Information regarding the Elyra S.1
There are three major classes of so-called "superresolution" optical microscopes. Localization microscopy goes by acronyms such as PALM or STORM. STED is a technique used exclusively on Leica microscopes. The technique that we selected for the UA is called Structured illumination microscopy (SIM). While there are several vendors with SIM systems, we selected the Zeiss Elyra S.1 based on a strong on-site demonstration held at the University of Arizona from Dec 2013 through Jan 2014. Dr. David Elliott (now retired, Cellular & Molecular Medicine) submitted what became a successful NIH Shared Instrumentation Grant. The UA Office of Research, Innovation & Impact (RII) leveraged the grant into a request for proposal in the summer of 2015 that ultimately brought three new high-end instruments to the University.
Learn more about superresolution online from Zeiss. The JCB had a very nice Superresolution review article in 2010.
Advantages of SIM
- 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 improves the image resolution in both XY (lateral) and Z (axial). The others may have better XY resolution, but their axial resolution is usually similar to that found in a confocal microscope (~1.5 times the wavelength).
- SIM can use the same antibody labeling or fluorescent protein 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 proteins.
- SIM can image up to 10-15um into tissue. The others typically have depth limitations of 10um or less.
Limitations of SIM
- 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. (See additional information below)
- Because of the amount of intense light used to acquire SIM images, fluorophores that bleach easily do not work well with this technique.
Preparing samples for SIM
While sample preparation is similar to what might be done for widefield, deconvolution or confocal microscopes, a higher attention to the small details will give the best results. The truth is that attention to these sorts of details will improve image quality in all types of fluorescence microscopy. Doug Cromey has expanded on a sample preparation handout originally prepared by Zeiss Imaging Specialist, Bryant Chhun. See: "Sample Preparation Guidelines for Superresolution Structured Illumination (SR-SIM)"
The #1.5H high performance coverslips can be challenging to find. Doug Cromey has a handout regarding coverslips that includes links to vendors of the the #1.5H thickness.
- Keep it clean! - Excess mounting media, fingernail polish, oil from a different brand of microscope, or fingerprints on the coverslip will adversely effect the imaging. Please make sure the coverslip is scrupulously clean before bringing your sample to the SIM microscope.
- Mounting media - Since we are pushing the optical limits, everything has an effect on the final image, including the refractive index (RI) of the sample mounting media. Bubbles in the mounting media are not good, be careful when assembling your slide. If you seal the edges of the coverslip with fingernail polish. Make sure it is dry before you come to the SIM microscope.
- Mounting sectioned tissue - With all of the superresolution methods, the closer the sample is to the coverslip, the better the images are. To get the best SR-SIM images you may want to mount your sections on the coverslip - please be aware that #1.5H coverslips are typically not coated or "charged". This means that the sections could float off during immuno-staining. There are a number of ways to improve "stickiness", including a thin coating of poly-L-lysine.
- Test your samples on a fluorescence microscope first to ensure that they work - SIM microscopy needs bright samples with good signal-to-noise to capture images that can be processed. If your sample is not bright when imaged by other methods, images captured using SIM will not provide the clarity needed to obtain the best resolution.
- The SIM microscope is fluorescence based, and uses specific laser lines and filters - Make sure that the dyes you will be using match the capabilities of the SIM instrument. The instrument has 405, 488, 561*, and 642 laser lines. Filter specifications are on the instrument page or in the sample preparation handout. If you have questions about the suitability of your fluorescent dyes, please contact Doug Cromey. *Please note that the 561nm laser line is a bit longer of a wavelength than the 543nm laser that is used on many of the campus confocal microscopes.
Working with your acquired data
We highly recommend the purchase of an external hard disk drive in the 2-4TB size range. A single unprocessed z-stack can be several GB in size.
The Elyra S.1 uses a proprietary file format (as do most of the high-end microscope vendors) to store the multi-dimensional data the instrument acquires. There are several options for opening the SIM processed data files:
- Download and install the free Zeiss ZEN lite software for Microsoft Windows. Please note, this is a large software file, so we will have copies of it available in the facility.
- Another option is to use the free FIJI (Windows/MacOS/Linux) software (a variation of ImageJ that was intended to work better with 3D data).