The Zeiss Elyra S.1 uses the technique of structured illumination (SR-SIM) to achieve resolutions up to two times better than that of a confocal, deconvolution or standard fluorescence microscope. Resolutions in XY of 110nm and in Z of 300nm are possible (resolution is wavelength and sample-preparation dependent), allowing users to image a volume in the sample that is 1/8 that of previous techniques.

The good news is that this super-resolution technique is a not-too-difficult jump from the dyes and sample prep techniques used for a confocal or deconvolution microscope. The Elyra S.1 can image four fluorescent colors, roughly corresponding to DAPI, FITC/GFP, Rhodamine/RFP, and CY5 (see below). SR-SIM can image to depths of 10-15 microns into a sample. Note: SR-SIM does not work well (or at all) with dimly fluorescent samples or samples that photo-bleach.

The Elyra S.1 has the following microscope objectives:

• Plan-Neofluar 10x/0.30
• Plan-APO 40x/1.4 Oil
• Plan-Apochromat 63x/1.40 Oil
• Plan-Apochromat 63x/1.30 Oil/Water/Glycerine KORR
• Alpha Plan-APO 100x/1.46 Oil

The laser lines are 405, 488, 561, and 642nm. Imaging can be performed somewhat faster using a single multi-band cube, or with 4 sequential cubes to reduce spectral overlap:

• EF LBF 405/488/561/642
• EF BP 420-480 / BP 495-550 / LP 655
• EF BP 495-550/BP 570-620
• BP 420-480/LP 655

This Windows 10 workstation (HP Z820 with 8 Xeon cores and 128GB of RAM) is set up to run HCImage (Hamamatsu). HCImage can be configured with macros that allow the software to do semi-automated image analysis (using thresholding and segmentation) of image data from the DMI6000 microscope, or images from other sources. For calibrating images from other optical microscopes, Mr. Cromey has a stage micrometer that users can borrow. Numerical data from image analysis measurements can be exported as CSV files for further analysis (CSV files can be imported into MS Excel).

To use the workstation, contact Mr. Cromey first to discuss your image analysis needs. The workstation is free to use, however, if a macro needs to be built for your lab’s analysis, there may be several hours of billable labor involved to write it.

Other possible uses of this workstation include video editing and working with large files that might be too big to work with on standard lab computers/laptops. With the assistance of our IT team, the data directory on the Zeiss Axio Observer 7 with Apotome III and the Leica DMI6000 have been mapped so that they can be read on this workstation (read only access), allowing users to pull their data directly to this computer worktation if needed.

Software currently installed on this workstation (Jan 2023):

• HCImage 4.7 (initially purchased by the SWEHSC, P30 ES006694)
• ImageJ (FIJI) (open source image analysis software)
• Cell Profiler (open source image analysis software)
• Qu-Path (open source whole slide image analysis software)
• Free viewer software (Zeiss ZEN Blue, Nikon Elements viewer, Leica LAS X, Olympus viewer software, Aperio Scanscope)
• Adobe Creative Cloud (including Photoshop, Premier Pro) - (NOTE: you may need to have a license for these associated with your UA netID, if needed you can request access to Doug Cromey's license)
• Image data management resources: 
  • OMERO.insight (open source software for connecting to the campus OMERO server)
  • CISCO AnyConnect (required for the UA VPN)
  • Glencoe NGFF-converter (capable of batch transforming specific proprietary file formats into OME-TIF files, which may be required before they can be uploaded to the campus OMERO server)

This mulitfunction, fully-motorized inverted microscope can capture both 24bit color transmitted light images, and 16bit epi-fluorescence images (up to 4 channels).

The microscope includes a 5Mpix camera for color brightfield images (Leica DCF450), as well as in DIC (Nomarski), and Polarization [Note: crossed polars only, this is not a full-on POL microscope]. The sCMOS greyscale camera (Hamamatsu Flash 4.0) can capture 4Mpix images in 16bits at up to 30fps. The microscope has fluorescence cubes for dyes similar to DAPI, FITC/GFP, Rhodamine/TRITC/RFP, and CY5 (to check your dyes with our filters, see our page at FPbase). The microscope has 2.5x, 5x, 10x, 20x, 40x dry objectives and 40x, 63x, 100x oil objectives, as well as a 1.0/1.6x optivar for intermediate magnifications (the 2.5x and 100x oil objectives are available, but not routinely mounted on the microscope - please contact Doug Cromey if you need these objectives). The stage can accommodate microscope slides (both the standard 1x3 and the larger 2x3), multiwell plates*, and small culture dishes (35mm, 70mm)*. We have a BiopTechs Delta-T live cell culture dish controller to allow for long-term live cell imaging at 37 degrees C.

Capturing images is fairly easy and the Leica LAS-X software is able to allow users to stitch together multiple fields of view, creating a much larger image (color or greyscale cameras).

In addition to the Leica software we have added stereology software to this microscope. Stereology is a rigorous form of mathematical sampling for image analysis. Use of this software will require additional training.

* low mag images through plastic dish bottoms are possible, but higher magnification image capture or DIC/POL images require the use of a coverslip thickness (#1.5, 0.170mm) glass bottom on the dish.