Super-resolution Microscopes

The second class of super-​resolution methods involves spatially selective deactivation of fluorophores in samples using a so-​called 'donut beam'. In STimulated Emission Depletion (STED) microscopy, one minimizes the volume of fluorescence emission at the focal point, thus enhancing the achievable resolution. Depending on settings and labeling, a resolution of up to ~50nm could be achieved. Our in-​house setup is the following:

Aberrior STEDYCON

The Abberior STEDYCON setup combines a super-intuitive acquisition software with a compact but capable 2D STED system capable of achieving down to 30 nm resolution (external page specific dyes required). With only a few clicks needed between a sample overview and the first STED image, this system offers the easiest transition from a point-scanner confocal into the super-resolution world.

• Microscope base: Zeiss Axio Observer Z1 (inverted)
• Available excitation lasers: 405, 488, 561 and 640 nm
• Available STED lasers: 775 nm
• Detectors: 3 filter-based APDs capable of photon counting and gated detection
• Objectives: 100x

The last family of super-​resolution methods at SCF, Single-​Molecule Localization Microscopy (SMLM) can dramatically improve spatial resolution over standard diffraction-​limited imaging techniques, with localization precision in the single nm range. To achieve this, the emission of distinct molecules is separated over time (through photoswitching, photoactivation or photoconversion), allowing the localization of individual molecules. The SCF is equipped with a MINFLUX Single Molecule Localization Microscope capable of achieving 1-3 nm localization precision in 3D and ultra-​fast single-​molecule tracking up to 10 kHz.

Abberior MINFLUX (MINFLUX)

The Abberior MINFLUX microscope defines an entirely new class of super-​resolution methods that combines the strengths of the single molecule localization family and STED microscopy. Individually activated fluorescence-emitting molecules are localized through an iterative scanning approach using an excitation beam with a dark center, which reduces the number of emitted photons required per localization.

MINFLUX can routinely achieve single-​digit nm localization precision along x, y and z axis. Thanks to its photon efficiency, multiple localizations can be generated during a single emission event. This, together with the variety of metrics acquired during the localization process, enables post-​processing statistical analysis and filtering.

The instrument can operate in two modalities:

• Localization mode: Either 2D or 3D, the emitters in the field of view are localized sequentially one by one, until the underlying structure is reconstructed.
• Tracking mode: A single fluorophore is tracked in 3D over time with a frequency up to 10 kHz yielding the track of the movement of this molecule with an extraordinary high spatial and temporal resolution.
  

Setup:

• Microscope base: Olympus IX83 (inverted)
• Available lasers (point-​scanner confocal mode): 405, 488, 561 and 640 nm
• Available lasers (MINFLUX mode): 561 and 640 nm
• Detectors: 5 filter-​based APDs. 2-​Color MINFLUX can be performed for some compatible dye pairs
• Active sample stabilization system with sub-​nm precision
• Objectives: 20x, 40x and 100x oil immersion (for MINFLUX mode)

More information on the manufacturer website external page here.