cellular & Molecular Medicine

Doug Cromey, M.S., 626-2824
Microscopy core facility manager

Online SIM Images:

Educational:

Overview:
Superresolution is a broad term used to describe several relatively recent techniques that have allowed optical microscopes to obtain image resolutions better than the current optical limit (one-half the wavelength of light being used, the "Abbe limit"). These techniques are typically referred to by the acronyms; PALM/STORM, STED, or SIM. For an excellent, not overly technical, review that explains and compares these techniques, please see: A guide to super-resolution fluorescence microscopy (2010), JCB 190:165-175.

All of these techniques require specialty and often quite expensive instrumentation. In addition, careful attention to sample preparation becomes crucial as these techniques push the limits of optics.

 

Structured Illumination Microscopy (SIM):
After evaluating the different superresolution techniques, we feel that a SIM instrument is the best fit for a shared instrument facility that would meet the diverse needs of the UA research community. Our reasoning:

  • SIM is the only technique that allows for up to 4 color fluorescence images at superresolution. The others are limited to 2 colors at superresolution and additional colors will be either at epifluorescent or confocal resolutions.
     
  • SIM is the only technique that improves the image resolution in both XY (lateral) and Z (axial). The others may have better XY resolution, but their axial resolution is similar to that found in a confocal microscope (~1.5 times the wavelength).
     
  • SIM can use the same antibody labeling or fluorescent protien labeling protocols already in use in most UA labs that are using epifluorescence, deconvolution, confocal, or multiphoton microscopes. The others require specialty dyes or photoactivatable fluorescent protiens. (See below for more on sample prep)
     
  • SIM can image up to 10-20um into tissue. The others have depth limitations of 10um or less.

 

The benefit of SIM superresolution microscopy:

  • Demonstration SIM image (sample from the Carol Gregorio lab) showing routine epifluorescence, deconvolved, and structured illumination of the same field of view.
     
  • Current optical microscope technologies are limited to lateral (XY) resolutions of approximately one-half the wavelength used to image the sample. As an example: the 480nm light typically used to excite Fluorescein (Alexa488, eGFP) and imaged through a 63x/1.4NA objective provides a lateral resolution of approximately 240nm with an axial resolution of ~720nm (assuming carefully optimized sample prep). Using SIM would yield a 2x improvement in lateral and axial resolution (at 480nm excitation the resolution would be lateral ~120nm, axial ~300nm). This improved resolution means that the XYZ volume (voxel) being imaged is 8x smaller than what is imaged using a confocal microscope. The smaller voxel size means that colocalization accuracy is greatly improved.
     
  • The kinds of biological structures that can be imaged in greater detail at these resolutions include:
    • Structural protiens
    • Vesicles
    • Dendritic spines
    • Bacteria and other microorganisms
    • Cilia and microvilli
    • Mitochondria
    • Chromosomal interactions
    • Resolving proteins that are close to each other

 

Limitations of SIM superresolution microscopy

  • In general the superresolution techniques are not fast. SIM needs to take multiple images at each focal plane and then multiple focal planes (Z stack). These images are then mathematically processed to obtain the final superresolution images. SIM is not well suited for live cell imaging because of the slow image acquisition process. Fixed cells or sectioned tissue are more appropriate samples for a SIM instrument.
     
  • All of the superresolution techniques expect careful attention to sample preparation. This means using the newer high-performance #1.5H coverslips (lower variability in thickness), fewer choices with mounting media, and samples mounted on the coverslip rather than the slide (including sectioned tissue). The coverslip needs to be very clean, samples should be fresh (not subject to freeze/thaw cycles) and most likely you will only be able to mount one coverslip/slide.
     
  • Because of the amount of intense light used to acquire SIM images, fluorophores that bleach easily do not work well with this technique.

A tip of the hat to the Microscopy Unit at A•Star Institute of Medical Biology (Singapore) for most of the SIM sample prep tips.

     

     

© 2013, Arizona Board of Regents
Douglas Cromey, Cellular & Molecular Medicine, University of Arizona College of Medicine