Superresolution microscope

Superresolution microscopes are designed to provide details that go beyond the limit of resolution, around 200nm, imposed by diffraction of classic microscopes - confocal or widefield.

With the appropriate experimental set up, you can resolve objects below 120nm. The superresolution technique available in IRR are Structural Illumination Microscopy (SIM) and Total Internal Reflection Fluorescence (TIRF) microscopy.

SIM works by illuminating the same object repetitively with a moving grid or point pattern of light.  The combined signals are mathematically processed in Fourier space, with out of focus light discarded and higher resolution light reconstructed.  Structured illumination and processing in Fourier space are not restricted to high magnification imaging, they can also be implemented at low magnification.

In TIRF microscopy, laser excitation of the sample is at a critical angle such that the laser light runs parallel to the interface between coverslips and the specimen (usually culture medium).  As only the first 100-200nm from the coverslip are illuminated, and the rest of the sample stays in darkness, a high signal to noise ratio is achieved.  TIRF is best used for events localised at the plasma membrane.

Zeiss Elyra

The Zeiss Elyra is a dual camera microscope capable of structured illumination at low magnification (Apotome mode, 10x, 20x, and 40x lenses) and in SIM and TIRF modes at high magnification (63x).  It is capable of very fast scanning in Leap mode (volume) and Burst mode (single focal plane).  The Elyra has four lasers at 405, 488, 561, and 642nm.  There are two filter sets that allow combinations of DAPI, GFP, RFP, and Cy5 imaging.  Contact us before usage to check for compatibility.  

Special applications and features:

  1. Apotome and  and SIM mode microscopy delivers clean and crisp images of samples
  2. TIRF mode for superresolution by the basal plasma membrane
  3. Superresolution of samples below 100nm in SIM mode
  4. Superresolution of events by the plasma membrane near the coverslip in TIRF mode
  5. Leap mode for fast volume acquisition
  6. Burst mode for very fast slice acquisition in one or two concomitant channels
  7. Live Imaging in environmental chamber

Note that the Elyra strictly requires glass bottom dishes (no polymer) or coverslips.  The facility is happy to provide these at low cost for your pilot experiments.

 

Location: IRR Imaging Hub, contact IRR.Imaging@ed.ac.uk for access

 

For other superresolution techniques, we advise to contact our colleagues at IGC who co-host ESRIC, the Edinburgh Superresolution Imaging Consortium. 

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