Zeiss Elyra 7 with SMLM and SIM super-resolution

 

The AMI super-resolution fluorescence microscope is capable of capturing incredible biological detail. Visiting researchers can use the super-resolution microscope to gain insight into sub-cellular architecture, protein assemblies, and biological interactions. Samples containing synaptic junctions, pathogen-host interactions, organelle structure, and many others, can be imaged with one of two super-resolution approaches.

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Structured Illumination Microscopy

Structured Illumination Microscopy (SIM) employs frequency information to calculate a final image with two-times greater resolution than a standard fluorescence microscope. This results in an eight-fold improvement in all three dimensions. On top of being capable of imaging samples in 3-D, SIM can also be used to capture live-cell processes with low-to-moderate photobleaching and/or photodamage. The AMI super-resolution microscope has an incubator that allows samples to be imaged in a temperature- and CO₂-controlled environment. This makes the microscope perfect at studying live cell cultures in enhanced detail with 120-80nm resolution.

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Single Molecule Localisation Microscopy

Single Molecule Localisation Microscopy (SMLM) over-comes the natural resolution limit by calculating the location of single, clearly separated signals produced by single molecules. Detecting spatially separate fluorescence signals within biological samples which are complex and dense is where the magic of SMLM becomes apparent. Using a variety of methods (i.e., PALM, STORM, dSTORM, PAINT, etc.—collectively called "single molecule localisation"), detection of signal can be separated in time rather than in space. This leads to the "blinking" of molecules which can be captured across many thousands or hundreds-of-thousands of sequential images. A more precise location of each individual blink can be calculated to produce resolutions of 70-20nm. Following reconstruction, samples can be seen with detail approaching macromolecular scales. A prerequisite for this is that the sample is fixed so that no spatial changes occur during the long acquisition process. This makes SMLM most suitable at imaging thin samples that have been fixed. 

 

Instrument specifications

• Inverted microscope configuration

• Auto focus for time-lapse acquisitions of live samples

• Contrast methods:

  • bright-field transmission

  • differential interference contrast (DIC)

  • fluorescence

• Environment control:

  • Temperature range of 20-42°C

  • Up to 5% CO₂

  • Relative humidity

• Objectives:

  • 100× 1.46 NA oil-immersion with correction collar and DIC

  • 63× 1.46 NA oil-immersion TIRF

  • 63× 1.4 NA oil-immersion with DIC

  • 40× 1.2 NA water-immersion with correction collar

  • 25× 0.8 NA multi-immersion

  • 10× 0.5 NA

• Laser excitation:

  • 405nm 50mW ■

  • 488nm 500mW ■

  • 561nm 500mW ■

  • 642nm 500mW ■

• Emission filter sets:​

  • Zeiss FL Filter Set 77 HE (GFP■/mRFP■/Alexa 633■)​

  • Zeiss FL Filter Set 25 (400■/495■/570■)

• Camera pco.edge sCMOS monochrome camera

• Andor EM-CCD camera iXon Ultra 897 monochrome camera

• Automated stage control with piezo X-, Y-, and Z-control

 

Instrument capabilities for live cells (SIM)

• Resolution: down to ~100nm (~60nm with SIM²)

• Speed: down to 250ms an image, and 3-10s for full 3-D volumes

• Depth: max. ~100µm

• Field of view: 2mm to 200µm

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Instrument capabilities for fixed samples (SMLM)

• Resolution: down to ~20nm

• Speed: 5 minutes to 3 hours per field of view

• Depth: max. ~40µm

• Field of view: 2mm to 200µm

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Instrument uses

• Optical sectioning ("Apotome")

• TIRF

• SMLM

• SIM

• Live-cell imaging

• Enhanced SIM/SIM deconvolution (i.e., "SIM²")

• Tiling and stitching (for large fields of view)

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